Discerning the variation in fungal-treated wheat straw as ruminant feed

The use of white-rot fungi in bioprocessing of lignocellulosic biomass such as wheat straw into valuable ruminant feed ingredients is promising. Fungi are capable of degrading lignin – a cell wall polymer that negatively affects ruminal degradability of the biomass. For this purpose, fungi, such as C. subvermispora, P. eryngii and L. edodes, have been identified as potential species. The vast availability of strains of these species, as well various wheat straw types and conditions lead to four main research questions: (1) Do strains of the same fungal species differ in their extent of lignocellulose degradation and the subsequent improvement in the ruminal degradability of wheat straw? (2) How do high potential strains/species vary in utilizing a certain nutrient, such as protein? (3) Do different fungal strains/species possess unique delignification mechanisms? (4) Do various wheat straw types have effects on how fungi degrade the cell wall components?

As a start to the screening trial, in Chapter 2, the bioprocessing capabilities of two strains of C. subvermispora, CS1 and CS2, were compared. Both fungi were capable of selectively degrading lignin and improve in vitro gas production (IVGP) of wheat straw, with CS1 showing a higher potential than CS2. The two fungi also differed in their growth pattern and enzymatic activities, as well as in the degree of selectivity in lignin degradation. There was no correlation between the activities of the lignin-degrading enzymes and the extent of lignin degradation, indicating that more complex mechanisms are involved in modifying the cell wall. In Chapter 3, the IVGP was used as a decisive screening parameter for the best performing fungi. A number of strains of the fungal species C. subvermispora, P. eryngii and L. edodes were screened based of their IVGPs. All strains that belonged to the same species showed a distinctive pattern of growth and changes in the pH of substrate. Nonetheless, large variations of their bioprocessing capabilities were observed. Out of the 32 fungal strains studied, 17 strains showed a significantly higher (P < 0.05) IVGP, compared to the control after 7 weeks of treatment. The best performing strains for each species were selected. They were (in order of IVGP for each species): CS1, CS6 and CS12 (C. subvermispora); PE6, PE2 and PE3 (P. eryngii); LE8, LE7 and LE10 (L. edodes).

Two high-ranked strains for each species in Chapter 3 were subjected to three follow-up studies on variation in protein solubilization and modification of lignin structures. In Chapter 4, different fractions of crude protein (CP) and in vitro protein fermentation of wheat straw treated with six fungal strains (2 strains per species) were evaluated. All fungal strains caused a relative enrichment of the CP content (23.3 to 30.9%) in wheat straw. There was no quantitative difference in the total amount of CP among different fungal-treated straws. However, the CP fraction provided a unique insight into the variation among fungi in solubilizing protein. The results showed that fungi increased the soluble protein fractions and decreased the cell wall bound protein. Only wheat straw treated with P. eryngii and L. edodes strains resulted in a higher protein availability for fermentation in the rumen (30.2 to 47.1%). C. subvermispora strains on the other hand, did not improve the protein availability. To validate the established relationship between IVGP and the cell wall contents, determined using the Van Soest method (Procedure A), more specific analyses of the cell wall (Procedure B) were used in Chapter 5. In Procedure B, pyrolysis (Py-GC/MS) and alditol-acetates were used to determine Py-lignin and carbohydrates content, respectively. Procedure A showed a higher correlation coefficient of lignin/carbohydrate to the IVGP, compared to Procedure B (Pearson’s r of -0.84 vs. -0.69, respectively). The large differences between methods indicate disparities in the mass recovery of cell wall components, which complicates the understanding of degradation of cell wall components and its effect on IVGP. Procedure A is more indicative of the cell wall solubility – which explained the IVGP well, rather than indicating the actual cell wall degradation by fungi. The more specific Procedure B better elucidates the structure, which helps with the understanding of variations among fungal strains in modifying the cell wall. As for the cellulose (and glucan) contents, neither procedure resulted in an accurate quantification of the cellulose due to interference of fungal biomass. A contribution of pure fungal biomass to the total IVGP was expected, since it showed higher IVGP than its corresponding treated wheat straw. Hence, specific analyses of the lignin and carbohydrates contents are required for accurate description of the ruminal degradability of wheat straw.

The combined pyrolysis-GC/MS and 2D-nuclear magnetic resonance (NMR) analysis in Chapter 6, revealed fundamentally different delignification mechanisms among fungal species. It was proposed that the cleavage of Cβ-O-aryl linkages as the main mechanism for C. subvermispora, besides Cα-Cβ cleavage. While the predominant mechanism for L. edodes and P. eryngii was through the cleavage of Cα-Cβ linkages. In Chapter 7, the persistence of the high potential strains of C. subvermispora and L. edodes in improving the ruminal degradability of wheat straw of different maturities and cultivars was studied. The degradation of lignin by both fungi was more pronounced on mature straw (MS3; ~89.0%), compared to the MS1 (~70.7%). In contrast, cellulose was less degraded on MS3 compared to MS1 (~8.3 vs. 14.6%). The ‘preference’ of fungi in the delignification of a more mature straw was confirmed by Py-GC/MS. A higher abundance of the S-unit compounds was found on MS3, which made the mature straw more susceptible to fungal attack. A variable extent of degradation of lignin (47 to 93.5%) was observed when both fungi were grown on different straw cultivars. The high potential strains of C. subvermispora and L. edodes were able to improve the IVGP of various straw types. However, the magnitude of IVGP was only affected by different cultivars, but not by straw maturity.

Research presented in this thesis discerned the biological variation of different fungal strains/species in modifying the cell wall contents, producing variable effects on the ruminal degradability of wheat straw. The new finding on the capability of some fungi to solubilize protein from the cell wall is important, which could pave the way for more advanced and in-depth studies in the future. The high potential strains selected in Chapter 3 were proven capable of improving the nutritive value of various batches of wheat straw, indicating the potential of bringing this eco-friendly pretreatment method into practice.