Towards a tailor-made enzyme cocktail for saccharification of mildly steam pretreated Norway spruce (Biomass structure guides enzyme supplementation)
Doctoral thesis, 2024
The use of spruce in biorefineries (refineries converting biomass into bioproducts) is limited due to its recalcitrance to enzymatic saccharification (process of breaking down polymers into monomers using enzymes). To overcome this issue, harsh steam pretreatment conditions can be applied, even though they result in yield losses caused by (hemi-)cellulose solubilization and lignin condensation. The aim of this thesis was to build the foundation for tailor-made cocktails for the enzymatic saccharification of mildly steam pretreated spruce, using structural knowledge of the material before and after enzymatic hydrolysis. Spruce was steam pretreated under different conditions ranging from low to high severities. Advanced analytical techniques revealed diverse hemicellulose content and structural characteristics following the different pretreatment procedures.
Based on the structural changes, a cellulolytic cocktail was supplemented with enzymes that targeted different structures in steam pretreated spruce. Lytic polysaccharide monooxygenases were supplemented to enhance the hydrolysis of highly ordered cellulose. Hemicellulases were supplemented to remove the hemicellulose shield and increase accessibility of enzymes to cellulose. Finally, the rearrangement of lignin in less severely steam-treated materials was tested by laccase supplementation. Changes in saccharification yields were detected in relation to supplementation strategy and led to unique alterations in the structural characteristics of the residual material. Enzymes secreted by the fungus Thermothielavioides terrestris grown on different steam pretreated materials were studied. Even minor changes in the structure of spruce affected composition of the fungal secretome, indicating that each steam pretreated substrate required a different enzyme ratio for effective degradation. The secreted enzymes were tested as a supplement to the cellulolytic cocktail, resulting in increased saccharification.
Overall, this thesis highlights the importance of combining structural knowledge before and after hydrolysis with understanding of enzymatic function. Together, they can drive the development of tailor-made cocktails for more efficient lignocellulose saccharification.
Based on the structural changes, a cellulolytic cocktail was supplemented with enzymes that targeted different structures in steam pretreated spruce. Lytic polysaccharide monooxygenases were supplemented to enhance the hydrolysis of highly ordered cellulose. Hemicellulases were supplemented to remove the hemicellulose shield and increase accessibility of enzymes to cellulose. Finally, the rearrangement of lignin in less severely steam-treated materials was tested by laccase supplementation. Changes in saccharification yields were detected in relation to supplementation strategy and led to unique alterations in the structural characteristics of the residual material. Enzymes secreted by the fungus Thermothielavioides terrestris grown on different steam pretreated materials were studied. Even minor changes in the structure of spruce affected composition of the fungal secretome, indicating that each steam pretreated substrate required a different enzyme ratio for effective degradation. The secreted enzymes were tested as a supplement to the cellulolytic cocktail, resulting in increased saccharification.
Overall, this thesis highlights the importance of combining structural knowledge before and after hydrolysis with understanding of enzymatic function. Together, they can drive the development of tailor-made cocktails for more efficient lignocellulose saccharification.
steam explosion
filamentous fungi
laccase
pretreatment
LPMOs
softwood
enzymatic hydrolysis
Conference room KB 10, floor 10, Kemigården 4, Gothenburg
Opponent: Richard P. Chandra, Adjunct Professor of Biology, Trinity Western University, Canada
Author
Fabio Caputo
Chalmers, Life Sciences, Industrial Biotechnology
Understanding the impact of steam pretreatment severity on cellulose ultrastructure, recalcitrance, and hydrolyzability of Norway spruce
Biomass Conversion and Biorefinery,;Vol. 14(2024)p. 27211-27223
Journal article
Overall structural changes and cellulose ultrastructure after enzymatic hydrolysis of mildly steam pretreated Norway spruce
Investigating the role of AA9 LPMOs in enzymatic hydrolysis of differentially steam-pretreated spruce
Biotechnology for Biofuels and Bioproducts,;Vol. 16(2023)
Journal article
Elucidating Thermothielavioides terrestris secretome changes for improved saccharification of mild steam-pretreated spruce
Biotechnology for Biofuels and Bioproducts,;Vol. 17(2024)
Journal article
Insights into the combined action of laccase and LPMOs for steam-pretreated spruce saccharification
Spruce is a common and abundant wood in Sweden and Nordic countries and can be used in many applications as well as starting material in refineries converting biomass into biochemicals (so called biorefineries). The tough structure of spruce makes it hard to break down into sugars by enzymes (a process called enzymatic saccharification) without applying harsh treatments which also result in the loss of valuable wood components. To overcome unwanted side effects of harsh treatments, milder treatment can be applied even though enzymatic saccharification of milder pretreated materials needs to be improved.
The goal of this work was to use a detailed determination of the material's structure before and after treatment with different enzymes to improve the understanding of how different enzymes contribute to saccharification in milder treated spruce. Such understanding could build the foundations for future tailor-made enzyme-mixtures that overcome the challenges associated with these processes. Spruce was treated with steam under different conditions that ranged from mildly to severely treatments. The analyses indicated that the treated materials’ structure differed depending on the level of pretreatment. With this knowledge, a commercial enzyme cocktail was supplemented with enzymes targeting specific parts of the pretreated spruce resulting in varying levels of sugar released and specific alterations on the spruce structure. Filamentous fungi are microorganisms that by secreting enzymes can grow on wood which is relevant to study in order to improve enzymatic saccharification. The response of the filamentous fungus Thermothielavioides terrestris to the different pretreated spruce samples was analyzed. Surprisingly, even small changes in the structure of the wood led to large differences in the types of enzymes the fungus produced. This shows that each type of pretreated material needs its own specific enzyme cocktail for efficient breakdown.
In conclusion, this research shows how combining an understanding of spruce structure with enzyme knowledge can help develop tailor-made enzyme cocktails for breaking down tough plant materials like spruce into starting materials for subsequent use in biorefineries. This approach could eventually reduce the need for harsh treatments, lower the amount of enzymes needed, improve yields in biorefineries and make the overall process more sustainable and cost-effective.
The goal of this work was to use a detailed determination of the material's structure before and after treatment with different enzymes to improve the understanding of how different enzymes contribute to saccharification in milder treated spruce. Such understanding could build the foundations for future tailor-made enzyme-mixtures that overcome the challenges associated with these processes. Spruce was treated with steam under different conditions that ranged from mildly to severely treatments. The analyses indicated that the treated materials’ structure differed depending on the level of pretreatment. With this knowledge, a commercial enzyme cocktail was supplemented with enzymes targeting specific parts of the pretreated spruce resulting in varying levels of sugar released and specific alterations on the spruce structure. Filamentous fungi are microorganisms that by secreting enzymes can grow on wood which is relevant to study in order to improve enzymatic saccharification. The response of the filamentous fungus Thermothielavioides terrestris to the different pretreated spruce samples was analyzed. Surprisingly, even small changes in the structure of the wood led to large differences in the types of enzymes the fungus produced. This shows that each type of pretreated material needs its own specific enzyme cocktail for efficient breakdown.
In conclusion, this research shows how combining an understanding of spruce structure with enzyme knowledge can help develop tailor-made enzyme cocktails for breaking down tough plant materials like spruce into starting materials for subsequent use in biorefineries. This approach could eventually reduce the need for harsh treatments, lower the amount of enzymes needed, improve yields in biorefineries and make the overall process more sustainable and cost-effective.
Subject Categories
Industrial Biotechnology
ISBN
978-91-8103-109-6
Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 5567
Publisher
Chalmers
Conference room KB 10, floor 10, Kemigården 4, Gothenburg
Opponent: Richard P. Chandra, Adjunct Professor of Biology, Trinity Western University, Canada