Pretreatment of pine and poplar particleboards with Pleurotus ostreatus (Jacq.) P. Kumm: wood structure decomposition, potential of solid fuel and biogas production

doi:10.21203/rs.3.rs-5048807/v1

The study evaluated the effects of biological pretreatment on comminuted pine and poplar shavings and particleboards with urea-formaldehyde resin (UF), utilising Pleurotus ostreatus. The pretreatment notably reduced lignin content by 6.8–8.3%, enhancing the biomass's mechanical and agglomeration properties, thus confirming the initial hypotheses. Values for the specific compaction work of treated biomass were higher than those of raw biomass (24.03 kJ·kg^–1 vs. 21.70 kJ·kg^–1), correlating with the production of denser pastilles (1014 kg·m^–3 vs. 959 kg·m^–3). After pretreatment, enhanced structural properties of the biomass facilitated increased methane yields, showing up to a 3.7-fold increase for pine and 2.9-fold for poplar UF particleboards. This research advances the potential for developing recycling and biogas technologies, offering novel insights into UF degradation via fungal pretreatment. The findings underscore the necessity for further detailed studies to analyse changes in resin content post-pretreatment and their impact on the properties of wood materials.

comminuted shavings mechanical properties

pressure agglomeration

pastille

fibrous substances

methane yield

The production of particleboards globally has nearly doubled from 1995 to 2021, reaching 103,955,051 m³, highlighting a growing challenge in waste management for this type of biomass (FAOSTAT 2024). Mainly, waste particleboards containing UF are significant challenges for the environment and recycling technology (Ihnat et al. 2020). Following the Directive of the European Parliament and the Council (2008/98/EC), which emphasises waste prevention, preparation for reuse, recycling, and other recovery methods over disposal, the management of particleboard waste has become increasingly crucial (EP 2008). Nevertheless, existing disposal methods, such as landfilling or thermochemical conversion, present considerable limitations and disadvantages (Karade 2010).

UFs, although widely utilised because of their low cost and effective binding properties (Baharuddin et al. 2023), present significant challenges in removing wood waste. Consequently, recycled biomass often shows deteriorated mechanical properties, restricting its further application (Besserer et al. 2021). Moreover, the storage of particleboard waste poses environmental hazards bringing the risk of soil contamination and the gradual release of formaldehyde into the atmosphere(Wang et al. 2020).

Given these challenges, it is essential to explore new methods for managing particleboard waste that are both effective and environmentally sustainable. A promising approach is the biological pretreatment using white rot fungi such as the oyster mushroom (Pleurotus ostreatus) (Bellettini et al. 2019). These fungi can delignify lignocellulosic biomass, potentially enhancing the accessibility of cellulose and hemicellulose for subsequent biodegradation (Kainthola et al. 2021). Oyster mushrooms can degrade particleboards containing UF by breaking down hemicellulose, cellulose, and lignin, aided by specific microbial species, particularly Actinomycetales (Liu et al. 2022). The enzymatic capabilities of oyster mushrooms and their potential for biodegradation of industrial wastewater, including lead tolerance and biosorption of heavy metals (Bhatnagar et al. 2021), showcase their broad potential. These fungi produce a range of industrially significant enzymes in paper production (Skočaj et al. 2018), such as cellulase, xylanase, lipase, peroxidase, amylase, protease, laccase (which degrades lignin) (Kumar et al. 2009), and manganese peroxidase (Bánfi et al. 2015). However, there is no information about enzymes produced during the degradation of UF. Enzyme activity varies with the different growth stages of fungi and in response to other substrates (Devi et al. 2024). Spent edible mushroom substrate is an economical and convenient resource for sustainable bioethanol production with efficient recovery of lignocellulolytic enzymes (Devi et al. 2022). Although UF acts as a binder in board production and provides fungal resistance (Amini et al. 2018), the oyster mushroom, being a robust fungus, has the potential to decompose, keeping in mind the mentioned mycoremediation potential. While the pretreatment with white rot fungi has been extensively studied concerning agricultural waste (Kainthola et al. 2021), applying it to waste particleboard containing synthetic resins is a novel area.

In managing waste from particleboards, the impact of biological pretreatment on the mechanical properties of comminuted biomass plays a crucial role, particularly in influencing the pressure agglomeration process. The oyster mushroom is a promising agent for the biological pretreatment of pine or poplar wood shavings intended for pellet production. Research confirms its efficacy in breaking down lignocellulosic components and enhancing substrate biodegradation (Girmay et al. 2016). Mechanical properties such as shear stress, cohesion, and both internal and external friction angles are influenced by biological pretreatment. This alteration can enhance the characteristics of pressure agglomeration (Lee et al. 2022). Moreover, studies have indicated that biological pretreatment reduces the average particle size of the biomass, which facilitates improved densification and results in pellet strength (Olughu et al. 2021).

Anaerobic fermentation presents an alternative approach, yet it cannot be excluded that UFs negatively impact microorganisms responsible for methane production, thereby possibly limiting the efficiency of this process (Wang et al. 2011). Previous studies have identified potential applications for oyster mushrooms as a biological pretreatment for pine or poplar chips in biogas production (Kumar and Sharma 2017). Notably, pretreatment with the fungus Peniophora incarnate T‑7 has been shown to reduce lignin content by up to 70% in poplar wood, resulting in a sevenfold increase in the yields of glucose, xylose, and biofuel (Ma et al. 2021). The oyster mushroom demonstrates potential as a biological pretreatment for wood chips, with proven lignin degradation, enhanced enzymatic saccharification, and increased biomethane yield from the spent fungal substrate (Vasilakis et al. 2023). However, further research is required to fully understand its impact on producing solid fuel and biogas and refine the processes for commercial applications.

The scientific question posed by this research is: Can biological pretreatment with the oyster mushroom effectively decompose pine and poplar shavings, particularly those in particleboards containing UF? Will pretreatment enhance the mechanical properties of the comminuted biomass, thus improving the strength of pastilles and the efficiency of biogas production? This study aims to identify the specific alterations in biomass's structure and chemical composition following biological pretreatment and to explore how these changes enhance pressure agglomeration processes and anaerobic fermentation efficiency. The primary scientific objective is to acquire novel, insightful information on the role of the oyster mushroom and evaluate the effectiveness of its use in decomposing particleboard shavings, its impact on the mechanical properties of biomass, and the efficacy of pressure agglomeration and biogas production processes. Ultimately, the research seeks new insights and knowledge regarding the potential advantages of biologically processing particleboards for producing solid fuels and biogas.

The research hypotheses formulated for this study are as follows:

Biological pretreatment within a specified timeframe using the Pleurotus ostreatus leads to significant decomposition of lignin and hemicelluloses in wood shavings and particleboards, thereby increasing the availability of cellulose and enhancing the mechanical properties of the comminuted biomass.
Structural and chemical alternation during the pretreatment of wood and particleboard with Pleurotus ostreatus will improve the processed material's mechanical properties, thus increasing compaction capacity during the pressure agglomeration process, resulting in greater pastilles strength. The material alternation will also facilitate more effective anaerobic fermentation in biogas production.

The article's novelty resides in its unique approach to addressing the ecological utilisation of particleboard waste containing synthetic resins. Previous research has predominantly centred on mechanical and chemical recycling methods and transforming particleboard waste, whereas biological pretreatment with white rot fungi has not yet been harnessed for these materials. Moreover, this study offers a comprehensive analysis of the impact of biological pretreatment on the mechanical properties of the waste, the efficiency of the pressure agglomeration process, and the parameters of biogas production. This represents a substantial contribution to the advancement of environmentally friendly technologies.

2.1. Material

The source material comprised two types of shavings: one was dedicated to producing particleboard inner layer and consisted mainly of pine wood. These shavings were later called “pine shavings”. The second type was poplar shaving from sawmill waste specially shredded to produce the inner layer in multilayer particleboards. These pine and poplar shavings were obtained from the Research and Development Centre for Wood-Based Panels, Ltd. in Czarna Woda. A detailed analysis of the physicochemical parameters of these shavings is outlined in the first article (Tryjarski et al. 2023), with the densification process and strength of agglomerates discussed in the second article of this series (Tryjarski et al. 2024). For the present study, wood shavings and particleboards with UF were comminuted using a knife mill (LMN-100, Alchem Group Ltd., Toruń, Poland) equipped with a 3 mm mesh sieve to ensure a uniform particle size distribution. Each batch type, including shavings and particleboards with UF, underwent 17 weeks of biological pretreatment using the Pleurotus ostreatus. The two wood species, TW; pine (PI), and poplar (PO), were differentiated into product types PT; SH shavings, and PB particleboards, and material state MT as raw (R) and after fungal pretreatment (T). Eight averaged samples were designated as follows: POSR – comminuted raw poplar shavings, POST – comminuted poplar shavings after pretreatment, POBR – comminuted raw poplar particleboards, POBT – comminuted poplar particleboards after pretreatment, PISR – comminuted raw pine shavings, PIST – comminuted pine shavings after pretreatment, PIBR – comminuted raw pine particleboards, PIBT – comminuted pine particleboards after pretreatment. An analysis of physicochemical properties was conducted for these eight materials to facilitate the interpretation and elucidation of features influencing pressure agglomeration and anaerobic fermentation.

2.2. Procedure for pretreatment of material with Pleurotus ostreatus

The medium, jars, and jar seals were sterilised in an autoclave at 126°C. Material samples weighing between 48–49 g, with moisture increased to 20–30% by the addition of demineralised water, were placed in polyamide nets and polyethylene bags, which were hermetically sealed. These samples were then sterilised using ionising radiation at a dose of 28 kGy at the Institute of Nuclear Chemistry and Technology (Andres and Wojciechowska 2017).

Pleurotus ostreatus mycelium, obtained from MycoLabs (MycoLabs Aleksandra Jarczok, Pszczyna, Poland), was inoculated into 0.5-litre preserving jars filled with a thick layer of Malt Extract Agar medium (Pol-Aura Ltd., Morąg, Poland). The reason to pour a lot of medium into jars was to prevent overdrying. These previously sterilised containers provided a secure environment for further mycelial growth. Lids, sealed with cellulose cotton wool encased in sterile gauze, were fitted to the jars, which were then placed in a bacteriological incubator (Incudigit 80, JP Selecta, Barcelona, Spain). A vessel of water was included to maintain humidity. The multiplication of the mycelium occurred at 24°C; upon complete coverage of the medium surface, the sample grids were introduced into the jars. These were then put into a climate chamber (2MXM, PHU Refrigeration, Warsaw, Poland), controlled by computer software to ensure optimal climatic conditions. The fungal pretreatment process extended over 17 weeks at a temperature of 28°C and a relative air humidity of 97%. Additionally, two water vessels were placed within the chamber to prevent sample drying, as verified in preliminary multi-week studies. Based on the literature (Li et al. 2018), the relative air humidity set at 75% proved too low, reducing the material's moisture from 24–10% after eight weeks. According to the literature, the required conditions for cultivating oyster mushrooms is 80–90% humidity, with temperatures ranging from 24–28°C (Setiawati et al. 2021), or even 80–100% and 20–30°C (Soares et al. 2022). However, for material pretreatment, lower relative humidity levels of 65–85% are expected (Mustafa et al. 2016). The effect of temperature and relative humidity on fungal activity is well-documented (Aghajani et al. 2018).

After the biological pretreatment, the compacted material samples were removed from bags by shaking and rubbing, and the material was rubbed to break agglomerates roughly. The material was air-dried for four weeks, and averaged samples from approximately a dozen identical original samples were prepared for further study. This pretreatment aimed to influence wood and particleboard’s physicochemical properties and prepare the material for pressure agglomeration and biogas production. By degrading materials’ components, Pleurotus ostreatus can potentially increase the material's porosity (Kumar et al. 2021), facilitating further agglomeration and enhancing biogas yield by improving substrate availability for anaerobic fermentation microorganisms.

2.3. Physical properties of comminuted material

For comminuted shavings and particleboards in both the raw state and after fungal pretreatment, physical parameters were determined to interpret better and explain differences in criterion parameters relative to the tested factors. These parameters included moisture content relative to the wet basis (MC), particle size distribution, external friction angle (δ), and higher heat value (HHV). Each of the eight averaged samples was stored in individual,

The MC was determined for samples weighing 2 g using the MA50/1.R moisture analyser (Radwag, Radom, Poland). The measurements were conducted over an extended period.

The particle size distribution of comminuted materials, with each sample weighing 80, 40, 70, 70, 20, 50, 100, and 50 g for PISR, PIST, PIBR, PIBT, POSR, POST, POBR, POBT, respectively, was conducted using a vibrating separator (LAB-11-200/UP, Eko-Lab, Brzesko, Poland), adhering to the ANSI/ASAE S319.4 standard. A set of sieves with mesh sizes of 0.15, 0.212, 0.3, 0.425, 0.6, and 0.85 mm and a bottom pan were used. These mesh sizes were chosen based on preliminary tests to ensure the middle sieve contained the most mixture, aiming to achieve a particle size distribution close to a normal distribution. A detailed description of the method used to perform this distribution and calculate the distribution parameters is available in the article about the physicomechanical properties of raw and crushed shavings (Tryjarski et al. 2023). Considering the research aims and article length constraints, the particle size distribution analysis included the geometric mean of particle size (x_g).

The angle δ for comminuted pine and poplar particleboards, both in raw form and after fungal pretreatment, was determined using a thin layer of material resting on a chromium steel plate. Three dies with opening diameters (d_m) of 6, 8, and 10 mm were made of chromium steel for pressure agglomeration. The plane's inclination, where the particle movement on the plate surface was recorded, was measured using an electronic protractor with an accuracy of 0.1°. The heat of combustion for all material samples was determined for dried material, as described in the article on pressure agglomeration of raw and comminuted shavings (Tryjarski et al. 2024).

2.4. Mechanical properties of the material: strength and flowability

The strength and flowability of comminuted pine and poplar particleboards, both in raw form and after fungal pretreatment, were determined using a direct shear tester. The tester featured an internal shear diameter d_m of 60 mm and a height of 170 mm. The procedure was carried out following the requirements of Eurocode 1 (Eurocode 1 2006) and is described in detail in the article on the physicomechanical properties of raw and comminuted shavings (Tryjarski et al. 2023).

2.5. Crude fibre, detergent fractions, and lignocellulosic

Chemical reagents were purchased from ANKOM Technology Corporation (USA, NY). Weighting was conducted with a moisture analyser with an accuracy of 0.1 mg. Crude fibre and detergent fractions were determined according to ANKOM 200 fibre analyser manual (A200 manual; Method 8 2022). Samples were kept with MC levels below 10% because moisture can dilute the added acid (NREL 2008). The material was comminuted in a knife mill to mitigate the risk of overheating. Particle sizes were maintained below 2 mm, with the size x_g below 1 mm, and the mixture was not sieved to avoid altering the overall sample integrity, as finer particles contain more mineral components.

Analytical samples and control alfalfa (ALF-2022-001) were sealed in mesh bags (F57, ANKOM Technology) with an electric welder. Sample mass was 0.95–1.00 g for crude fibre (CF) determination and 0.45–0.50 g for neutral detergent fibre (NDF), acid detergent fibre (ADF), and acid detergent lignin (ADL). Each set of 22 samples of the tested materials included two control samples and one empty bag to establish a correction factor for the empty bag (C₁) or the ashed bag (C₂). Since the tests were conducted in two series, two bags, instead of three, were randomly used for two biomass samples to assess all eight distinct materials within a single series while maintaining consistent test conditions.

During the determination of CF, samples were first extracted in a 0.255N sulphuric acid (H₂SO₄) solution, followed by a 0.313N sodium hydroxide (NaOH) solution. Both extractions were conducted at 100°C for 40 min using an ANKOM 200 fibre analyser (ANKOM Technology Corporation, USA, NY). After each extraction and the removal of the solution, the samples were rinsed twice in demineralised water at temperatures between 50–90°C for 5 min, and excess water was removed by placing the bags on filter paper. The bags were then soaked in a beaker with acetone for 5 min, air-dried on wire mesh, and finally dried in a laboratory dryer (SLW 115, Pol-Eko Aparatura, Wodzisław Śląski) at 102 ± 2°C for 4 h. Bags containing samples and the empty control bag were put into crucibles and ashed in a muffle furnace (FCF 5SM, Czylok, Jastrzębie Zdrój) at 600°C for 2 h. Crucibles and ash were cooled in a desiccator and weighed. The CF content was calculated from Eq. 1, and the correction for the empty bag after ashing (m_ab) from Eq. 2 (Table 1).

To determine the NDF content, samples with bags were extracted in a mixture of 0.01 g·ml^–1 sodium sulphite and alpha-amylase at 2 ml·l^–1 in a neutral detergent solution (ND) for 75 min. After extraction, the samples were rinsed twice with alpha-amylase at 0.002 g·ml^–1 in demineralised water at 70–90°C for 5 min, then in hot demineralised water for 5 min. After rinsing and drying, the samples were soaked in acetone for 5 min. The samples were air-dried and finally in a laboratory dryer, similar to the CF determination process. The dried samples were cooled in desiccant punches and weighed. The NDF content was calculated from Eq. 3, and the correction for the empty bag after extraction (drying) (C₄) from Eq. 4 (Table 1).

For the ADF determination, samples were extracted with AD for 60 min. After extraction, the samples were rinsed three times in demineralised water at a temperature of 70–90°C in five-minute intervals, with the pH of the water checked using litmus paper after the final rinse. The rest of the procedure was the same as for NDF determination.

The ADL determination followed the ADF process. Dry samples were immersed with 72% (w/w) sulphuric acid and agitated thirty times over three hours at thirty-minute intervals. Extracted samples were thoroughly rinsed under running water until all traces of acid were eliminated, as verified by the pH of the rinse water using litmus paper. The water was removed from the samples by immersing them in acetone for 3 min, then drying them in a laboratory dryer at 105 ± 2°C for four hours. The dried samples were cooled in desiccant punches, weighed, and ashed in crucibles in a muffle furnace at 525°C for three hours. The crucibles with ash were cooled down in a desiccator and weighed. The ADL was calculated according to Eq. 1. The correction for an empty bag after ashing (C₂) was derived from Eq. 2 (Table 1). The results from the biomass tests for CF, NDF, and ADF were compared with those for the control sample. For CF of crushed poplar particleboard after biological pretreatment, the results were validated in interlaboratory tests conducted at the Institute of Animal Sciences WULS in Warsaw, with relative errors from these studies below 1%.

The cellulose content was calculated from the difference between ADF and ADL, as per Eq. 5, hemicellulose from the difference between NDF and ADF, as per Eq. 6, and lignin content is equivalent to ADL, as per Eq. 7 (Table 1).

Table 1

Summary of equations used to determine CF, *NDF*, *ADF*, *ADL*, and biogas production parameters (the remaining equations for determining the analysed parameters can be found in articles on the physical properties of biomass (Tryjarski et al. 2023) and pelleting (Tryjarski et al. 2024)
Parameter	Equation	No.	Source
CF; ADL, %	\(\:CF\left(ADL\right)=\frac{100\left[{m}_{4}-\left({m}_{1}{C}_{2}\right)\right]}{{m}_{2}}\)	1	(A200 manual; Method 8 2022)
Correction of empty bag after ashing C₂, –	\(\:{C}_{2}=\frac{{m}_{ab}}{{m}_{1}}\)	2
NDF; ADF, %	\(\:NDF\left(ADF\right)=\frac{100\left[{m}_{3}-\left({m}_{1}{C}_{1}\right)\right]}{{m}_{2}}\)	3
Correction of empty bag after extraction C₁, –	\(\:{C}_{1}=\frac{{m}_{db}}{{m}_{1}}\)	4
Cellulose, %	\(\:cellulose=ADF-ADL\)	5	(Wolfrum et al. 2009)
Hemicellulose, %	\(\:hemicellulose=NDF-ADF\)	6
Lignin, %	\(\:lignin=ADL\)	7
Volume of dry biogas, ml	\(\:{V}_{d,N}=V\bullet\:\frac{(p-{p}_{w})\bullet\:{T}_{N}}{{p}_{N}\bullet\:T}\:;\:V=\frac{{m}_{NaCl}}{{\rho\:}_{NaCl}}\)	8	VDI 4630 2016
Water vapor pressure in biogas, hPa	\(\:{p}_{w}=6.11231\bullet\:{e}^{(17.5043\bullet\:\frac{{t}_{c}}{\text{241,2}}+{t}_{c})}\)	9	VDI 4630 2016
Corrected concentration of the dry biogas component, %	\(\:{C}_{d,corr}={C}_{{CH}_{4}({CO}_{2},\:{O}_{2},\:{H}_{2})}\bullet\:\frac{100}{{C}_{{CH}_{4}}+{C}_{{CO}_{2}}+{C}_{\:{O}_{2}}+{C}_{\:{H}_{2}}}\)	10	VDI 4630 2016, modified

CF, crude fibre, %; ADL, acid detergent lignin, %; m₁, m₂, m₃, m₄, m_db, m_ab, m_NaCl, the mass of the empty bag, sample, bag with sample after extraction (drying), mass loss of the bag with sample after ashing, mass of the empty bag after extraction (drying), mass loss of the empty bag after ashing, and saturated sodium chloride solution, respectively, g; C₁, C₂, correction of the empty bag after extraction and ashing, respectively, –; NDF, neutral detergent fibre, %; ADF, acid detergent fibre, %; ρ_NaCl, the density of a saturated sodium chloride solution, 1.158 ml·g^–1; p, p_N, p_w, p_u, the pressure of the biogas phase at the time of reading, normal; p_N = 1013 hPa, the vapour pressure of the water as a function of the temperature of the ambient space, hPa; T, T_N, the temperature of the ambient space and normal; T_N = 273 K, respectively, K; t_c, the temperature of the biogas at the volume meter, °C; C_{CH4(CO2, O2, H2)}, concentration in biogas of methane, carbon dioxide, oxygen, and hydrogen, respectively., %; C_d,corr, corrected concentration of the dry biogas component, %.

2.6. Physical properties of substrates before and after anaerobic fermentation

The total solid (TS) was calculated based on the determined MC of the material. For volatile solid (VS) determination, randomly dried inoculum and anaerobic fermentation residues were collected from glass jars and milled to pass 1,02–2 mm sieves. VS was determined as mass loss during ash determination of material based on the PN-EN ISO 18122:2016-01 standard.

2.7. Pressure agglomeration of comminuted material

The compaction tests for comminuted pine and poplar particleboards, both in their raw form and after fungal pretreatment, were conducted using a universal TIRAtest testing machine equipped with chrome steel dies. These dies featured a closed chamber with an external diameter of 30 mm, internal diameter d_m of 6, 8, and 10 mm, and a height of 200 mm. While tests for raw and comminuted shavings encompassed a broader range of outputs, including pellets, pastilles, and granules (Tryjarski et al. 2024), this article focused specifically on the impact of fungal pretreatment by exclusively producing pastilles from crushed particleboards, aligning with the study's aims. The process of pressure agglomeration of materials during pastilles formation, along with equations for determining the parameters of pastilles compaction and strength under radial and axial loading, were detailed in the article about pressure agglomeration of raw and comminuted shavings (Tryjarski et al. 2024).

2.8. Anaerobic fermentation and biogas yield and composition

Biogas yield tests were conducted using the VDI 4630:2016-11 standard (VDI 4630 2016). Blends containing agricultural inoculum (INOC) were prepared to maintain a mass ratio of VS from substrates to inoculum not exceeding 0.5. The 2 l bioreactors, with 1800 g blends each, were checked for airtightness, and the air was expelled over the solution's surface by nitrogen purging. The bioreactors were put in water baths (Fig. 1) and connected to glass containers filled with a saturated sodium chloride (NaCl) solution to prevent the dissolution of carbon dioxide (CO₂) from the biogas.

Three samples were prepared for each material type, along with controls of INOC and a blend of INOC with microcrystalline cellulose (CM) as a reference substance. The bioreactors were submerged above blend level in a water bath maintained at 37°C ± 1°C by a thermostat. Biogas production was monitored daily by weighting the displaced sodium chloride solution on an accurate electronic scale to 0.1 g. The biogas mass was then calculated using a conversion factor of 0.8635 derived from the inverse value of sodium chloride density. Following this, the biogas composition was analysed using two gas analysers. The DP-28 BIO gas analyser (Nanosens Ltd., Wyskogotowo, Poland) measured the percentages of methane (CH₄), carbon dioxide (CO₂), oxygen (O₂), and hydrogen (H₂) in the biogas over 75 s. The Gas Data GFM 400 gas analyser (OMC Envag Ltd., Warsaw, Poland) assessed hydrogen sulphide (H₂S) concentration in ppm (ml·m^–3) within the biogas, requiring at least 50 s for measurement. These measurements were taken sequentially. During the biogas efficiency tests, the temperature and air pressure in the laboratory were recorded daily. The actual volume of produced biogas or methane was converted to the volume of dry gas or methane under standard conditions using Eq. 8. Water vapour pressure (p_w) was calculated using the Magnus equation, Eq. 9, and the corrected concentration of biogas components in dry gas was derived from Eq. 10 (Table 1). The yield of biogas (Y) and methane (Y_CH4) was expressed in the dry gas volume per unit of VS.

2.9. Statistical analysis

Most measurements were conducted in triplicate. The assessment of lignocellulosic compounds in the materials was carried out in two series, adhering to the recommendations of the NREL method (NREL 2008). During pressure agglomeration, 20 pastilles were produced, with 8–10 each subjected to radial and axial compressive loading and further analysed.

Dependent variables were verified against the assumptions. The Kolmogorov-Smirnov (K-S), Lilliefors-corrected (K-S-L), and Shapiro-Wilk (S-W) tests were applied to assess the normality of distributions, alongside Levene and Brown-Forsyth tests for homogeneity of variance. Based on the K-S test results, the distributions of variables were found to conform to the normal distribution. The results of the Levene and Brown-Forsyth tests confirmed the homogeneity of variance across the analysed factors.

The impact of the pretreatment of comminuted shavings and particleboard with oyster mushroom, wood species, and product type (shavings or particleboard) on the physicochemical properties of the materials was analysed. Additionally, the influence of diameter d_m on the pressure agglomeration process and the strength of agglomerates under radial and axial loading was evaluated. The effects of the biological pretreatment on the efficiency of anaerobic fermentation, as indicated by methane yield and biogas composition, were also assessed. Statistical analyses were conducted using multivariate analysis of variance (MANOVA) with the Fisher-Snedecor F test. Differences between mean values were evaluated using the Tukey test at a significance level of p ≤ 0.05, using Statistica version 13.3.

3.1. Physicomechanical properties of comminuted biomass

After the fungal pretreatment, particle size was slightly reduced, particularly in the case of comminuted shavings. Differences between wood species were marginal, at the edge of statistical error. The particles of comminuted shavings and particleboards had an average size of 0.44 mm ± 0.06 mm, with a median of 0.43 mm. The size x_g was notably higher for raw pine comminuted shavings at 0.61 mm (Table 2).

The impact of fungal pretreatment on the material's mechanical properties from comminuted particleboards was statistically significant in most instances. These changes were more pronounced for pine than for poplar. Notably, the results concerning the strength σ_c were particularly striking, where for raw pine and poplar particleboard materials, they were 33.7 kPa and 42.1 kPa, respectively. After pretreatment, they increased to 48.7 kPa and 64.4 kPa, respectively.

Table 2

Parameters (mean values and ± standard deviations, SD) of physicomechanical properties of pine and poplar comminuted shavings and particleboards in the raw state and after 17 weeks of pretreatment with *Pleurotus ostreatus*; MC, moisture content; x_g, the geometric mean of particle size; δ, angle of external friction for wood comminuted material-chrome steel; σ₁, major consolidating stress; σ_c, unconfined yield strength; ff_c, flow index; τ, shear stress; c, cohesion; φ_e, the effective angle of internal friction; φ_c, angle of internal friction for determined flowability; φ_lin, linearised angle of internal friction; *HHV*, higher heating value; CF, crude fibre; *NDF*, neutral detergent fibre; *ADF*, acid detergent fibre; *ADL*, acid detergent lignin
Parameter	Pine				Poplar
	shavings		particleboard		shavings		particleboard
	raw	treated	raw	treated	raw	treated	raw	treated
x_g, mm	0.61^b* ± 0.01	0.42^a ± 0.02	0.42^a ± 0.02	0.42^a ± 0.01	0.51^b ± 0.03	0.44^a ± 0.01	0.43^b ± 0.01	0.40^a ± 0.03
MC, %	5.80^a ± 0.06	6.60^b ± 0.05	5.11^a ± 0.04	6.73^b ± 0.01	5.57^a ± 0.07	5.65^b ± 0.03	5.31^a ± 0.08	6.60^b ± 0.03
δ, °			30.5^a ± 2.5	34.6^b ± 0.9			30.5^a ± 2.5	32.4^b ± 2.0
σ₁, kPa			123.2^a ± 2.5	161.6^b ± 12.2			117.5^a ± 20.7	141.7^b ± 11.3
σ_c, kPa			33.7^a ± 3.9	48.7 ^b± 7.3			42.1^a ± 9.5	64.4^b ± 13.8
ff_c, –			3.70^a ± 0.46	3.39^a ± 0.62			2.99^a ± 1.1	2.31^a ± 0.68
τ, kPa			34.8^a ± 0.4	46.1^b ± 1.6			34.9^a ± 0.7	44.4 ^b± 1.2
c, kPa			9.25^a ± 1.25	11.19^a ± 2.39			12.74^a ± 4.38	17.57^a ± 5.44
φ_e, °			37.3^a ± 0.1	45.4^b ± 1.2			35.9^a ± 3.2	42.9^b ± 1.2
φ_c, °			23.1^a ± 1.5	29.9 ^b± 3.0			20.0^a ± 5.4	23.9^a ± 3.5
φ_lin, °			32.5^a ± 1.0	41.0^b ± 3.1			28.7^a ± 5.6	33.7^a ± 5.0
HHV, MJ·kg^–1	18.37^b ± 0.18	17.50^a ± 0.50	18.72^b ± 0.07	17.23^a ± 0.27	17.74^a ± 0.29	16.89^a ± 1.05	17.61^b ± 0.39	16.65^a ± 0.41
CF, %	75.8^b ± 0.4	65.9^a ± 0.6	68.2^b ± 0.7	52.3^a ± 0.5	67.7^b ± 0.4	64.2^a ± 0.7	63.5^b ± 0.7	48.0^a ± 1.7
NDF, %	93.6^b ± 0.9	85.0^a ± 0.7	88.4^b ± 0.4	75.1^a ± 0.6	93.9^b ± 1.0	91.1^a ± 1.5	91.5^b ± 0.9	81.9^a ± 1.0
ADF, %	81.5^b ± 0.3	78.5^a ± 0.2	71.7^b ± 0.7	66.4^a ± 0.7	78.7^b ± 0.7	77.0^a ± 0.5	72.5^b ± 0.7	64.3^a ± 0.2
ADL, %	37.8^b ± 1.8	30.6^a ± 1.7	38.4^b ± 2.1	31.6^a ± 2.2	26.7^b ± 1.8	21.3^a ± 2.1	24.9^b ± 1.2	16.6^a ± 1.3
Cellulose, %	43.7^a ± 1.8	47.9 ^b± 1.7	33.3^a ± 2.6	34.8^a ± 2.5	52.0^a ± 1.9	55.7^b ± 2.5	47.6^a ± 1.9	47.7^a ± 1.3
Hemicellulose, %	12.1^b ± 0.7	6.5^a ± 0.6	16.7^b ± 1.0	8.7^a ± 1.3	15.2^a ± 1.6	14.2^a ± 1.9	18.9^a ± 1.6	17.6^a ± 0.9
Lignin, %	37.8^b ± 1.8	30.6^a ± 1.7	38.4^b ± 2.1	31.6^a ± 2.2	26.7^b ± 1.8	21.3^a ± 2.1	24.9^b ± 1.2	16.6^a ± 1.3

* Values in each row between raw and treated material with the different letters are statistically significant at p < 0.05.

The angle δ for both pine and poplar raw particleboards was consistently 30.5°, but after fungal pretreatment, it increased by almost 4° and by 2°, respectively. All three types of internal friction angles (φ_e, φ_c, and φ_lin) were higher for the comminuted pine particleboard compared to the poplar one, and their increase post-treatment was statistically significant (for poplar: φ_c and φ_lin close to significant). Following fungal pretreatment, a more substantial increase in stresses σ₁ was observed in pine particleboard, by 31%, than in poplar one, by 21%. Similarly, stresses τ increased by 32% and 27%, respectively, which was also statistically significant. The impact of pretreatment on the cohesion of the materials was less pronounced, with increases of 21% and 38% for the respective species, with a broad scatter of values rendering these differences not statistically significant. The flow coefficient decreased post-treatment by 8.4% for pine particleboard and 22.7% for poplar particleboard, indicating that the poplar particleboard experienced a more significant reduction of this coefficient than pine. The enhancement in mechanical parameters, particularly friction angles (δ, and φ), c, and τ and σ₁ stresses following fungal pretreatment, suggests a potential influence on pressure agglomeration processes, potentially facilitating the production of more strength pastilles. Fungal pretreatment enhances the cohesion between material particles, as corroborated by the literature (Ballen Sierra et al. 2023).

The HHV for pine and poplar wood decreased after fungal pretreatment by almost 1 MJ·kg^–1, with the reduction being more pronounced for pine. Notably, the HHV of raw pine particleboards containing UF was initially the highest at 18.72 MJ·kg^–1, but after pretreatment, it dropped to 17.23 MJ·kg^–1, representing an 8% decrease. These reductions are primarily attributed to lignin degradation and other fibrous compounds. Evidence of the interdependencies among the analysed parameters was provided by partial correlations between HHV and CF, NDF, ADF, and ADL, which were 0.876, 0.579, 0.709, and 0.818, respectively, indicating a strong correlation of HHVs with the structural components of the biomass. What is more, supported by the statement that lignin's lower heating value (related to HHV) was 23–27 MJ·kg^–1, compared to 19 MJ·kg^–1 of cellulose and hemicellulose, as detailed in the form of a regression equation (Demirbas 2004). Interestingly, a more significant decrease in HHV was observed for particleboards than for shavings alone, suggesting that the fungus could degrade the UF present in the boards.

Fungal pretreatment caused a statistically significant increase in the material MC, notably in particleboards, where the MC rose from 5.2–6.6% on average. Comparable changes were observed across both wood species. Pine shavings exhibited an increased MC from an initial 5.8–6.6%, while poplar shavings maintained a similar level at 5.6%. This noticeable rise in MC for pine and poplar post-treatment demonstrates that fungal pretreatment can effectively enhance the material's water absorption capacity. This aligns with findings from research (Wenny Surya Murtius et al. 2024). The oyster mushroom, recognised for its ability to degrade lignin and partially hemicellulose, along with other wood components, alters the cellular structure of the material (Badu et al. 2011), thereby increasing its porosity (Kumar et al. 2021), which in turn facilitates moisture absorption (Banik et al. 2017).

The differences observed in MC between pine and poplar and between wood and particleboard materials may stem from the distinct properties of these materials. Pine and particleboard materials seem more susceptible to fungal pretreatment, particularly in lignin degradation, which can lead to notable changes in water absorption. The dynamics of moisture absorption may also relate to the wood's inherent structure and chemical composition, and fungal decay may have a relatively more substantial influence on pine. Assuming that UF acts as a hydrophobic agent, it restricts moisture absorption from the air, explaining the lower MC in comminuted raw particleboards with UF (5.2%) compared to wood shavings (5.6%). The impact of fungal pretreatment on MC was more pronounced in UF-bonded particleboards than in shavings alone, suggesting that the Pleurotus ostreatus had to overcome the UF barrier, likely resulting in resin degradation. Changes in MC aligned with alterations in CF, NDF, ADF, and ADL content, as evidenced by Pearson correlation coefficients of − 0.971, − 0.927, − 0.903, and − 0.510, respectively. Since lignocellulosic compounds are connected with fibre content, some correlations were observed. Interestingly, a strong correlation existed between NDF and hemicelluloses (r = 0.857). It is worth mentioning that NDF is equivalent to the content of all wood structural components together, not sole hemicelluloses. Lignin content was directly linked to ADL but positively correlated with CF (r = 0.679). Following Pleurotus ostreatus pretreatment, ADL content decreased significantly, beneficially increasing cellulose content, indicated by a negative correlation coefficient between these variables (r = − 0.906). Statistically significant correlations were observed in most pairs of parameters, except between NDF and ADF associated with ADL. The more porous structures, capable of absorbing increased moisture, concurrently reduced the concentration of TS, leading to lower CF, NDF, ADF, and ADL values. The ADL content in pine materials was consistent between raw shavings and particleboards at 37.8% and 38.4%, respectively, and for treated materials at 30.6% and 31.6%. For poplar materials, ADL content was notably lower than in pine ones, with absolute differences of 11.1% for raw shavings, 9.3% for treated shavings, 13.5% for raw particleboards, and 15.0% for treated particleboards. The reduction in ADL content after fungal pretreatment confirmed its effectiveness in degrading lignin and was consistent across raw-treated material pairs, showing a more beneficial effect on particleboards of both wood species. The decreases in NDF and ADF content suggest partial degradation of holocellulose polymers, potentially advantageous for fermentation processes, as highlighted in the literature (Wan and Li 2012). The influence of fungal pretreatment was more substantial on changes in NDF (hemicellulose, cellulose, lignin) content than ADF (cellulose, lignin). It was more significant for particleboards than shavings and pine than poplar.

3.2. Pressure agglomeration of biomass and pastille strength

Fungal pretreatment and the choice of wood species significantly influenced many parameters describing the pressure agglomeration of particleboard materials in pastille formation. Variables such as pastille density (ρ_a), pastille ejection pressure (p_u), specific work of ejection (L_v), and the diameter (d_m) significantly shaped the physical properties of the agglomerates (Table 3). However, the effects of wood species on compaction work (L_s) and the impacts of fungal pretreatment on linear agglomerate expansion (R_l) and piston displacement during pastille ejection (s_u) were not statistically significant.

Table 3

Results of the analysis of variance and mean values with SD for specific compaction work (L_s), degree of compaction (I_s), compaction susceptibility coefficient (k_s), agglomerate (pastille) expansion index concerning its length (R_l) and diameter (R_d), maximum pressure to push the agglomerate out of the die opening (p_u), specific work to push the agglomerate out of the die opening (L_v), piston displacement to push the agglomerate out of the die opening (s_u), single agglomerate density (ρ_a), concerning wood species (TW), material pretreatment (MT), and die opening diameter (d_m)
Factor	L_s, kJ·kg^–1	I_s, –	k_s, J·m³·kg^–2	R_l, %	R_d, %	p_u, MPa	L_v, kJ·kg^–1	s_u, mm	ρ_a, kg·m^–3
TW	0.6175	< 0.0001	< 0.0001	< 0.0001	0.0001	< 0.0001	< 0.0001	0.0499	< 0.0001
d_m	< 0.0001	< 0.0001	< 0.0001	< 0.0001	< 0.0001	< 0.0001	< 0.0001	< 0.0001	< 0.0001
MT	< 0.0001	< 0.0001	< 0.0001	0.3743	0.0450	0.0011	0.0048	0.1327	< 0.0001
Mean and ± SD for wood species (TW)
Pine	22.89^a* ± 2.61	3.28^a ± 0.46	363.3^b ± 84.7	14.35^b ± 7.01	3.94^b ± 2.01	0.65^b ± 0.31	0.038^b ± 0.026	2.43^b ± 1.05	1002.3^b ± 40.8
Poplar	22.84^a ± 3.16	3.96^b ± 1.15	288.5^a ± 96.0	11.80^a ± 6.46	3.81^a ± 1.98	0.29^a ± 0.25	0.019^a ± 0.021	2.23^a ± 2.21	970.0^a ± 52.1
Mean and ± SD for die opening diameter (d_m, mm)
6	19.96^a ± 1.55	3.64^b ± 1.04	248.5^a ± 68.7	16.57^b ± 4.11	6.65^c ± 0.33	0.59^b ± 0.46	0.020^a ± 0.018	1.43^a ± 0.83	956.0^a ± 64.3
8	22.89^b ± 1.73	3.73^c ± 0.90	368.2^b ± 86.1	17.54^b ± 4.65	2.65^b ± 0.21	0.44^a ± 0.22	0.049^b ± 0.030	3.82^c ± 1.87	1000.2^b ± 31.3
10	25.75^c ± 1.69	3.49^a ± 0.87	361.1^b ± 87.6	5.11^a ± 2.54	2.33^a ± 0.33	0.38^a ± 0.25	0.016^a ± 0.010	1.74^b ± 1.22	1002.3^b ± 29.7
Mean and ± SD for material pretreatment (MT)
Raw	21.70^a ± 2.46	2.85^a ± 0.22	389.8^b ± 71.5	12.88^a ± 7.40	3.84^a ± 2.01	0.43^a ± 0.36	0.026^a ± 0.022	2.41^a ± 1.24	958.5^a ± 49.1
Treated	24.03^b ± 2.83	4.39^b ± 0.73	262.1^a ± 76.7	13.27^a ± 6.27	3.91^b ± 1.98	0.52^b ± 0.30	0.031^b ± 0.028	2.26^a ± 2.11	1013.8^b ± 30.8

* Values between factor levels with different letters are statistically significant at p < 0.05.

Work (L_s), compaction susceptibility coefficient (k_s), linear (R_l) and diametric expansion (R_d), pressure (p_u), work (L_v), displacement (s_u), and density (ρ_a) were statistically higher for pine than poplar. It was observed that the harder pine shavings were more challenging to densify compared to the more plastic poplar shavings, underscoring the importance of the raw material’s physical properties on the agglomeration process. Efficient densification of the more robust pine wood required higher die heights than those for poplar wood (Monedero et al. 2015), indicating the significant role of wood mechanical properties in the pressure agglomeration process, potentially affecting the efficiency and costs of pastilles production.

In producing pastilles from the particleboard materials, the diameter d_m played a crucial role in determining the work L_v and the physical characteristics of the resulting agglomerate. It was observed that increasing the d_m from 6 mm to 8 mm significantly affected the required compaction work while producing higher-density pastilles. This increase in density was advantageous, as denser agglomerates from the same type of biomass typically exhibit better structural stability and are less prone to disintegration during transport and use. The comparison material doses were 0.2, 0.3, and 0.5 g for d_m of 6, 8, and 10 mm. Under such conditions, the height of the compacted material sample was inversely proportional to the d_m. Consequently, reducing the compaction height led to producing pastilles with a higher density, as layers with a reduced height (thickness) are more readily compacted. The mechanism of better compaction of a thinner material layer stems from the reduced effect of the material's elasticity compared to a thicker layer. The comminuted shavings and particleboards were more easily rearranged, filling voids and displacing air between particles, and then overcoming elasticity, they underwent plastic deformation under the sudden increase in the compaction pressure gradient, resulting in the need for more significant work L_s. During compaction, feedback from the bottom of the die increased the transverse pressure from the deformation, which was proportional to the axial pressure (Miao et al. 2015).

Pastilles expansion, or the ability to return to their original shape after the load has been removed, tended to be inversely proportional to the d_m. This implies that more minor d_m led to more significant expansion, which may be undesirable for maintaining the cohesion of the agglomerates. Conversely, susceptibility k_s, or the ease with which the material compresses, was proportional to the die orifice diameter, suggesting that larger holes facilitate compaction while reducing the required pressures. Pastille expansion was linked to the compaction mechanism and the plastic deformation of particles, which is more favourable for those compacted in thinner layers. Thus, particles plastically bound by mechanical bridges in larger-diameter pastilles underwent less expansion.

Fungal pretreatment increased the work L_s required to produce pastilles from 21.70 kJ·kg^–1 to 24.03 kJ·kg^–1, and the results show that this additional energy contributed to the production of pastilles with a significantly higher density ρ_a, 1014 kg·m^–3 instead of 959 kg·m^–3 (by 5.8%). The pretreated material was more susceptible to compaction than the raw material, as it required less work to achieve a change in density ρ_a, with susceptibility k_s values of 262 J·m³·kg^–2 and 390 J·m·kg^–2, respectively. Pastilles' R_d and R_l expansion were slightly more significant for pretreated materials, indicating subtle differences in their physical properties resulting from the fungal pretreatment process. However, the R_l change was not statistically significant.

For comparison, the work L_s for noncomminuted pine shavings was higher at 42.7 kJ·kg^–1, and the density ρ_a was lower at 994 kg·m^–3, while for poplar shavings, these values were 32.7 kJ·kg^–1 and 978 kg·m^–3, respectively (unpublished authors' data). This is because, at the exact dosage, larger shavings were characterised by higher material elasticity and more significant shape deformations. Shape deformations cause proportional lateral pressures, increasing friction on the agglomerate's lateral surface and the die opening. Since the comminuted biomass after biological pretreatment exhibited higher angles δ and φ and more extensive c, along with a lower flow index than the raw material (Table 2), overcoming these higher resistances and performing more work L_s was necessary. These phenomena were also observed during the production of feed pellets (Hejft 2002).

Differences in pressure agglomeration parameters can be attributed to changes in the fibre content of the biomass. The degradation of fibres by the fungus affected the structure of lignocellulosic compounds, facilitating the formation of pastilles with greater efficiency and density. Correlations between the work L_s and fibre content, such as CF and ADF, alongside density ρ_a and NDF and ADF, suggest a relationship between the physicochemical properties of the materials during the agglomeration process.

Pastilles made of raw and fungal pretreated materials and subjected to radial and axial compressive loading exhibited varying values of modulus of elasticity (E), specific energy of compression until the agglomerate cracks (E_j) and maximum tensile strength during pastille cracking (σ_p). The modulus E was not statistically significantly affected by the diameter d_m (p = 0.8057, Table 4). Generally, pastilles with higher strength σ_p exhibited a lower modulus E. Axially loaded pastilles displayed strengths σ_p an order of magnitude greater than those loaded radially, with noticeable differences in the impact of various factors on strength parameter values. Given that pastilles show lower resistance to radial than axial loads, radial pressures are more significant when handling these products; thus, the interpretation of results will focus on this aspect. Pastilles made from pine particleboard demonstrated more resistance to radial compressive deformation compared to those made from poplar particleboards, with strengths σ_p of 2.14 MPa and 0.32 MPa, respectively, and energies E_j of 1.07 mJ·mm² and 0.18 mJ·mm², respectively.

The strength parameters of the pastilles during radial compression decreased significantly with increasing diameter d_m, while the modulus E tended to increase, although without statistically significant differences. Although the density ρ_a and work L_s corresponded with the diameter d_m, the pastilles' resistance to radial compressive loads was inversely proportional to this d_m. Larger diameter pastilles deformed more easily, requiring less energy, and generated lower maximum stresses until the pastilles cracked. This could be attributed to the lower homogeneity of the material compacted in larger diameter d_m, as the effect of friction δ between the material and the hole surface increased radially from the axis, locally weakening the internal structure of the pastilles.

Table 4

Results of analysis of variance, mean values, and SD for pastilles modulus of elasticity (E), specific agglomerate deformation energy (E_j), and maximum tensile strength during agglomerate (pastille) cracking (σ_p), considering wood species (TW), material pretreatment (MT), type of agglomerate load (TL), and die opening diameter (d_m) for raw and fungal pretreated particleboards
Factor	E, MPa	E_j, mJ·mm^–2	σ_p, MPa
Radial compression – all agglomerates
TW	< 0.0001	< 0.0001	< 0.0001
TL	< 0.0001	< 0.0001	< 0.0001
d_m	0.8057	< 0.0001	< 0.0001
MT	0.0160	< 0.0001	< 0.0001
Mean and ± SD for wood species (TW)
Pine	22.38^b* ± 19.52	55.99^a ± 63.17	45.60^a ± 46.15
Poplar	8.49^a ± 6.20	65.31^b ± 71.94	50.22^b ± 51.47
Mean and ± SD for type of agglomerate load (TL)
RC	24.65^b ± 18.42	0.62^a ± 0.66	1.23^a ± 1.31
AC	6.21^a ± 2.48	121.29^b ± 43.74	94.90^b ± 19.64
Mean and ± SD for die opening diameter (d_m, mm)
6	15.57^a ± 14.22	35.88^a ± 37.26	40.43^a ± 41.03
8	15.73^a ± 17.41	69.47^b ± 72.59	52.57^c ± 53.25
10	15.01^a ± 16.52	76.61^c ± 79.10	50.72^b ± 51.09
Mean and ± SD for material pretreatment (MT)
Raw	16.57^b ± 16.64	51.61^a ± 57.40	40.45^a ± 41.27
Treated	14.29^a ± 15.41	69.70^b ± 75.83	55.36^b ± 54.54
Axial compression (AC) – pastille
TW	0,9999	< 0.0001	< 0.0001
d_m	< 0.0001	< 0.0001	< 0.0001
MT	< 0.0001	< 0.0001	< 0.0001
Mean and ± SD for wood species (TW)
Pine	6.22^a ± 2.47	110.9^a ± 43.7	89.0^a ± 21.3
Poplar	6.22^a ± 2.50	130.5^b ± 42.5	100.1^b ± 16.7
Mean and ± SD for die opening diameter (d_m, mm)
6	9.15^c ± 1.90	71.0^a ± 16.6	79.5^a ± 16.8
8	5.43^b ± 0.74	138.3^b ± 30.7	103.9^c ± 18.3
10	4.09^a ± 0.67	152.7^c ± 28.4	100.4^b ± 15.1
Mean and ± SD for material pretreatment (MT)
Raw	5.69^a ± 1.90	102.8^a ± 36.2	80.2^a ± 15.1
Treated	6.74^b ± 2.86	138.6^b ± 44.2	109.0^b ± 12.1
Radial compression (RC) – pastille
TW	< 0.0001	< 0.0001	< 0.0001
d_m	0.1365	0.0040	< 0.0001
MT	0.0034	< 0.0001	< 0.0001
Mean and ± SD for wood species (TW)
Pine	38.5^b ± 15.2	1.07^b ± 0.67	2.14^b ± 1.31
Poplar	10.8^a ± 7.8	0.18^a ± 0.14	0.32^a ± 0.22
Mean and ± SD for die opening diameter (d_m, mm)
6	22.0^a ± 17.9	0.72^b ± 0.82	1.40^c ± 1.57
8	26.0^a ± 19.9	0.60^ab ± 0.59	1.24^b ± 1.30
10	25.9^a ± 17.5	0.54^a ± 0.51	1.06^a ± 1.00
Mean and ± SD for material pretreatment (MT)
Raw	27.5^b ± 17.7	0.40^a ± 0.13	0.74^a ± 0.25
Treated	21.8^a ± 18.8	0.84^b ± 0.87	1.73^b ± 1.70

* Values between factor levels with different letters are statistically significant at p < 0.05.

Pastilles from fungal pretreated materials exhibited more than double the strength σ_p and energy E_j. They displayed lower modulus E values than those made from raw materials. The modulus E did not show clear consistency with the values of fibrous structure CF, NDF, ADF, and ADL, nor with hemicellulose, cellulose, and lignin, highlighting the complex influence of material chemical composition on the elasticity of the pastilles. Interestingly, a significant correlation was observed between the energy E_j and strength σ_p and the fibre content. The highest values of strength σ_p correlation coefficients were noted for CF, NDF, and ADL at 0.956, − 0.944, and 0.881, respectively, suggesting that lignin content significantly affects the mechanical strength of the pastilles. Higher lignin content was associated with increased strength, likely owing to its natural binding and material stiffening properties. Furthermore, the pastilles strength increased inversely with cellulose content, while hemicellulose had no significant impact on E_j and σ_p.

In conclusion, these results verify that biological pretreatment with Pleurotus ostreatus can beneficially influence the physicochemical properties of particleboard materials, enhancing its density and efficiency in the pressure agglomeration process, which has implications for solid biofuels and the composites industry. The research findings substantiate the hypothesis that biological pretreatment improves pressure agglomeration properties and opens new avenues for further experiments.

3.3. Anaerobic fermentation, methane yield, and biogas composition

The comminuted materials exhibited very high values of TS (94.08% ± 0.64%) and VS (99.23% ± 0.20%), theoretically suggesting a favourable biogas yield. Comparing raw materials, these values were higher for particleboards than wood shavings. After fungal pretreatment, there was a statistically significant reduction, particularly noticeable in particleboards. This indicates that the fungus more extensively decomposed the UF-containing particleboards than the shavings without UF. For pretreated pine shavings, both TS and VS decreased significantly, from 94.20–93.40% and from 99.49–99.03%, respectively (Table 5), demonstrating the effectiveness of the fungal degradation process.

According to the findings (Kupryś-Caruk et al. 2023), the inoculum had a low TS content of 2.12% but a high ash content of 37.45% in TS, resulting in a relatively low VS content of 62.55%. Consequently, after blending approximately 13 g of materials with the inoculum, the contents of TS_me and VS_me in the blends were consistent, at 2.77% ± 0.01% and 71.29% ± 0.05%, respectively. However, in the blends with materials after biological pretreatment, TS_me and VS_me contents were lower than with raw materials, indicating decomposition by the fungus. After anaerobic fermentation, the contents of TS_mf and VS_mf in the blends decreased significantly to 2.13% ± 0.10% and 58.07% ± 1.06%, respectively, reflecting relative reductions of 23% and 19%. The decreases of TS_mf in blends with pretreated materials were smaller than those with raw ones. In contrast, the cutbacks in VS_mf were more significant, evidencing effective bioconversion of the treated organic matter (Karthikeyan et al. 2024) by specialised microorganisms during anaerobic fermentation (Chojnacka and Moustakas 2024). This suggests the transformation of biomass components during fungal pretreatment into more volatile (Piątek et al. 2016) and readily degradable forms (Wainaina et al. 2019), advantageous for anaerobic fermentation. This was facilitated by changes in the physicochemical properties of the materials by Pleurotus ostreatus, significantly reducing lignin content (Fig. 2), a significant barrier in biomass degradation and fermentation processes. Selective lignin degradation increased cellulose accessibility to enzymes that decompose organic matter. At the same time, hemicellulose reduction may have enhanced the porosity and water absorption of the material (Manyi-Loh and Lues 2023), supporting fermentation processes. These changes in the lignocellulosic structure enhanced the biogas production potential of the processed materials.

Table 5

Parameters (mean values and ± SD) of physicochemical properties of pine and poplar comminuted shavings and particleboards, raw and after 17 weeks of pretreatment with *Pleurotus ostreatus* and results of tests of yield biogas and its composition; TS, TS_me, TS_mf, total solids in the comminuted material, the blends of comminuted material and inoculum before and after fermentation; VS, VS_me, VS_mf, volatile solid in the comminuted material, the blend of comminuted material and inoculum before and after fermentation; Y, Y_CH4, yield in terms of volatile solid, biogas and methane, respectively; C_{CH4(CO2, O2, H2)}, concentration of methane, carbon dioxide, oxygen and hydrogen, in biogas, respectively
Parameter	Pine				Poplar
	shavings		particleboard		shavings		particleboard
	raw	treated	raw	treated	raw	treated	raw	treated
TS, %	94.20^a* ± 0.07	93.40^b ± 0.06	94.89^a ± 0.05	93.27^b ± 0.01	94.43^a ± 0.08	94.35^a ± 0.03	94.69^a ± 0.09	93.40^b ± 0.03
VS, %	99.49^b ± 0.02	99.03^a ± 0.06	99.36^a ± 0.01	98.91^b ± 0.01	99.32^a ± 0.01	99.30^a ± 0.04	99.38^b ± 0.08	99.07^a ± 0.02
TS_me, %	2.78^a ± 0.01	2.78^a ± 0.01	2.78^a ± 0.01	2.78^a ± 0.01	2.70^a ± 0.11	2.78^a ± 0.01	2.76^a ± 0.04	2.78^a ± 0.01
VS_me, %	71.36^b ± 0.01	71.24^a ± 0.01	71.32^b ± 0.01	71.22^a ± 0.01	71.32^a ± 0.01	71.31^a ± 0.01	71.32^b ± 0.02	71.25^a ± 0.01
TS_mf, %	2.13^a ± 0.19	2.16^a ± 0.07	2.00^a ± 0.31	2.09^a ± 0.06	1.98^a ± 0.01	2.13^b ± 0.06	2.22^a ± 0.14	2.28^a ± 0.02
VS_mf, %	58.1^a ± 0.2	57.8^a ± 0.1	58.4^b ± 0.1	57.3^a ± 0.1	57.1^a ± 0.3	56.8^a ± 0.6	60.0^b ± 0.1	58.9^a ± 0.1
Y, ml·g^–1	59.0^a ± 6.1	194.9^b ± 3.9	46.8^a ± 3.0	184.7^b ± 3.1	218.1^a ± 4.2	295.4^b ± 4.1	119.7^a ± 4.8	380^b ± 1.5
Y_CH4, ml·g^–1	34.3^a ± 5.2	108.3^b ± 3.1	29.3^a ± 1.4	109.1^b ± 2.0	138.8^a ± 6.6	169.3^b ± 1.2	71.0^a ± 5.0	206.7^b ± 1.5
C_CH4, %	66.6^b ± 0.4	64.9^a ± 0.9	69.8^b ± 0.3	64.4^a ± 1.8	65.0^b ± 0.1	62.8^a ± 0.1	64.3^b ± 0.2	62.2^a ± 0.4
C_CO2, %	26.3^a ± 0.5	29.4^b ± 1.3	21.7^a ± 0.4	28.5^b ± 1.4	28.3^a ± 0.2	31.5^b ± 0.1	28.9^a ± 0.2	31.7^b ± 0.2
C_O2, %	0.87^a ± 0.18	0.82^a ± 0.18	0.95^a ± 0.05	1.36^a ± 0.65	0.90^a ± 0.17	0.69^a ± 0.04	0.97^a ± 0.18	1.04^a ± 0.13
C_H2, %	6.23^b ± 0.34	4.92^a ± 1.24	7.52^b ± 0.05	5.74^a ± 0.2	5.78^b ± 0.07	4.98^a ± 0.03	5.78^b ± 0.11	5.04^a ± 0.03

* Values in each row between raw and treated material with the different letters are statistically significant at p < 0.05.

The biogas yield of 711 ml·g^–1 VS from microcrystalline cellulose fell within the acceptable range of 745 ml·g^–1 VS ± 10% per the VDI 4630 standard. The maximum biogas yield was substantially higher from materials subjected to fungal pretreatment than raw materials (Table 5), indicating that Pleurotus ostreatus effectively modified substrates, enhancing substrates’ availability for fermentative microorganisms. Additionally, degradation of UF resin during fungal pretreatment cannot be excluded. Although the methane content in biogas was inversely proportional to the biogas yield (r = − 0.801), the methane and biogas yields were in accordance (r = 0.998). The dynamics of the cumulative methane yield (Fig. 3) mirrored that of the biogas. The highest rate, achieved on the fourth day of anaerobic fermentation and sustained the longest (until day 26) with a methane production efficiency of 207 ml·g^–1 VS, was characteristic of pretreated poplar particleboards with UF. Compared to raw particleboards at 71 ml·g^–1, this was a 2.9-fold increase. In contrast, the smallest increase (1.2-fold) was observed for poplar shavings, though the methane yields were relatively the highest in the raw state and after pretreatment, at 139 ml·g^–1 VS and 169 ml·g^–1 VS, respectively. The most significant increase (3.7-fold) occurred for pine particleboards, but the methane yields for raw and pretreated states were much lower, at 29 ml·g^–1 VS and 109 ml·g^–1 VS, respectively. These yields were comparable to those for pine shavings, at 34 ml·g^–1 and 108 ml·g^–1 VS, respectively, representing a 3.2-fold increase.

In general, methane yields were higher from poplar than from pine materials. However, the effect of Pleurotus ostreatus pretreatment was more pronounced on pine than on poplar shavings and particleboards compared to raw materials of both wood species. The higher methane yields from poplar materials can be attributed to their significantly lower lignin content than pine, at 26.7% vs. 37.8% for shavings and 24.9% vs. 38.4% for particleboards, respectively (Table 2). After biological pretreatment, the absolute decrease in lignin content was 5.4% and 7.3% for pine and 8.3% and 6.8% for poplar, respectively. The relative decreases were similar, with average reductions in lignin content slightly higher in pine materials at 7.03%, compared to 6.86% in poplar materials. The correlation coefficient between methane yield and lignin content was negative, at − 0.848. The effect of this material pretreatment was more significant than rice straw with P. ostreatus, which reduced lignin content by 3.3% (Datsomor et al. 2022). In the study with four Pleurotus spp. fungi (P. diamor, P. eryngii, P. sajor-caju, P. citrinopileatus) on corn stover, lignin content was reduced by 1.85–3.32% (Wang et al. 2021). Two AA14 members of the LPMO from the white rot fungus Pycnoporus coccineus were found to significantly enhance wood saccharification efficiency by oxidatively cleaving highly resistant xylan-coated cellulosic fibres (Couturier et al. 2018). Small secreted proteins, together with lignocellulose-degrading enzymes, were elucidated to be integral components of the secretome and were hypothesised to also play a role in saprophytes and function in P. ostreatus as components of the ligninolytic system (Feldman et al. 2017). Converting lignocellulosic biomass to biofuels requires a critical pretreatment process to break down the recalcitrant lignin structure. For this purpose, several known ligninolytic enzyme assays were conducted, and the activity of lignin peroxidase, laccase, dye-decolorising peroxidase, and aryl-alcohol oxidase on the decomposition of an empty cluster of oil palm fruit was detected, confirming their ability to degrade lignin (Riyadi et al. 2020). No results were found on the treatment of UF-bonded wood.

The biogas’ methane, carbon dioxide, and hydrogen concentrations closely correlated with lignin content with correlation coefficients of 0.892, − 0.916, and 0.912, respectively. The inverse correlation of these biogas components was with cellulose, while hemicellulose showed no consistency with these components. The oxygen content in the biogas was below one percent (except for PIBT, which showed slightly more significant variability), and no correlation with lignocellulosic compounds or the effect of biological pretreatment was found. After pretreatment with Pleurotus ostreatus, the proportions of methane and hydrogen in the biogas decreased, while that of carbon dioxide increased significantly. This suggests that the quality of biogas slightly deteriorated after fungal pretreatment, as indicated by the negative correlation coefficient between methane content in biogas and material pretreatment method (r = − 0.641), but generally, the quality of the biogas, characterised by a high methane content of 62–70% and low carbon dioxide levels (around 30%), was excellent, with hydrogen typically comprising 5–6%. No hydrogen sulphide was detected in the biogas, except in isolated instances during the first five days of anaerobic fermentation and the process with raw poplar shavings between the 10th and 22nd days, at a gas meter detection limit, i.e., 10 ppm.

In summary, the results confirmed the hypothesis that biological pretreatment with Pleurotus ostreatus altered the physicochemical properties of materials, particularly particleboards, positively affecting anaerobic fermentation processes. Reducing lignin and hemicellulose content increased biogas yield, with significant implications for future industrial applications.

This research investigated comminuted pine and poplar shavings and UF-bonded particleboards pretreated with the fungus Pleurotus ostreatus, known for its capacity to degrade lignin and hemicellulose. We assessed the physicochemical properties of the materials, examined the impact of fungal decomposition on the pressure agglomeration process and anaerobic fermentation, and explored how this bioconversion affected pastilles strength, methane yield, and biogas composition. The fungal degradation of lignocellulose components, notably lignin, improved the mechanical properties of the materials, enhanced the density and compressive strength of pastilles, and increased the availability of cellulose and hemicellulose for fermentation processes, significantly boosting biogas efficiency.

The findings justified and confirmed Hypothesis 1: the application of Pleurotus ostreatus significantly reduced the lignin content in the UF-bonded materials (by 6.8–8.3%), underscoring the potential for decomposing materials traditionally considered resistant to biodegradation. The results also indicated effective organic matter decomposition by the fungus, as evidenced by reduced TS and VS values in pine and poplar materials: wood shavings and particleboards. The work L_s for pretreated materials was higher than for raw ones (24.03 kJ·kg^–1 vs. 21.70 kJ·kg^–1), corresponding with pastilles of significantly higher density ρ_a (1014 kg·m^–3 vs. 959 kg·m^–3). Hypothesis 2 was also substantiated, as chemical and physical modification of wood and particleboard materials under fungal influence facilitated pressure agglomeration, producing more cohesive and robust pastilles. The biologically modified biomass from particleboards demonstrated enhanced methane production efficiency, especially for poplar particleboards, where efficiency increased from 71 ml·g^–1 VS to 207 ml·g^–1 VS, suggesting enhanced substrate availability for fermentation microbes.

The novelty of this study lies in employing white rot fungi in particleboard pretreatment to convert them into biofuels, a method not previously documented in the literature. The results suggest the potential of such pretreatment for practical industrial applications. However, as per Melvin Kranzberg's principle (Zhan 2022), even technology aimed at positive outcomes has drawbacks and challenges. Scaling up this technology involves challenges such as the lengthy duration required for effective fungal decomposition and specific conditions for fungal growth, notably pasteurisation (Sánchez 2010). These limitations highlight the necessity for further, more detailed studies to optimise the processes and reduce decomposition time and costs while maintaining biodegradation efficiency.

In conclusion, this research provides valuable insights into the potential advantages of biologically pretreating particleboard waste containing synthetic resins. It highlights its efficacy in enhancing both biomass's mechanical properties and biogas production's efficiency. These findings pave the way for sustainable particleboard recycling of UF-bonded particleboards, contributing to developing eco-friendly wood waste management strategies.

ADF – acid detergent fibre (%)

ADL – acid detergent lignin (%)

c – cohesion (kPa)

C₁, C₂, correction of the empty bag after extraction and ashing, respectively

C_{CH4(CO2, O2, H2)} – concentration of methane, carbon dioxide, oxygen, and hydrogen in biogas, respectively (%)

C_d_,corr – corrected concentration of the dry biogas component (%)

CF – crude fibre (%)

C_H2S – hydrogen sulphide concentration in biogas (ppm)

d_a, d_m – diameter of the agglomerate and the die opening, respectively (mm)

E – modulus of elasticity in agglomerate compression (MPa)

E_j – specific energy of compression until the agglomerate cracks (mJ·mm^–2)

ff_c – flow index

HHV – higher heating value (MJ·kg^–1)

I_s – degree of biomass compaction

k_s – coefficient of shavings' susceptibility to compaction (J·m³·kg^–2)

L_s, L_v – Specific work; compacting and pushing the agglomerate out of the die opening, respectively (kJ·kg^–1)

m₁, m₂, m₃, m₄, m_db, m_ab, m_d – mass; empty bag, sample, bag with sample after extraction (drying), bag with sample after ashing, empty bag after extraction (drying), empty bag after ashing, single material dose, and saturated sodium chloride solution, respectively (g)MC –moisture content (% w.b.)

NDF – neutral detergent fibre (%)

p, p_N, p_w, p_u – pressure of the biogas phase at the time of reading, normal; p_N = 1013 hPa, vapour pressure of the water as a function of the ambient temperature (hPa) and a maximum of pushing the agglomerate out of the die opening, respectively (MPa)

R_l, R_d – agglomerate expansion rates relative to its length and diameter, respectively (%)

s_u – piston displacement to push the agglomerate out of the die opening (mm)

T, T_N – temperature; ambient and normal; T_N = 273 K, respectively (K)

t_c – temperature of the biogas at the volume meter (°C)

t_s – anaerobic fermentation time (h)

TS, TS_me, TS_mf – total solids in the comminuted material, blends of comminuted material and inoculum before and after fermentation (%)

V, V_d_,N – volume of the biogas ready off and dry biogas in the normal state, respectively (ml)

VS, VS_me, VS_mf – volatile solid in the comminuted material, blends of comminuted material and inoculum before and after fermentation (%)

x_g – geometric mean of particle size (mm)

Y, Y_CH4 – yield; volatile solid, biogas, and methane, respectively (ml·g^–3)

δ – angle of external friction for wood comminuted shavings-chrome steel (°)

ρ_a, ρ_NaCl – density; single agglomerate (kg·m^–3) and saturated sodium chloride solution (g·ml^-1), respectively

σ, σ₁ – stress; maximum normal, major consolidating, respectively (kPa)

σ_c – unconfined yield strength (kPa)

σ_p – maximum tensile strength (MPa)

τ – shear stress (kPa)

φ, φ_c, φ_e, φ_lin, – the angle of internal friction; general, for determined flowability, effective, linearised, respectively (°)

Abbreviations of descriptive factors

INOC – inoculum

PIBR – comminuted raw pine particleboards

PIBT – comminuted pine particleboards after Pleurotus ostreatus pretreatment

PISR – comminuted raw pine shavings after cutting-milling

PIST – comminuted pine shavings after Pleurotus ostreatus pretreatment

POBR – comminuted raw poplar particleboards

POBT – comminuted poplar particleboards after Pleurotus ostreatus pretreatment

comminuted

POST – comminuted poplar shavings after Pleurotus ostreatus pretreatment

MT – material state (raw, R, after Pleurotus ostreatus pretreatment, T)

PT – product type (shavings, SH, particleboard, PB)

TL – load type (axial compression, AC, radial compression, RC)

TW – wood species (pine, PI, poplar, PO)

Conflicts of interest

The authors declare that there are no financial or commercial conflicts of interest.

Author Contribution

Author Contributions: Conceptualization, P.T. and A.L.; methods, P.T. and A.L.; software, P.T. and A.L.; formal analysis, P.T. and A.L.; investigation, P.T., A.L., and S.W.; resources, P.T. and A.L; data curation, P.T. and A.L.; writing—original draft preparation, P.T.; writing—review and editing, A.L. and A.S.; visualization, P.T. and A.L.; project administration, A.L.; supervision, A.L. All authors have read and agreed to the published version of the manuscript.

Acknowledgements

This research is part of a doctoral dissertation supported by the Ministry of Science and Higher Education in Poland. The authors thank their colleagues from the Department of Biosystems Engineering for technical assistance in the research.

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No competing interests reported.

Pretreatment of pine and poplar particleboards with Pleurotus ostreatus (Jacq.) P. Kumm: wood structure decomposition, potential of solid fuel and biogas production

Status:

Version 1

Abstract

Figures

1. Introduction

2. Material and methods