Fungal treatment of lignocellulosic biomass

Summary PhD thesis Sandra J.A. van Kuijk Carbohydrates in plant cell walls are highly fermentable and could be used as a source for ruminant nutrition or biofuel production. The presence of lignin in cell walls hampers the utilization of these carbohydrates and should thus be removed. In this thesis, the possibilities of a fungal treatment of lignocellulosic biomass are investigated. A review of the scientific literature focusing on the potential of fungal treatments to increase the utilization of lignocellulosic biomass in ruminants feed ingredients is presented in Chapter 2. A prerequisite to the effective use of high lignocellulose feed ingredients is lignin removal, since lignin is negatively correlated to in vitro rumen degradability. Selective lignin degrading fungi have proven to increase in vitro rumen degradability with Ceriporiopsis subvermispora and Pleurotus eryngii showing the greatest potential. The effectiveness of fungal treatment is not only dependent on the choice of fungal strain, but also on the choice of substrate and culture conditions. Based on the literature review, four different edible fungal species, i.e. Ganoderma lucidum, Lentinula edodes, P. eryngii and P. ostreatus were chosen to treat four different substrates, i.e. miscanthus, rice straw, wheat straw and wood chips. The results of these fungal-substrate combinations are described in Chapter 3 and confirm that fungal species, substrate and incubation time are important factors in fungal treatment. The most promising fungus-substrate combinations are L. edodes treatment of wheat straw and L. edodes treatment of wood chips. These two fungus-substrate combinations were used in a more detailed characterization of changes in lignin upon fungal treatment. In Chapter 4, a study is described where L. edodes treated wheat straw and wood chips are analyzed by pyrolysis coupled to gas chromatography and mass spectrometry (Py-GC/MS) confirming the selective lignin degradation as determined with the detergent fiber analysis. Structural changes in lignin were observed with preferential degradation of syringyl (S) lignin units over guaiacyl (G) lignin units by L. edodes. Upon fungal delignification, a number of degradation products of lignin were observed. The building blocks in the original lignin consist of phenolic groups with 3 C-atoms in the side chain, while degradation products consist of phenolic groups with 0 to 2 C-atoms in the side chain. The ratio between side chain degradation products and original compounds was increasing in both wheat straw and wood chips upon L. edodes treatment. Besides side chain degradation, L. edodes modified the Cα-atom of the side chain by oxidation. Although correlations were found, a clear relationship between lignin composition and in vitro rumen degradability could not be demonstrated. The safety of a fungal treatment of ruminant feed ingredients requires the used fungus to have a Generally Regarded As Safe (GRAS) status. The literature data compiled in Chapter 2 indicates C. subvermispora, which does not have a GRAS status, as one of the most promising fungi for fungal treatment. For this reason this fungus was included in the remaining chapters. This fungus was researched in Chapter 5 with the substrates wheat straw and wood chips and compared to L. edodes. Both fungi selectively degraded lignin and improved in vitro rumen degradability and the amount of sugar released upon enzymatic saccharification. L. edodes continuously grew on wheat straw and wood chips while degrading lignin and hemicellulose at the same time. C. subvermispora colonized the wheat straw within the first week of treatment and starts degrading lignin and hemicellulose thereafter. Growth continued again after 5 weeks, which was accompanied by cellulose degradation. On wood chips, C. subvermispora stopped growing after the first week of treatment, while lignin degradation continued until 4 weeks of treatment. From 5 weeks onwards, no chemical changes were observed in wood chips. One of the explanations for this lack of change is the dense structure of the wood as observed with light microscopy. Both fungi degraded hemicellulose simultaneously with lignin. The loss of carbohydrates during fungal treatment and the long treatment times of up to 8 weeks represent a major disadvantage of fungal treatment of lignocellulosic biomass. In Chapter 6, the incubation of C. subvermispora and L. edodes with wheat straw and wood chips were supplemented with urea to stimulate growth, and manganese and linoleic acid to stimulate lignin degradation via the enzyme manganese peroxidase produced by the fungi. Addition of manganese increased the selectivity of C. subvermispora treatment of wheat straw within the first 4 weeks of treatment. Addition of 150 µg manganese per g substrate improved lignin degradation and in vitro rumen degradability the most. A combination of manganese and linoleic acid did not show synergistic effects. In Chapter 7 the particle size of wheat straw and wood chips, and the amount of C. subvermispora or L. edodes at the start of the treatment was varied. The amount of fungus added at the start of the treatment did not have an effect on colonization rate, lignin degradation or in vitro rumen degradability. L. edodes treatment of wheat straw chopped to 2 cm particles resulted in a higher lignin degradation and in vitro rumen degradability compared to L. edodes treatment of wheat straw chopped to a length of 0.5 cm. The particle size of wood chips did not have an effect on C. subvermispora treatment. In C. subvermispora treated wheat straw and L. edodes treated wood chips, a reduced growth was observed, which was unexpected based on results from previous experiments. A toxic compound to fungi (fungicide) was identified. This thesis describes the potential of fungal treatment to increase utilization of lignocellulosic biomass. Fungal treatment resulted in an increased in vitro rumen degradability, and thus an increased cellulose accessibility. The same theory applies for biofuel production in which fungal treatment results in an increased accessibility of cellulose for enzymes. The major disadvantages of this low cost, relatively simple and environmentally-friendly biotechnological treatment are the loss of carbohydrates during the relatively long process of fungal incubation. Future studies should focus on optimization of the fungal treatment to enable large scale application.