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Investigations of the importance of the redox environment in LPMO-supported bioconversion of pretreated lignocellulose
Umeå University, Faculty of Science and Technology, Department of Chemistry.
2024 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Undersökningar av redoxmiljöns betydelse vid LPMO-understödd biokonversion av förbehandlad lignocellulosa (Swedish)
Abstract [en]

Achieving high yields in enzymatic saccharification of cellulose is a critical step in biochemical conversion of pretreated lignocellulosic biomass. Sugar formed during saccharification serves as substrate for fermenting microorganisms producing bio-based fuels and chemicals. An oxidoreductase, lytic polysaccharide monooxygenase (LPMO), has recently gained attention for its potential to act synergistically with conventional hydrolytic enzymes catalyzing the deconstruction of cellulose. This investigation has focused on LPMO-supported enzymatic saccharification of cellulose, exploring the process conditions, particularly the redox environment, affecting LPMO-supported saccharification of biomass. The involvement of LPMO necessitates reevaluation of industrial process configurations, especially in terms of aeration strategies. The impact of aeration on saccharification and fermentation, for example through potential side effects on fermentation inhibitors generated during the pretreatment, is not well understood, and the aim of the investigations has been to shed light on that gap of knowledge.

The role of lignin as a reductant in LPMO-supported enzymatic saccharification was investigated, focusing on both lignin in the solid fraction and water-soluble lignin degradation products in the liquid fraction. A novel experimental set-up with controlled gas addition (six parallel reactions, three with air and three with N₂) was used to regulate the redox environment. Glucose production was consistently higher in reactions with air. Both lignin in the solid fraction and degradation products in the liquid fraction efficiently supported LPMO catalysis.

The benefits of continuous aeration in LPMO-supported enzymatic saccharification were weighed against the negative effects associated with high solids loadings in reaction mixtures. Studies in the range 12.5% to 17.5% water-insoluble solids (WIS) showed that the positive effects of aeration to support LPMO were larger than the negative effects of high solids loadings. Notably, glucan conversion with aeration at 17.5% WIS exceeded that obtained with N₂ at 12.5% WIS. Additionally, doubling the enzyme dosage was less effective in enhancing glucan conversion than using aeration rather than N₂. These findings demonstrate the significant potential of continuous aeration to boost LPMO activity when using high solids loadings in biomass conversion.

A hybrid hydrolysis and fermentation (HHF) process, incorporating an initial pre-hydrolysis phase with aeration at a relatively high temperature, was compared to simultaneous saccharification and fermentation (SSF). Using steam-exploded softwood as substrate, pre-hydrolysis with aeration improved glucan conversion in HHF, but the overall conversion remained modest. Extending the aeration period from 24 h to 48 h slightly enhanced saccharification but had a negative impact on the subsequent fermentation with Saccharomyces cerevisiae yeast. Thus, under the experimental conditions used, HHF with aeration led to increased glucan conversion, but the benefits were not sufficient to achieve an ethanol yield and productivity that was comparable to those achieved using SSF.

The potential negative impact of aeration on subsequent fermentation was investigated further in studies of the liquid phase of steam-exploded softwood. Compared to parallel N₂ control reactions, aeration caused a more inhibitory environment for S. cerevisiae yeast. Although the concentrations of some inhibitors, such as furfural, decreased during aeration, there was a slight but consistent increase in the concentrations of formaldehyde, a phenomenon that could, at least partially, explain increased inhibition. Sulfite detoxification was effective regardless of aeration. Laccase treatment showed mixed effects on fermentability, which could be attributed to the treatment causing an overall decrease invthe content of phenolic inhibitors, but also formation of more toxic substances from relatively harmless precursors.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2024. , p. 77
Keywords [en]
Lignocellulose bioconversion, lytic polysaccharide monooxygenase (LPMO), lignin, cellulose, enzymatic saccharification, yeast
National Category
Biocatalysis and Enzyme Technology Bioprocess Technology
Identifiers
URN: urn:nbn:se:umu:diva-229512ISBN: 978-91-8070-499-1 (print)ISBN: 978-91-8070-500-4 (electronic)OAI: oai:DiVA.org:umu-229512DiVA, id: diva2:1896934
Public defence
2024-10-11, Lilla Hörsalen, KB.E3.01, KBC-huset, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2024-09-19 Created: 2024-09-11 Last updated: 2024-09-12Bibliographically approved
List of papers
1. Comparison of solid and liquid fractions of pretreated Norway spruce as reductants in LPMO-supported saccharification of cellulose
Open this publication in new window or tab >>Comparison of solid and liquid fractions of pretreated Norway spruce as reductants in LPMO-supported saccharification of cellulose
2022 (English)In: Frontiers in Bioengineering and Biotechnology, E-ISSN 2296-4185, Vol. 10, article id 1071159Article in journal (Refereed) Published
Abstract [en]

The role of lignin in enzymatic saccharification of cellulose involving lytic polysaccharide monooxygenase (LPMO) was investigated in experiments with the solid and liquid fractions of pretreated Norway spruce from a biorefinery demonstration plant using hydrothermal pretreatment and impregnation with sulfur dioxide. Pretreated biomass before and after enzymatic saccharification was characterized using HPAEC, HPLC, Py-GC/MS, 2D-HSQC NMR, FTIR, and SEM. Chemical characterization indicated that relatively harsh pretreatment conditions resulted in that the solid phase contained no or very little hemicellulose but considerable amounts of pseudo-lignin, and that the liquid phase contained a relatively high concentration (∼5 g/L) of lignin-derived phenolics. As judged from reactions continuously supplied with either air or nitrogen gas, lignin and lignin fragments from both the solid and the liquid phases efficiently served as reductants in LPMO-supported saccharification. When air was used to promote LPMO activity, the enzymatic conversion of cellulose after 72 h was 25% higher in reactions with pretreated solids and buffer, and 14% higher in reactions with pretreatment liquid and microcrystalline cellulose. Research in this area is useful for designing efficient saccharification steps in biochemical conversion of lignocellulosic biomass.

Place, publisher, year, edition, pages
Frontiers Media S.A., 2022
Keywords
cellulose, enzymatic saccharification, lignin, lignocellulose bioconversion, lytic polysaccharide monooxygenase, Norway spruce, pretreatment liquid, reductant
National Category
Bioenergy
Identifiers
urn:nbn:se:umu:diva-202231 (URN)10.3389/fbioe.2022.1071159 (DOI)000904323600001 ()36582841 (PubMedID)2-s2.0-85145009929 (Scopus ID)
Funder
Swedish Energy Agency, 47516-1Bio4Energy
Available from: 2023-01-10 Created: 2023-01-10 Last updated: 2024-09-11Bibliographically approved
2. LPMO-supported saccharification of biomass: effects of continuous aeration of reaction mixtures with variable fractions of water-insoluble solids and cellulolytic enzymes
Open this publication in new window or tab >>LPMO-supported saccharification of biomass: effects of continuous aeration of reaction mixtures with variable fractions of water-insoluble solids and cellulolytic enzymes
2023 (English)In: Biotechnology for Biofuels and Bioproducts, E-ISSN 2731-3654, Vol. 16, no 1, article id 156Article in journal (Refereed) Published
Abstract [en]

Background: High substrate concentrations and high sugar yields are important aspects of enzymatic saccharification of lignocellulosic substrates. The benefit of supporting the catalytic action of lytic polysaccharide monooxygenase (LPMO) through continuous aeration of slurries of pretreated softwood was weighed against problems associated with increasing substrate content (quantitated as WIS, water-insoluble solids, in the range 12.5–17.5%), and was compared to the beneficial effect on the saccharification reaction achieved by increasing the enzyme preparation (Cellic CTec3) loadings. Aerated reactions were compared to reactions supplied with N2 to assess the contribution of LPMO to the saccharification reactions. Analysis using 13C NMR spectroscopy, XRD, Simons’ staining, BET analysis, and SEM analysis was used to gain further insights into the effects of the cellulolytic enzymes on the substrate under different reaction conditions.

Results: Although glucose production after 72 h was higher at 17.5% WIS than at 12.5% WIS, glucan conversion decreased with 24% (air) and 17% (N2). Compared to reactions with N2, the average increases in glucose production for aerated reactions were 91% (12.5% WIS), 70% (15.0% WIS), and 67% (17.5% WIS). Improvements in glucan conversion through aeration were larger (55–86%) than the negative effects of increasing WIS content. For reactions with 12.5% WIS, increased enzyme dosage with 50% improved glucan conversion with 25–30% for air and N2, whereas improvements with double enzyme dosage were 30% (N2) and 39% (air). Structural analyses of the solid fractions revealed that the enzymatic reaction, particularly with aeration, created increased surface area (BET analysis), increased disorder (SEM analysis), decreased crystallinity (XRD), and increased dye adsorption based on the cellulose content (Simons' staining).

Conclusions: The gains in glucan conversion with aeration were larger than the decreases observed due to increased substrate content, resulting in higher glucan conversion when using aeration at the highest WIS value than when using N2 at the lowest WIS value. The increase in glucan conversion with double enzyme preparation dosage was smaller than the increase achieved with aeration. The results demonstrate the potential in using proper aeration to exploit the inherent capacity of LPMO in enzymatic saccharification of lignocellulosic substrates and provide detailed information about the characteristics of the substrate after interaction with cellulolytic enzymes.

Place, publisher, year, edition, pages
BioMed Central (BMC), 2023
Keywords
Biochemical conversion, Biodegradation, Enzymatic saccharification, Enzyme, High substrate loading, Lignocellulose, LPMO, Lytic polysaccharide monooxygenase
National Category
Biocatalysis and Enzyme Technology
Identifiers
urn:nbn:se:umu:diva-215919 (URN)10.1186/s13068-023-02407-y (DOI)2-s2.0-85174602336 (Scopus ID)
Funder
Swedish Energy Agency, P47516-1Swedish Research Council, 2020-05318Bio4Energy
Available from: 2023-11-02 Created: 2023-11-02 Last updated: 2024-09-11Bibliographically approved
3. Comparison of simultaneous saccharification and fermentation with LPMO-supported hybrid hydrolysis and fermentation
Open this publication in new window or tab >>Comparison of simultaneous saccharification and fermentation with LPMO-supported hybrid hydrolysis and fermentation
2024 (English)In: Frontiers in Bioengineering and Biotechnology, E-ISSN 2296-4185, Vol. 12, article id 1419723Article in journal (Refereed) Published
Abstract [en]

Enzymatic saccharification is used to convert polysaccharides in lignocellulosic biomass to sugars which are then converted to ethanol or other bio-based fermentation products. The efficacy of commercial cellulase preparations can potentially increase if lytic polysaccharide monooxygenase (LPMO) is included. However, as LPMO requires both a reductant and an oxidant, such as molecular oxygen, a reevaluation of process configurations and conditions is warranted. Saccharification and fermentation of pretreated softwood was investigated in demonstration-scale experiments with 10 m3 bioreactors using an LPMO-containing cellulase preparation, a xylose-utilizing yeast, and either simultaneous saccharification and fermentation (SSF) or hybrid hydrolysis and fermentation (HHF) with a 24-hour or 48-hour initial phase and with 0.15 vvm aeration before addition of the yeast. The conditions used for HHF, especially with 48 h initial phase, resulted in better glucan conversion, but in poorer ethanol productivity and in poorer initial ethanol yield on consumed sugars than the SSF. In the SSF, hexose sugars such as glucose and mannose were consumed faster than xylose, but, in the end of the fermentation >90% of the xylose had been consumed. Chemical analysis of inhibitory pretreatment by-products indicated that the concentrations of heteroaromatic aldehydes (such as furfural), aromatic aldehydes, and an aromatic ketone decreased as a consequence of the aeration. This was attributed mainly to evaporation caused by the gas flow. The results indicate that further research is needed to fully exploit the advantages of LPMO without compromising fermentation conditions.

Place, publisher, year, edition, pages
Frontiers Media S.A., 2024
Keywords
cellulase, hybrid hydrolysis and fermentation, lignocellulose bioconversion, LPMO, lytic polysaccharide monooxygenase, simultaneous saccharification and fermentation, yeast
National Category
Bioenergy Other Chemistry Topics
Identifiers
urn:nbn:se:umu:diva-228133 (URN)10.3389/fbioe.2024.1419723 (DOI)001275918600001 ()39055343 (PubMedID)2-s2.0-85199325807 (Scopus ID)
Funder
Swedish Research Council, 2020-05318Swedish Energy Agency, P47516-1Swedish Energy Agency, P2022-00569Bio4Energy
Available from: 2024-08-05 Created: 2024-08-05 Last updated: 2024-09-11Bibliographically approved
4. Bioconversion of pretreated softwood: effects of aeration on inhibitors and fermentability using saccharomyces cerevisiae yeast
Open this publication in new window or tab >>Bioconversion of pretreated softwood: effects of aeration on inhibitors and fermentability using saccharomyces cerevisiae yeast
(English)Manuscript (preprint) (Other academic)
National Category
Biocatalysis and Enzyme Technology Bioprocess Technology
Identifiers
urn:nbn:se:umu:diva-229518 (URN)
Available from: 2024-09-11 Created: 2024-09-11 Last updated: 2024-09-12

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