Optimizing fungal treatment of lignocellulosic agro‐industrial by‐products to enhance their nutritional value

Abstract This study delves into the dynamic interaction between various fungal strains, substrates, and treatment durations to optimize the nutritional value of these by‐products. Six fungi, including Penicillium chrysogenum, Fusarium sp., Fusarium oxysporum, Fusarium solani, Penicillium crustosum, and Cosmospora viridescens, were evaluated across three substrates: wheat straw (WS), cedar sawdust (CW), and olive pomace (OP) over treatment periods of 4, 8, and 12 weeks. The study discerned profound impacts of these fungi across multiple parameters, including cellulose variation (C.var), lignin variation (L.var), and in vitro true digestibility variation (IVTD.var). Our results demonstrated that the various fungi had a significant effect on all parameters (p < .001). Noteworthy, F. oxysporum and F. solani emerged as exemplars, displaying notable lignin degradation, cellulose liberation, and IVTD enhancement. Importantly, P. crustosum demonstrated substantial cellulose degradation, exhibiting optimal efficacy in just 4 weeks for all substrates. Notably, F. sp. excelled, yielding favorable results when treating WS. P. chrysogenum achieved optimal outcomes with 8‐week treatment for WS. Both Fusarium sp. and P. chrysogenum exhibited slight cellulose release, with remarkable reduction of WS lignin compared to other substrates. Especially, WS and OP displayed superior digestibility enhancements relative to CW. It should be noted that the treatment duration further shaped these outcomes, as prolonged treatment (12 weeks) fostered greater benefits in lignin degradation and digestibility, albeit with concomitant cellulose degradation. These findings underscore the intricate balance between fungal strains, substrates, and treatment durations in optimizing the nutritional value of lignocellulosic agro‐industrial by‐products. The outcomes of this study lead to the enhancement in the overall value of by‐products, promoting sustainable livestock feed and advancing agricultural sustainability.


| INTRODUC TI ON
The exponential growth of industrialization and globalization, coupled with population growth, has dramatically accelerated the expansion of agricultural and industrial operations worldwide (Wei et al., 2022;Yafetto et al., 2023).This has resulted in significant production of lignocellulosic biomass, which amounts to approximately 181.5 billion tons annually, of which 8.2 billion tons is used (Mujtaba et al., 2023).Morocco is characterized by its vibrant agro-industrial landscape, which contributes significantly to its gross domestic product (GDP), with agriculture accounting for nearly 13%.Morocco is a notable producer of various agricultural by-products, including industrial (such as olive, tomato, wasted potatoes (Chauhan et al., 2023), and wood by-products) and agricultural (such as wheat straw) wastes amounting to around 4 million tons per year.According to the Ministry of Energy Transition and Sustainable Development, Department of Energy Transition ( 2023), the technical energy potential is about 13.4 million MWh (megawatt hour) per year.In parallel with Morocco's contributions, China plays a pivotal role as a colossal producer, contributing 126 million metric tons of wheat straw and accounting for 25% of the world's cotton production.The United States leads in the production of agricultural residues, contributing an impressive 80% of the global total, along with a substantial 25% of the world's sugarcane bagasse.In addition, Europe plays a significant role in the production of oat straw, accounting for a remarkable 64% of the world's total (Velvizhi et al., 2023).Unfortunately, a significant portion of these agro-industrial by-products receive inadequate posttreatment and are often incinerated or disposed of in makeshift landfills.This situation raises significant environmental and sustainability concerns.
The cell wall fraction of lignocellulosic biomass, which includes carbohydrates such as cellulose and hemicellulose (Tufail et al., 2021) and pectin, and the nonstructural or cell content fraction, which contains carbohydrates such as starch, sugars (watersoluble carbohydrates; WSC), organic acids (OA), fructans, lipids, and proteins, have significant potential for sustainable ruminant production (Sufyan et al., 2024).Neutral detergent fiber (NDF) can be partially degraded to volatile fatty acids by ruminal microorganisms (fungi, bacteria, etc.) to provide energy for the organisms.
Additionally, adjusting the ratio of nonfiber carbohydrates to neutral detergent fiber (NFC/NDF) can influence ruminal fermentation and microbial type in ruminants (Ma et al., 2022).Lignocellulosic feeds provide an alternative to conventional feedstuffs such as cereals and oilseeds, demonstrating their promise to promote environmentally friendly and economically viable practices (Chai et al., 2022;Cheng & Whang, 2022).This alternative is particularly relevant in regions facing challenges due to prolonged drought, such as Morocco (Castellani et al., 2017).However, studies have revealed that the utilization of these agro-industrial by-products in ruminant feed is suboptimal, primarily due to their low nutritional value (Bentil, 2021;Rouches et al., 2019;Sajid et al., 2022).
In response to this nutritional challenge, researchers have explored various methods to enhance the nutritional value of lignocellulosic biomass, with a particular focus on biological approaches using ligninolytic fungi.The biological approach offers significant advantages, such as environmental friendliness and economic affordability, over chemical and physical methods (Pantet al., 2022).
Ligninolytic fungi, such as Ganoderma lucidum, Lentinula edodes, Pleurotus eryngii, Pleurotus ostreatus, Trichoderma reesei, Mucor indicus (Iyyappan et al., 2023), as well as Penicillium and Fusarium (Benatti & Polizeli, 2023), have shown promise in enhancing the digestibility of lignocellulosic substrates.However, despite this potential, there is a notable gap in the literature regarding universally accepted criteria for assessing the effectiveness of these fungi in improving the nutritional value of lignocellulosic biomass.In particular, these studies have not clearly defined generally accepted criteria for assessing the efficacy of the fungi used, or if such criteria exist, they have been applied in a limited manner.For instance, achieving high digestibility from a particular biomass after treatment with a specific fungus is undoubtedly a positive outcome.However, it is not sufficient to confirm the overall effectiveness of the fungus in biomass treatment.The potential of fungi lies in their ability to enhance nutritional value by improving digestibility, decreasing lignin content, reducing cellulose crystallinity, and maintaining or improving the different nutrient contents (e.g., CP, cellulose, and NFC) in lignocellulosic biomass.To determine true efficacy, it is imperative to conduct trials on different lignocellulosic materials, taking into account critical factors, such as the initial biomass components (including cellulose, lignin, NFC, and CP) and treatment duration.This comprehensive approach ensures a more accurate assessment of the versatility and applicability of the fungal treatment to different feedstock sources.While basidiomycetes have demonstrated considerable potential, it's essential to expand the scope of the investigation to include ascomycete fungi as well (Grace Barrios-Gutiérrez et al., 2023).Exploring a broader range of fungal species can lead to a more holistic understanding of the potential applications of ligninolytic fungi in biomass treatment.Such inclusivity can uncover novel fungal candidates that may offer unique advantages or synergistic effects when applied to different lignocellulosic materials.The primary objective of this study is to establish a comprehensive set of criteria for the effective selection of fungi in the treatment of agroindustrial by-products, with the overarching goal of enhancing their nutritional value.To achieve this objective, we conducted a thorough comparison and evaluation of six different fungal strains: Fusarium solani, Fusarium oxysporum, F. sp., Penicillium chrysogenum, Cosmospora viridescens, and Penicillium crustosum.This evaluation encompassed three different types of lignocellulosic materials, namely wheat straw, olive pomace, and cedar sawdust.Furthermore, the study spanned three treatment durations of 4, 8, and 12 weeks.
Wheat straw was obtained from fields in El Hajeb region of Morocco, while OP was obtained from the Olea Food Company in Meknes, Morocco, and CW was obtained from the Ifrane National Park in Morocco.CW and WS were ground to a particle size of 0.5-1.5 cm, while OP particles were 2-5 mm long.This variation in substrate size was chosen to accurately represent their natural origin in the field.
The substrates were then soaked in water at room temperature for 3 days to ensure complete water penetration.The excess water was then drained off, and the substrates were autoclaved at 121°C for 20 min (van Kuijk et al., 2015).
A quantity of substrate, containing approximately 30 g of dry matter, was weighed and placed in 250 mL glass bottles closed with cotton to allow contamination-free air exchange.The bottles were then autoclaved at 121°C for 20 min and were kept at room temperature (25 ± 3°C) until use.A total of 72 bottles were prepared for the combinations of three substrates, six fungi, and four treatment times.All 72 combinations were replicated in three time periods.

| Spawn preparation for different fungi
The fungi used were selected based on their utilized carbon source.
Two fungi, C. viridescens, and P. crustosum, were selected from a culture medium with only cellulose (C) as a carbon source.Two other fungi, F. oxysporum and, F. sp., were selected from a culture medium with only lignin (L) as a carbon source.Finally, F. solani and P. chrysogenum were selected from a culture medium containing both C and L as carbon sources.The fungi were identified in the laboratory and stored in glycerol at −20°C (Nait M'Barek et al., 2019).
Sterilized sorghum grains were inoculated with a 10-mm disk of colonized agar culture.The inoculated grains were then incubated at 25°C for 15 days, allowing for the complete colonization of all grains by fungal mycelium.Subsequently, the resulting spawn, comprising sorghum grains fully colonized by mycelium, was used to treat the substrates (van Kuijk et al., 2017).

| Experimental setup
Lignocellulosic materials were subjected to fungal treatment using the fungal spawn.At the start of the experiment, 11 ± 0.2 w/w % DM (dry matter) of prepared spawn consisting of colonized sorghum grains was added to the substrate placed in the bottles.The weights of the substrate and spawn were accurately measured.The spawn was mixed aseptically with the substrate using sterile spoons and tweezers to ensure even distribution.The inoculated substrates were then placed in 250 mL glass bottles and incubated at 26°C under 70%-80% relative humidity (Chaitanoo et al., 2021;van Kuijk, del Río, et al., 2016).All fungal treatments were performed simultaneously and repeated three times at different periods.

| Sampling techniques and procedures
At 4-week intervals during the 12-week treatment period, a 250 mL glass bottle of each fungus-substrate combination was removed from the incubator.After manual mixing, the treated substrate was dried at 60°C for 72 h, then ground through a 1-mm sieve and subjected to chemical (e.g., fibers), physical (cellulose crystallinity), and biological (digestibility) analyses.
The choice of treatment duration-4, 8, and 12 weeks-was a critical aspect of our study design.It allowed us to gain insights into the short-term, mid-term, and long-term effects of fungal strain treatment on these agro-industrial by-products.

| Fiber composition analysis
Fibers were analyzed according to the Van Soest method (Van Soest et al., 1991).Acid detergent lignin (ADL) was defined as L, while C was determined as the difference between acid detergent fiber (ADF) and ADL.The variation or change in C and L (C.var and L.var) was calculated to evaluate the changes in the fibers compared to the control (Benaddou et al., 2023a(Benaddou et al., , 2023b)).L.var was calculated using the following formula (Equation 1): where ADL c is the ADL of the control within the same treatment duration and ADL t is the ADL of the treated sample with one of the fungi.
The control values were then excluded before statistical analysis.
C.var was calculated using the following formula to determine the change of C during treatment (Equation 2): where ADF c and ADL c are the ADF and ADL of the control within the same treatment duration and ADL t and ADF t are the ADL and ADF of the sample treated with one of the fungi. (1)

| In vitro digestibility assessment
For in vitro true digestibility (IVTD) measurements, rumen contents were collected from four Simmental bulls after slaughter at a local abattoir.The rumen contents (fluid and fibrous mat) were promptly transported to the laboratory in thermos containers, maintaining a temperature of 39.0°C.Upon arrival, the rumen contents were homogenized, filtered through two layers of cheesecloth under a carbon dioxide (CO 2 ) atmosphere, and incubated using an ANKOM DAISYII incubator.This specialized incubator is equipped with four rotating digestion jars and ensures consistent heat (39.0°C) and agitation.Each incubation jar received 1600 mL of buffer solution and 400 mL of rumen fluid as inoculum, along with 25 filter bags containing the samples (Ankom F57) (Gulecyuz, 2017).
Approximately 0.5 ± 0.05 g of the sample was placed into each filter bag.Subsequently, each filter bag was introduced into its respective incubation cylinders containing the solution.Before sealing, the cylinder was aerated with CO2 for 40 s.The sealed cylinders were then placed into the incubator for a 48-h incubation period.
Following incubation, the solution was drained, and the filter bags were cleaned with water and subsequently dried at 60°C for 72 h.
The NDF determination procedure was then followed to eliminate the remaining cell solubles (Abrahamsen et al., 2021;Tilley & Terry, 1963).The in vitro true digestibility (IVTD) of both treated and untreated samples was calculated using the formula (Equation 3): where: W1: Weight of the filter bag, W2: Weight of sample DM, W3: Final weight after NDF analysis, and C1: blank bag correction.
IVTD.var was used to assess the change (in %) in IVTD between the treated and control samples.

| Fungal saccharification
In this study, we employed the 3,5-dinitrosalicylic acid (DNSA) method to assess fungal saccharification, which is essential for evaluating the efficiency of fungal enzymes in converting complex carbohydrates into valuable reducing sugars (RS) (Boshagh, 2021.RS variation (RS.var) was calculated using the formula (Equation 4): where RS.var represents the percentage change in reducing sugars, RS t is the reducing sugars content in the treated substrate after 12 weeks, and RS c is the reducing sugars content in the control substrate.

| Quantifying crystallinity index
In this study, we used X-ray diffraction (XRD) to determine cellulose crystallinity through the Segal crystallinity index (CI).Diffractograms were meticulously recorded over the range of 10° to 30° at a scan rate of 2 per minute.The CI was calculated by evaluating the height ratio between the intensity of the crystalline peak (I002 -I am ) and the total intensity (I002).This calculation was performed after subtracting the background signal measured without cellulose (C) using the empirical equation developed by Segal et al. (1959) and further discussed by Varma et al. (2019) (Equation 5): Additionally, the Bragg equation was employed to determine the d-spacing (interplanar spacing) as follows (Equation 6): Here, 'λ' represents the incident X-ray wavelength (1.5406 Å), 'θ' signifies the peak position in radians, 'n' corresponds to the diffraction order, and 'd' denotes the d-spacing measured in Ångströms (Å).
To calculate the crystallinity index (CI) variation (CI.var) after 12 weeks, we used the following formula (Equation 7): where CI.var represents the percentage change in CI after 12 weeks.
CI t is CI of the treated substrate after 12 weeks.CI c is CI of the control (untreated) substrate.
The crude protein (CP) and ether extract (EE) contents were determined, as proposed by the Association of Official Analytical Chemists (AOAC) (1990).NFC was determined using Equation 8( Mertens, 1997): where NDF refers to neutral detergent fiber, EE refers to ether extraction, and CP stands for crude protein.

| Measuring enzymatic activity
Maize was dried at 60°C for 72 h before being milled and passed through a 1-mm screen.Enzymatic extraction was carried out using a modified version of the method described by Rodrigues et al. (2008).Laccase activity was determined using 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as substrate, and lignin peroxidase assay was determined using the azure B dye as a substrate.Manganese peroxide (MnP) activity was measured by monitoring the oxidation of Mn 2+ to Mn 3+ in 0.11 M sodium lactate, and the enzyme activities were expressed in IU/mL.Cellulase activity was determined, according to the Ghose method (Ghose, 1987).
It should be noted that all the activities detected at the beginning were checked at the beginning of each treatment period (at the beginning, after 4 weeks of treatment, and after 8 weeks of treatment) without quantification.

| Statistical methods and analysis
The experiments were performed in triplicate, and the sources of variation were identified as fungal species, substrates, blocks, and treatment duration.Normality and homogeneity of the variances were tested using the Shapiro-Wilk test and Bartlett's test, respectively.To evaluate the combined effect of fungus, substrate, and treatment duration on biomass degradation measurements (C.var, L.var, and IVTD.var), a three-way analysis of variance (ANOVA) was performed using R software (Alkarkhi & Alqaraghuli, 2020).Tukey's test was used for multiple comparisons at a significance level of 0.05.

| Fungal strains and substrate characteristics
The physicochemical characteristics of the substrates are shown in Table 1.The analysis revealed significant differences in fiber, CI, and RS among the different lignocellulosic biomass samples.As shown in Table 2, cellulase and ligninase activities were present in all fungi regardless of the selection medium, except for F. sp. and C. viridescens.

| Effect of fungi
The performance of selected fungal strains in degrading lignocellulosic substrates and improving nutritional value was assessed in this  Regarding L degradation, F. oxysporum and F. solani were the most efficient, removing more than 40% of the L, followed by F. sp.
with 25% compared to the control.Contrarily, P. crustosum and C.
viridescens did not show significant degradation of L from any of the substrates.They increased the amounts of lignin from the substrates (F = 52.52%and F = 36.32%,respectively).P. chrysogenum showed insignificant L reduction from OP and CW, but significant decomposition from WS (L.var = −26.65 ± 7.1%).
In terms of digestibility, F. oxysporum and F. solani emerged as the best fungi that significantly improved digestibility compared to the other fungi (mean of IVTD.var was 41.71 ± 13.6% and 30.35 ± 17.9%, respectively).C. viridescens and P. chrysogenum slightly increased digestibility for all substrates (the mean of IVTD. var was 11.90 ± 5.8% for both).F. sp. and P. crustosum showed insignificant improvement in digestibility (Figures 1, 2, and 3).Of the total increase in digestibility, F. oxysporum and F. solani accounted for a large proportion (F was 28.5% for both).

| Effect of substrate
The study investigated the effect of substrates, including WS, CW, and OP, on fungal treatment.First, there were significant differences in physicochemical composition (dry matter (DM), fibers, pH, CI, and RS) among the three substrates before treatment.The DM content varied with WS having the highest value (79.22 ± 2.00%), followed by OP (66.74 ± 1.20%) and CW (59.41 ± 1.90%).OP and CW had similar percentages of C and L (about 35% and 20%, respectively), whereas WS had a higher percentage of C (49.35%) and a lower percentage of L (11.4 ± 2.40%).
Second, after treatment, Figures 1 and 2 show that the substrates differed significantly in their IVTD.var,C.var, L.var, and ADF (p < .001)(Table 3).As shown in Figure 2, the study revealed that lignin and cellulose of CW were highly resistant to fungal attack followed by OP and WS.The NFC/NDF ratio of WS increased more than those of OP and CW.On the other hand, CP increased more in WS and OP than in CW.The digestibilities of treated WS and OP were significantly improved (the mean of IVTD.var was 22.65 ± 17% and 19 ± 17.26% respectively).Among the increased total digestibility (IVTD.var> 0) of

| Interaction fungus × substrate × treatment duration
The enhanced nutritional value of the treated biomass is typically associated with notable increases in cellulose availability, concurrent reductions in lignin content, and a substantial elevation in overall digestibility.As presented in Figure 5, F. solani and F. oxysporum significantly improved the nutritional value of the treated substrates compared to the other fungi and the control after up to 8 weeks of incubation.However, it should be noted that the speed of this improvement was very high in the case of WS, followed by OP, but this improvement was low in CW.After 8 weeks, F. oxysporum was found to have degraded cellulose in CW and OP but continued to increase cellulose in WS.For F. solani, a greater release of cellulose was observed in the CW after 8 weeks of incubation.There was no significant change in lignin up to 4 weeks of treatment compared with the control in all fungi.P. crustosum and C. viridescens had no significant effect on the change in percentage of lignin in OP and WS, but C. viridescens reduced lignin in CW by more than 50% compared with the control after 12 weeks of incubation.P. chrysogenum degraded WS lignin by 25% relative to the control during 8 weeks of treatment, but after this duration, the change in lignin was negligible.In terms of digestibility, F. oxysporum and F. solani increased the digestibility of all substrates exponentially over time.P. chrysogenum slightly (but not more than 20%) increased CW digestibility continuously up to 12 weeks of treatment, while attacking WS lignin only between 4 and 8 weeks.

| Variables correlations
As depicted in Figure 6, the inertia of the first dimensions indicates whether there are strong relationships between variables and suggests the number of dimensions that should be studied.The total   Note: Means (±SD), within the same row and the same parameter, lacking a common superscript differ (α = .05).
Cluster 2 includes individuals (the substrates treated by F. sp.

| DISCUSS ION
The present study aimed to evaluate the performance of six different fungi in improving the nutritional value of three lignocellulosic substrates (CW, WS, and OP).The fungi were subjected to treatment durations of 4, 8, and 12 weeks to assess their efficacy over a varying duration.Fungi can optimize the nutritional value of lignocellulosic biomass if they improve IVTD, increase cellulose content, reduce crystallinity index, lower lignin levels, enhance crude protein, and improve nonfiber carbohydrate (NFC) and RS content.

| Effect of fungi
The selected fungi display different abilities to break down the lignin barrier while demonstrating a different selectivity in their approach to cellulose processing (Figures 1 and 2).Additionally, these fungi showed differences in the release of NFC and the production of CP (Figure 4).Several fungi have developed the production of lignocellulolytic enzymes, creating a synergistic system capable of degrading the primary polymers found in lignocellulosic biomass.These  and 4, Table 4).These fungi share characteristics with  4).
The first group (P.crustosum and C. viridescens) exhibited limited lignin degradation, as indicated by the high value of L.var, which was lower than that observed in Lentinula edodes (van Kuijk et al., 2015) (Table 4), suggesting that the targeted fungi did not effectively break down lignin in the lignocellulosic materials possibly due to the absence of or low ligninolytic activity (Table 2).Despite the limited lignin degradation, this group demonstrated a remarkable capacity for cellulose degradation, as evidenced by the low value of C.var, exceeding that of Pseudotsuga menziesii (Alexandropoulou et al., 2017).
This implies that the fungi in this group modified the lignin structure rather than removing it.This modification allowed them to access the cellulose and hydrolyze it into nonfiber carbohydrates (NFC), especially RS through the action of endoglucanases (Table 2).
However, the low NFC and RS content in the substrate treated by these fungi indicates that the released NFC was not primarily utilized for enhancing nutritional parameters but rather converted into secondary metabolites, such as CO 2 (Ma et al., 2020).Additionally, the limited in vitro true digestibility (IVTD) (Figure 1) observed in this group suggests that despite their proficiency in cellulose degradation, the overall digestibility of the treated substrate remained restricted, possibly due to the redirection of NFC and RS toward secondary metabolic processes.2).The laccase and manganese peroxidase system is known for its effectiveness in degrading lignin Chen et al. (2012).This enzymatic system is highly efficient in the oxidation of both phenolic and nonphenolic components of lignin (Debnath & Saha, 2020; Manyapu The bioconversion yield of hydrolysis was 52% (based on total cellulose content)

| Effect of substrate
The lignocellulosic substrate tested in this study is characterized by a high content of lignified plant cell walls, which includes not only lignin but also hemicellulose and cellulose.Additionally, this lignocellulosic biomass contains only small amounts of ash, proteins, and nonfiber carbohydrates (NFC) (Table 1).The lignin content and composition vary among different feed ingredients, including residues of wheat, rice, corn, oil palm, cocoa, bamboo, sugarcane, water hyacinth, cedar, birch, and spruce (van Kuijk, del Río, et al., 2016), as well as olive pomace (Benaddou et al., 2023b).
Following a 12-week treatment, substantial variations in IVTD.
var, C.var, L.var, and ADF were noted across the substrates (p < .001).
Notably, the nutritional value of treated wheat straw (WS) exhibited a better improvement than that of cedar wood (CW).This difference can be attributed to various factors.First, the relatively short length of straw fiber cells plays a role, with wheat straw fiber cells averaging 1.18 mm in length and 13.60 μm in width (Singh et al., 2011), while softwood tracheids are approximately 3 mm in length and 20-35 μm in width.Additionally, the cellulose crystallinity index (CI) was higher in CW (CI = 54.5 ± 2.5%) compared to OP (CI = 41.2 ± 3.0%) and WS (CI = 33.4± 2.7%).The highly crystalline cellulose in CW, coupled with its elevated lignin content, may limit accessibility to enzymes, hindering the rate and efficiency of nutritional value improvement.
In contrast, the amorphous regions of WS cellulose offer a greater reactive surface area compared to crystalline regions.This enhanced reactive surface area provides more sites for enzyme action, facilitating cellulose degradation (Xu et al., 2019).As a result, WS proved to be more accessible for enzymes of most fungi used, leading to improvements in CP, NFC/NDF ratio, IVTD, and cellulose, thereby enhancing its nutritional value.Our study revealed that when exposed to olive pomace (OP) and cedar wood (CW), P. chrysogenum and F. sp.showed limited lignin breakdown.In contrast, previous studies with other fungi, such as Pityriasis versicolor and Trametes versicolor, demonstrated more effective lignin degradation in different wood types like Pinus yunnanensis (Qi et al., 2023;Wang et al., 2013).
However, the scenario changed when wheat straw (WS) was used with P. chrysogenum and F. sp.In this case, they exhibited significant lignin breakdown, comparable to the results observed with Pseudotsuga menziesii when treated with willow sawdust for 30 days in another study (Alexandropoulou et al., 2017).Even though F. sp.
can break down lignin, it didn't make the wheat straw easier to digest.This is puzzling.Our study also found that when F. sp.broke down lignin, it reduced the digestibility by about 25%.Breaking down lignin may have produced chemical inhibitors that make biomass less digestible, reducing its nutritional value.
Olive pomace (OP) is the solid residue that remains after oil is extracted from olives.These residues include pulp, skin, pit, and a small amount of residual oil.The substrate content provides an ideal environment for fungal growth, facilitating the breakdown of complex components such as lignin and cellulose.Fungi can utilize the carbon sources present in olive pomace and promote the release of enzymes that contribute to the breakdown of lignocellulosic materials.This natural decomposition process, driven by fungal activity, enhances the degradation of lignin and cellulose, ultimately affecting the value of olive pomace for various applications, including animal feed and other industrial processes (Innangi et al., 2017;Lammi et al., 2019;van Kuijk, Sonnenberg, et al., 2016).
Tolerance to variable conditions is important for nutritional value improvement by fungi.A successful fungus must be able to function effectively under different environmental conditions, such as temperature, humidity, pH, etc.In this study, a high pH tolerance was observed in F. oxysporum.The performance of F. oxysporum was maintained in different substrate pH levels (from 4.2 ± 0.4 to 6.0 ± 0.7).Adaptability to different substrates is also an important aspect.Efficient degradation of different types of substrates, such as wheat straw, cedar sawdust, and olive pomace, is a characteristic of the best performing fungus.According to the results, F. oxysporum and F. solani are adapted and perform optimally on a wider range of lignocellulosic materials (OP, CW, and WS).

| Effect of treatment duration
The selection of treatment durations ranging from 4 to 12 weeks was based on our aim to comprehensively evaluate the efficacy of the To study the associations between biomass changes(C.var,L.var, and   IVTD.var) and the different fungi, substrates, and treatment times, a principal component analysis (PCA) was carried out by R. The alluvial graph was plotted using Origin software to visualize the transitions and connections between the different variables (fungus, substrate, and treatment duration).The alluvial plot represented only positive observations (C.var > 0, L.var > 0, and IVTD.var > 0).The stacked bars represented variables, and the segments represented the frequency observations (F) in the data frame that belonged to that level.In other words, F represented the occurrence of factors (fungi, substrates, or duration treatment) in cellulose, lignin, or digestibility change.The colored streams between the stacked bars represented a group of observations corresponding to the value for each variable indicated by the stream, and the thickness of the stream indicated how many observations belonged to that group.To create the clustergram, we utilized the OriginPro software after standardizing the variables(L.var,C.var, and IVTD.var).The distance type employed was the Pearson correlation.The row names are composed successively of the fungus name, followed by the substrate treated by this fungus, and subsequently, the treatment duration (4, 8, or 12 weeks).For example, 'F.oxysporum-OP-8w' signifies olive pomace treated by F. oxysporum for 8 weeks.

TA B L E 1
Abbreviations: ND, not detected.

F
Visualizing the effectiveness of fungal treatment on the treated substrate over time using an alluvial diagram with positive observations.(a) Visualization of C.var; (b) visualization of L.var; and (c) visualization of IVTD.var.CW, cedar wood sawdust; F, frequency of observation; OP, Olive pomace; WS, wheat straw.treated substrates, the digestibility of WS makes up a large proportion (F = 41.75),followed by OP (F = 35.14%)and CW (F = 23.3%).

F
Clustergram of fungal treatment effects on different substrates over varied durations.dataset inertia is expressed by the first two dimensions, accounting for 93.56% of the total variability.Moving on to the hierarchical classification of individuals, the presence of three clusters was revealed: Cluster 1 consists of individuals (the substrates treated by P. crustosum and C. viridescens) sharing the following characteristics: High values for the variable L.var and low values for the variables C.var and IVTD.var (variables are sorted from the weakest).

F
Changes in the NFC/NDF ratio and crude protein content of lignocellulosic substrates (CW, cedar wood sawdust; OP, olive pomace; WS, wheat straw) treated by various fungal strains (F.solani, F. oxysporum, F. sp., P. chrysogenum, P. crustosum, and C. viridescens) over a 12-week duration.abc Difference in superscripts in the same substrate indicates significance at p < .05.TA B L E 3 Change in crystallinity index (CI.var)and reducing sugars (RS.var) in fungus-treated substrates after 12 weeks.
and P. chrysogenum) characterized by C.var, L.var, and IVTD.var whose values do not significantly differ from the mean.Cluster 3 comprises individuals (the samples treated by F. oxysporum and F. solani, especially the samples of WS and OP incubated for 12 weeks) characterized by: High values for the variables IVTD.var and C.var (variables are sorted from the strongest) and low values for the variable L.var.The Pearson correlation coefficient (r) between C, C.var, L, L.var, ADL, ADF, IVTD.var, and IVTD is presented in Figure 6.Positive correlations were observed between C and IVTD (r = .7),and between C.var and IVTD.var (r = 0.79).In contrast, negative correlations were found between L.var and C.var (r = −.69),L.var and IVTD.var (r = −.78), and ADL and IVTD (r = −.78).

F
Principal component analysis (PCA).(a) Variables factor map (PCA); (b) Individuals factor map (PCA); (d) Qualitative factor map (PCA); and (c) Ascending Hierarchical Classification of the individuals.The labeled factors, variables, and individuals are those the best shown on the plane.Ceriporiopsis subvermispora, Lentinula edodes, Hericium clathroides, Pleurotus ostreatus, and Pleurotus eryngii (Labuschagne et al., 2000; Liang et al., 2010; Makkar & Singh, 1992), and Pleurotus salmoneostramineus (Bickel Haase et al., 2024).Based on the PCA (Figures 5 and 6), the fungi used in our study can be classified into three distinct groups.The first group comprises P. crustosum and C. viridescens, the second group consists of F. sp. and P. chrysogenum, and the third group is composed of F. oxysporum and F. solani.This classification suggests that each group, consisting of two fungi, demonstrates a notable level of consistency in their performance when compared to the overall mean values of C.var, L.var, and IVTD.var,NCF/NDF, and CP.The minimal deviations from the mean values across these parameters indicate that the fungi within each group may exhibit relatively balanced and consistent enzymatic activity in cellulose and lignin degradation, as well as in enhancing overall digestibility.It is noteworthy that the enzyme activities and their effects on substrate composition and treatment duration differed from those observed in other studies (Table

F
The Pearson correlation between parameters.C, Cellulose; C.var, cellulose variation; IVTD, in vitro true digestibility; L.var, lignin variation.TA B L E 4Comparative analysis of fungal treatment of lignocellulosic substrates.
(Mól et al., 2023)ogenum exhibited β-glucosidase activity.β-glucosidasesforma heterogeneous group of hydrolytic enzymes with significant biological importance, acting on various substrates(Mól et al., 2023).Despite the lack of endoglucanase and β-glucosidase activity measured in F. sp., it was able to de- Saravanan et al. (2023)tional value.It is noteworthy that F. oxysporum and F. solani demonstrated a lower capacity to degrade wheat straw lignin compared to Ceriporiopsis subvermispora and Lentinula edodes, as reported byvan Kuijk, Sonnenberg, et al. (2016b)andMadadi (2017).On the contrary, F. oxysporum and F. solani exhibited a higher lignin degradation capacity for wheat straw compared to Pleurotus ostreatus, where the lignin reduction did not exceed 34% after 8 weeks of incubation, as reported bySaravanan et al. (2023).