Influence of white-rot fungi on chemical composition and in vitro digestibility of lignocellulosic agro-industrial residues

In this work, we tested the ability of white-rot fungi to alter the chemical composition of lignocellulosic agro-industrial residues so that they could be more susceptible to microbial degradation by rumen microorganisms, thus improving in vitro digestibility of neutral detergent fiber. Agro-industrial residues that were incubated for 60 days with Pleurotus ostreatus PLO06 or Lentinula edodes UFV73 showed significant changes in chemical composition, increasing (P<0.05) the crude protein content and the in vitro digestibility of neutral detergent fiber. Lignin content and structural carbohydrates decreased after fungal fructification in most substrates and an increase (P<0.05) in dry matter mineralization was also observed for residues treated with P. ostreatus PLO06. L. edodes UFV73 required a more balanced carbon/nitrogen ratio to grow on lignocellulosic substrates, and P. ostreatus PLO06 was in general, more effective than L. edodes UFV73 to alter the chemical composition and digestibility of the agroindustrial residues. Among the residues tested, eucalyptus bark showed the highest increase in digestibility after fungal growth. The marked increments in fiber digestibility obtained with substrates inoculated with either P. ostreatus PLO06 or L. edodes UFV73 indicate the usefulness of biological pretreatments to improve the nutritive value of low-quality lignocellulosic feedstuffs that could be incorporated into ruminant rations.

large amounts of residues with a complex chemical composition, in which cellulose, hemicellulose and lignin are the main constituents (Dashtban et al., 2009;Sánchez, 2009).
The disposal of lignocellulosic residues has often been associated with environmental pollution as well as sanitary and ecological effects (Dashtban et al., 2009), but several lignocellulosic residues could be useful to obtain products of great economic, energetic or nutritional value, such as chemicals, biofuels and animal feeds (Okano et al., 2006;Hasunuma et al., 2012).
Ruminant animals in particular have evolved a symbiotic relationship with biomass-degrading microbes that allows them to digest lignocellulosic biomass and at least some agricultural and industrial lignocellulosic residues are used as ingredients of cattle diets (Okano et al., 2006;Sánchez, 2010;Shrivastava et al., 2011).Nonetheless, the extension of digestion and nutritional value of these residues may be limited by their content of lignin and protein.Therefore, strategies for pretreatment of lignocellulosic biomass can improve the overall digestion and the nutritional value of agricultural and industrial residues that could be used in ruminant rations (Okano et al., 2006;Fazaeli, 2007).
The biological breakdown of lignocellulosic materials depends on the hydrolytic activity of enzymes produced by a variety of microbial species, especially bacteria and fungi (Sharma and Arora, 2010;Dong et al., 2013).White-rot fungi are well known for their ability to produce hydrolytic and oxidative extracellular enzymes and decompose lignocellulosic substrates (Philippoussis et al., 2011).In this regard, the genera Pleurotus and Lentinula belong to a group of widely distributed edible mushrooms with the ability to degrade several lingocellulosic substrates (Sánchez, 2010;Philippoussis et al., 2011).
P. ostreatus is a highly ligninolytic fungus with great potential for biotechnological applications due to its simple growth requirements and ability to secrete different oxidative enzymes, such as peroxidases and laccases, active against a variety of toxic substances and recalcitrant substrates (Sánchez, 2010;Shrivastava et al., 2011).Previous work demonstrated strain-specific variations in mushroom production and P. ostreatus PLO06 showed high activities of manganese peroxidase, xylanase and cellulase growing in a variety of complex and growth conditions (Luz et al., 2012).
The basidiomycete Lentinula edodes (Berkeley) Pegler (Lentinus edodes), known as shiitake, is the second most popular mushroom in the world (Rincão et al., 2012).L. edodes is valued for its nutritional and medicinal properties, culinary and industrial applications (Jiang et al., 2010) as well as biodegradation and biotransformation abilities (Lakhtar et al., 2010).Strains of L. edodes produce extracellular oxidizing enzymes capable of biodegrading lignin-related recalcitrant compounds, such as polyphenols.
P. ostreatus and L. edodes have been studied as an alternative to improve the nutritional value of lignincelulosic substrates as ruminant feeds (Tuyen et al., 2012;Dong et al., 2013;Tuyen et al., 2013).However, the majority of these previous studies focused only on substrates with a very limited range of neutral detergent fiber digestibility and lignin concentrations (Okano et al., 2006, Lynch et al., 2013).Furthermore, no previous study has been conducted to address the simultaneous use of lignocellulosic substrates for mushroom production and animal feeds.This technology could be profitable especially in small farms, where the use of abundant agro-industrial substrates could serve as a source of income to farmers who could commercialize the mushrooms harvested from fructified substrates and use these as substrates with greater nutritional value in ruminant feeds.
In the present study, we explored the ability of P. ostreatus PLO06 and L.edodes UFV73 to degrade lignin and other recalcitrant substrates at a high rate to: 1) determine their ability to improve the protein content of several agro-industrial residues with different chemical compositions and concentrations of digestible carbohydrates and 2) improve the in vitro digestibility of different lignocellulosic materials that could also be applied for mushroom production.
Although some substrates used in this study usually have low nutritional quality to be included directly into livestock diets, we hypothesized that the fungal biomass could improve the nutritional value of these residues by increasing the concentration of protein and digestibility of the substrates as well as reducing the lignin content of the lignocellulosic residues.

Microorganism and growth conditions
P. ostreatus PLO06 and L.edodes UFV73 were isolated from eucalyptus sawdust and were obtained from the culture collection maintained at the Mycorrhizal Associations Laboratory/BIOAGRO in the Universidade Federal de Viçosa (Viçosa, Brazil).These strains were selected because P. ostreatus PLO06 has simple nutritional requirements and both strains show high rates of substrate decomposition.Stock cultures were kept stored in potato dextrose agar plates (PDA, Merck, Darmstadt, Germany) and subcultured every six months.Working cultures were also stored in PDA plates and maintained at 4°C until use.

Inoculum and substrate preparation
Inoculum of P. ostreatus PLO06 and L. edodes UFV73 was prepared by transferring a 8 mm disc from the working cultures to a new PDA plate followed by incubation for seven days at 25C.The spawn was prepared in glass pots using pre-cooked and heatsterilized rice grains (121C).Spawn sterilization was performed three times for 2 h, during 48 h intervals.The spawn was inoculated with approximately 10 agar disks (each with 1 cm in diameter) containing mycelia.The cultures were then incubated at 25C for 20 days.After this incubation period, colonized rice grains were used (20 g samples) to aseptically inoculate polypropylene bags permeable to oxygen containing 1.5 kilograms of the heat-sterilized agro-industrial residues.Sterilization of the substrates used in this work was performed as described for the spawn.
P. ostreatus PLO06 was cultivated in eucalyptus bark, eucalyptus sawdust, corn cobs, sugarcane bagasse, coffee bark and coconut fiber while L.edodes UFV73 was grown in eucalyptus bark, eucalyptus sawdust, corn cobs and sugarcane bagasse supplemented with urea (0.5 %, w/w) to stimulate fungal growth.All the experiments were performed twice with at least two technical replicates.
Inoculated agro-industrial residues were kept in an incubation room maintained at room temperature (25C).After the residues had been fully colonized (approximately 60 days), the polypropylene bags were transferred to a fructification room kept at 20°C and with relative humidity of 90% to allow the production of fructification bodies (mushrooms).Control treatments (substrates without fungal inoculation) were prepared and sterilized as described above for the inoculated agro-industrial residues.Control treatments and fructified substrates (obtained after harvesting the mushrooms) were transferred to paper bags (approximately 1.5 Kg) and dried at 60  5 °C in an oven with forced ventilation.The residues were ground in a Willey mill, sieved through a mesh of 1 mm and 100 g of milled residue was packed individually in glass bottles of 200 g and stored at room temperature until being used for chemical and digestibility analysis.

Chemical analysis
After the samples were dried and milled, chemical analyses were performed to determine dry matter (DM), organic matter (OM), crude protein (CP), ether extracts (EE) and ash following the protocols described by AOAC (2000).The contents of neutral detergent fiber (NDF) were determined according to Mertens (2002), using thermostable amylase (Termamyl 120 L, Novozymes).The acid detergent fiber (ADF) and acid detergent lignin (sulfuric acid 72 %) were determined using sequential analyses proposed by Robertson and Van Soest (1981).The concentration of hemicelluloses was calculated as the difference between NDF and ADF performed sequentially on the same sample.Cellulose concentration was determined as the difference between ADF and ADL.

In vitro digestibility of neutral detergent fiber (IVDNDF)
The IVDNDF was determined by the method proposed by Tilley and Terry (1963), with some modifications, as follows.The ruminal fluid was collected (samples of 1000 mL) 2 h after feeding from cattle fistulated in the rumen and kept in the Dairy Cattle Facility of the Animal Science Department at the Federal University of Viçosa.Sampled animals graze on pasture and had ad libitum access to corn silage during the morning period.Ruminal digesta was filtered through four layers of gauze, stored in Thermo bottles and transported immediately to the laboratory, where the ruminal fluid was incubated at 39°C for about 30 min.Ruminal bacteria were collected anaerobically from the center of the flask.
The IVDNDF analysis was carried out in two stages.In the first stage, 350 mg samples of residues were weighed and mixed with 4 mL of rumen fluid in serum bottles followed by the addition of 32 mL of anaerobic McDougall buffer solution kept under CO 2 flux.The bottles were sealed with rubber stoppers and aluminum seals and were incubated at 39°C/120 rpm for 48 h.In the second step, following incubation for 48 h, the material was filtered in crucible filters that had been previously dried, weighed and washed three times with hot distilled water.After filtration, the crucibles were transferred to universal bottles and filled with 70 mL of neutral detergent solution.The bottles containing the crucibles were sterilized at 121°C for 15 min.After autoclaving, the crucibles were washed several times with hot water until the neutral detergent solution had been removed, followed by a final wash with 15 mL of pure acetone.The crucibles were transferred to the oven at 105°C until constant weight.All the analyses were performed twice in triplicate samples.The in vitro digestibility of neutral detergent fiber (%) was calculated based on the equations proposed by Tilley and Terry (1963).

Statistical analysis
All the experiments were performed with two biological replicates and triplicate samples.All the data regarding IVDNDF and sample chemical composition were subjected to analysis of variance (ANOVA) and significant differences were analyzed with the Tukey's test using the Statistical Analysis System and Genetics software (Ferreira, 2011).Differences among means with P<0.05 were considered statistically significant.

RESULTS
When lignocellulosic agro-industrial residues were inoculated with the white-rot fungus P. ostreatus PLO06, a decrease (P<0.05) in organic matter was often observed (Table 1).This effect was related with a greater mineralization of the organic matter, indicated by the increase (P<0.05) in ash concentration and greater in vitro digestibility of the fructified substrates (Tables 1 and  3).The only exception was the coconut fiber, where the fructified residue showed decreased mineralization (decrease in ash content).However, digestibility was still significant, which is coherent with the lower content of neutral detergent fiber (NDF) and acid detergent fiber (ADF) determined for this residue (Table 1).
Most inoculated residues also showed an increase (P<0.05) in the concentration of crude protein and ether extract, except for corn cobs in which differences in protein concentration and ether extract were not significant (Table 1).In the eucalyptus and coffee bark, non-inoculated and fructified residues had the same ether extract and crude protein content (P>0.05),respectively.
Except for the sugarcane bagasse, a greater degradation of NDF (P<0.05) was observed in the fructified residues, with an average decrease in fiber content of 20.57% (Table 1).In sugarcane bagasse and corn cobs, hemicellulose was the main fraction hydrolyzed by P. ostreatus PLO06 (P<0.05), with lignin being little affected by the activity of the fungi (Table 1).
When eucalyptus bark and sugarcane bagasse were supplemented with 0.5 % urea, a decrease (P<0.05) in organic matter was observed in the fructified residues inoculated with L. edodes UFV73.In the corn cobs    residue, differences (P>0.05) in chemical composition were not observed between the non-inoculated and fructified residues.The decrease in organic matter was directly related with the increase in ash content and this result was also observed for eucalyptus bark and sugarcane bagasse inoculated with L. edodes UFV73 (Table 2).
It was hypothesized that an increase in fungal biomass in the fructified residue could increase the concentrations of EE and CP in the residues.Except for the sugarcane bagasse, an increase in the concentration of CP (P<0.05) was observed in the fructified treatment of all residues.However, even in sugarcane bagasse a 27.5 % (P> 0.06) increase in CP was observed and the concentration of Table 3.In vitro digestibility of neutral detergent fiber of non-inoculated (NI) or fructified (F) lignocellulosic agro-industrial residues.Fructified residues were inoculated with either P. ostreatus PLO06 or L. edodes UFV73 and incubated until the production of fructification bodies (60 days).Residues inoculated with L. edodes UFV73 were supplemented with urea (0.5 %, w/w).EE remained the same (P>0.05)for all the residues inoculated with L. edodes UFV73 (Table 2).L. edodes UFV73 did not alter the chemical composition and digestibility of eucalyptus sawdust supplemented with 0.5% urea (Table 2).However, L. edodes UFV73 could alter the fiber composition of the agro-industrial residues eucalyptus bark and corn cobs, decreasing (P<0.05)major cell wall constituents (Table 2).In general, fructified residues had approximately 17.9% less NDF than non-inoculated residues and this effect also reflected in a lower ADF and cellulose content in most residues were the fungus was able to grow.Sugarcane bagasse was the only exception, since ADF and cellulose remained unchanged and hemicellulose contend reduced more than 57 % in the fructified residue (Table 2).Reduction in lignin content were only observed for eucalyptus bark, where the fructified residue showed a lignin content 79% lower than the non-inoculated substrate.

Residue
In vitro digestibility of neutral detergent fiber (IVDNDF) in the fructified residues was always greater than the non-inoculated substrate for P. ostreatus PLO06 (Table 3).Eucalyptus sawdust supplemented with 0.5% urea was the only residue treated with L. edodes UFV73 where digestibility was not affected (P>0.05).Digestibility of fructified eucalyptus bark inoculated with P. ostreatus PLO06 and L. edodes UFV73 increased approximately three-fold and eight-fold, respectively, compared to the non-inoculated substrate.The increase in digestibility varied from 13 to 200% for residues inoculated with P. ostreatus PLO06 and from 40 to 775% for residues inoculated with L. edodes UFV73.
Based on the fact that P. ostreatus PLO06 has simple growth requirements, substrate supplementation was not required for its growth in the lignocellulosic residues used in this work.However, L. edodes UFV73 could not develop well in residues with a high carbon/nitrogen ratio.Therefore, the supplementation of the residues with 0.5 urea or 20% rice bran (data not shown) was required for fungal growth.When the chemical composition and digestibility of agro-industrial residues that had been inoculated with P. ostreatus or L. edodes were compared, P. ostreatus showed a greater potential to improve the nutritional quality of the tested residues (Table 4).Increases in fiber degradation, protein content and digestibility were highly significant for both fungi, however, P. ostreatus was much better at degrading lignin and mineralizing the substrates (Table 4).

DISCUSSION
The white-rot fungi are among the most efficient microorganisms in depolymerization of complex substrates and lignin mineralization (Elisashvili et al., 2008;Sánchez, 2009).Edible mushroom are also highly appreciated for their nutritional, medicinal and gastronomic characteristics (Sánchez, 2010).Considering the growth of the mushroom market, there has been an increasing interest among farmers to produce mushrooms for commercial purposes using alternative lignocellulosic substrates (Sánchez, 2009).In this regard, the use of agro-industrial residues for mushroom production could be advantageous to reduce the environmental impact of these residues, to aggregate economical value into products of high nutritional value to humans (mushrooms) and to allow the use of such residues in animal rations.
In this work, we show that both P. ostreatus PLO06 and L. edodes UFV73 can alter the chemical composition and improve the digestibility of several agro-industrial residues of low nutritional value (Tables 1, 2, 3 and 4).We focused on the chemical composition and digestibility of the fructified residues (after the mushrooms being harvested) due to the fact that these substrates can be used for mushroom production directed to human consumption, which is a more profitable and noble destination for farmers.
Our results demonstrate that, on average, crude protein concentration was 56.5% greater after mushroom was harvested compared to non-inoculated agro-industrial residues (Table 4).In this regard, one could argue that if mushrooms are not destined to human consumption and the residues are used in animal rations with all the fungal biomass on it (including fruiting bodies), protein concentration could be much higher on these substrates.Additionally, other main components of the residues were also affected by fungal growth.
For P. ostreatus PLO06 and L. edodes UFV73, mineralization of most substrates was increased in the fructified residues, as indicated by greater ash content in the samples.The only exception was coconut fiber inoculated with P. ostreatus PLO06 and eucalyptus sawdust inoculated with L. edodes UFV73, which showed a reduction in ash content.P. ostreatus and L. edodes harbour many hydrolytic (CMCases, xylanases) and oxidative (laccases and maganese peroxidases) enzymes that decompose lignocellulosic biomass into low molecular weight molecules that can be further metabolized by the fungi.P. ostreatus usually grows well even in substrates with very low nutritional quality and high carbon/nitrogen ratios (Luz et al., 2012), whereas L. edodes has a high O 2 demand and requires more complex substrates for growth (Lee et al., 2012).
Losses of OM were also observed when the residues were inoculated with P. ostreatus PLO06 and L. edodes UFV73.The exception was coconut fiber inoculated with P. ostreatus PLO06 and eucalyptus sawdust inoculated with L. edodes UFV73.The result could be explained by a higher concentration of NDF, ADF and lignin in these residues, which was correlated with a decrease in fungal growth and reduced fruiting body formation (Gaitán-Hernández et al., 2011).
The white-rot fungi were able to degrade the structural and nonstructural components of different residues, reducing NDF, ADF and lignin.Analysis of the main fiber components cellulose, hemicelluloses and lignin indicated that cellulose and lignin were preferentially attacked by P. ostreatus PLO06 in eucalyptus sawdust, eucalyptus and coffee bark and coconut fiber.The relative increase in other plant cell components (cellulose) sometimes was observed (sugarcane bagasse) probably due to the preferential utilization of soluble sugars and structural carbohydrates more accessible to enzymatic degradation, such as hemicellulose, in the residues colonized by the fungus.Elisashvili et al. (2008) evaluated the activity of lignocellulolytic enzymes produced by L. edodes and Pleurotus spp.strains in submerged and solid-state fermentation of various lignocellulosic wastes.The authors reported wide differences among the substrates tested and related enzyme activity to an increase in fungal biomass and the degradation of fibrous components of the substrate, including cellulose, hemicelluloses and lignin.
Lignin, a major component of plant cell wall of agroindustrial residues, reduced in residues treated with P. ostreatus PLO06 and even L. edodes UFV73 could reduce the lignin content of the residues in 16%.In contrast to P. ostreatus, the low nitrogen (N) content of lignocellulosic residues can be a limiting factor for L. edodes growth in agro-industrial residues.Therefore, supplemental nitrogen (urea or rice bran) is often added to lignocellulosic residues to stimulate carbon mineralization from accessible plant constituents (cellulose) as well as complex substrates, such as lignin.
Lignin is highly recalcitrant in the rumen, mainly due to the anaerobic nature of this ecosystem.Kilpatrick et al. (2000) showed that lignin is often associated with cellulose in a 3-D matrix that prevents the access of microbial extracellular enzymes to the cellulose in the plant cell wall.Different species of white-rot fungi produce hydrolytic enzymes with distinct kinetic properties and the efficiency of cellulose degradation can vary even among different fungal strains (Baldrian and Valásková, 2008).Although the white-rot fungi have the potential to decompose lignin and depolymerize lignocellulose, hemicellulose and cellulose, the chemical composition of the substrate affects the extension of substrate utilization and its applicability in ruminant feeds (Okano et al., 2006;Baldrian and Valásková, 2008;Rahman et al., 2011;Shrivastava et al., 2011;Tuyen et al., 2012;Mahesh and Mohini, 2013;Lynch et al., 2013;Tuyen et al., 2013).
The content of lignin decreased in all residues inoculated with P. ostreatus, except in the sugarcane bagasse and corn cobs.According to Allison et al. (2009), substrates with low nitrogen levels stimulate the degradation of lignin by white-rot fungi, while substrate with high concentrations of nitrogen promotes the degradation of structural polysaccharides (cellulose and hemicellulose).
Traditionally, supplementation of agro-industrial residues for the cultivation of L. edodes is performed to decrease the C/N ratio to approximately 20-25, since high concentrations of nitrogen can inhibit mycelia growth and prevent complete colonization of the substrate, as well as reduce the ligninolytic activity of the fungus.In this work, we observed that L. edodes UFV73 growth was limited to the surface of the substrate and a more thoroughly colo-nization was never observed on this substrate.Therefore, changes in other parameters could not be observed.Differences in degradation and digestibility of the main fiber components in the fructified residues inoculated with L. edodes UFV73 might be explained by the unique cell wall assembly and chemical structure of the lignin and lignin-carbohydrate complex in these residues.
Most residues inoculated with P. ostreatus PLO06 and L. edodes UFV3 increased the levels of CP and the content of EE.Gaitán-Hernández et al. (2006) showed that barley straw inoculated with L. edodes IE-105 had 35% more CP than untreated controls after 57 days of incubation, but a 44% decrease in CP was reported for wheat straw inoculated with a different strain of L. edodes and incubated for 61 days at 25°C.A similar pattern was reported for the levels of EE.The authors explained these contradictory results based on the differences in concentration of soluble carbohydrates between the substrates, suggesting that some variability in chemical composition should be expected among different lignocellulosic substrates treated with white-rot fungi.
Analysis of the in vitro digestibility of neutral detergent fiber of fructified lignocellulosic residues (Table 3) revealed that the digestibility of lignocellulosic substrates inoculated with P. ostreatus PLO06 or L. edodes UFV3 was dramatically increased upon fungal fructification.
Differences for in vitro digestibility of neutral detergent fiber between residues inoculated with P. ostreatus PLO06 or L. edodes UFV73 (Table 3) can be explained considering two main factors: 1) differences in chemical composition of different substrates (Tables 1 and 2), in which the fibrous constituents (NDF, ADF and lignin) of plant cell walls are negatively correlated with digestibility (Velásquez et al., 2010); 2) differences in hydrolytic activity among the species of white-rot fungi used in the study.P. ostreatus and L. edodes are known to produce hydrolytic enzymes with distinct kinetic properties and the efficiency of cellulose degradation can vary even among different fungal strains (Baldrian and Valásková, 2008;Elisashvili et al., 2008).
Our data reveals that substrate digestibility is often correlated with the decrease in lignin content or an increase in crude protein concentration in the lignocellulosic residues, a characteristic that is highly desirable for the utilization of substrates in animal feeds (Okano et al., 2006;Rahman et al., 2011;Shrivastava et al., 2011;Tuyen et al., 2012;Mahesh andMohini, 2013, Lynch et al., 2013;Tuyen et al., 2013).
Eucalyptus bark was always the residue with the highest increase in digestibility (Table 3), regardless of the species of white-rot fungi that was used to inoculate the substrate.Non-inoculated eucalyptus bark showed a high contend of minerals and substantial amounts of lignin and protein compared to other substrates, and these features of the chemical composition might have a significant effect on fungal growth and degradation of insoluble substrates.
Based on these results, it appears that P. ostreatus PLO06 has advantages to improve the chemical composition and digestibility of low quality lignocellulosic agro-industrial residues, whereas L. edodes UFV73 would be advantageous for use in substrates with a more balanced carbon/nitrogen ratio.As such, P. ostreatus PLO06 has been recognized for its efficient biodegradation of different lignocellulosic substrates and more effective colonization of residues (Elisashvili et al., 2008;Luz et al., 2012), which could help us in preventing the growth of contaminants (saprophytic fungi) and improve its practical applications.

Conclusion
P. ostreatus PLO06 and L. edodes UFV73 were able to alter the physicochemical properties of major structural components contained in lignocellulosic residues so that they become more susceptible to degradation by rumen microorganisms.Overall, fructified substrates improved in protein concentration, showed lower fiber content and had greater in vitro digestibility of the neutral detergent fiber.The ash content revealed that lignin was being mineralized mainly by P. ostreatus PLO06, probably due to its pronounced oxidative activity.The use of lignocellulosic substrates for production of commercially valuable mushroom could be useful to aggregate biological value to residues that would be further incorporated into ruminant rations.

Table 4 .
Effect of white-rot fungi on chemical composition and digestibility of agro-industrial residues.Substrates inoculated with P. ostreatus or L. edodes were incubated for 60 days.Mushrooms were harvested (F) and the chemical composition and digestibility of the residues were compared with non-inoculated residues (NI).
Averages in the same line followed by different lower case letters for each fungus differs at 5 % probability by Tukey's test.DM = dry matter; OM = organic matter; CP = crude protein; EE = ether extract; NDF = neutral detergent fiber; ADF = acid detergent fiber; CEL = cellulose; HEM = hemicellulose; ADL = acid detergent lignin; IVDNDF = in vitro digestibility of neutral detergent fiber; NI = non-inoculated substrate; F = Fructified substrate.