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Biorefining of lignocellulose: Detoxification of inhibitory hydrolysates and potential utilization of residual streams for production of enzymes
Umeå University, Faculty of Science and Technology, Department of Chemistry.
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Lignocellulosic biomass is a renewable resource that can be utilized for the production of biofuels, chemicals, and bio-based materials. Biochemical conversion of lignocellulose to advanced biofuels, such as cellulosic ethanol, is generally performed through microbial fermentation of sugars generated by thermochemical pretreatment of the biomass followed by an enzymatic hydrolysis of the cellulose. The aims of the research presented in this thesis were to address problems associated with pretreatment by-products that inhibit microbial and enzymatic biocatalysts, and to investigate the potential of utilizing residual streams from pulp mills and biorefineries to produce hydrolytic enzymes.

A novel method to detoxify lignocellulosic hydrolysates to improve the fermentability was investigated in experiments with the yeast Saccharomyces cerevisiae. The method is based on treatment of lignocellulosic slurries and hydrolysates with reducing agents, such as sodium dithionite and sodium sulfite. The effects of treatment with sodium borohydride were also investigated. Treatment of a hydrolysate of Norway spruce by addition of 10 mM dithionite resulted in an increase of the balanced ethanol yield from 0.03 to 0.35 g/g. Similarly, the balanced ethanol yield of a hydrolysate of sugarcane bagasse increased from 0.06 to 0.28 g/g after treatment with 10 mM dithionite. In another study with a hydrolysate of Norway spruce, addition of 34 mM borohydride increased the balanced ethanol yield from 0.02 to 0.30 g/g, while the ethanol productivity increased from 0.05 to 0.57 g/(L×h). While treatment with sulfur oxyanions had a positive effect on microbial fermentation and enzymatic hydrolysis, treatment with borohydride resulted in an improvement only for the microbial fermentation. The chemical effects of treatments of hydrolysates with sodium dithionite, sodium sulfite, and sodium borohydride were investigated using liquid chromatography-mass spectrometry (LC-MS). Treatments with dithionite and sulfite were found to rapidly sulfonate inhibitors already at room temperature and at a pH that is compatible with enzymatic hydrolysis and microbial fermentation. Treatment with borohydride reduced inhibitory compounds, but the products were less hydrophilic than the products obtained in the reactions with the sulfur oxyanions.

The potential of on-site enzyme production using low-value residual streams, such as stillage, was investigated utilizing recombinant Aspergillus niger producing xylanase and cellulase. A xylanase activity of 8,400 nkat/ml and a cellulase activity of 2,700 nkat/ml were reached using stillages from processes based on waste fiber sludge. The fungus consumed a large part of the xylose, the acetic acid, and the oligosaccharides that were left in the stillages after fermentation with S. cerevisiae. In another study, the capability of two filamentous fungi (A. niger and Trichoderma reesei) and three yeasts (S. cerevisiae, Pichia pastoris, and Yarrowia lipolytica) to grow on inhibitory lignocellulosic media were compared. The results indicate that the two filamentous fungi had the best capability to utilize different nutrients in the media, while the S. cerevisiae strain exhibited the best tolerance against the inhibitors. Utilization of different nutrients would be especially important in enzyme production using residual streams, while tolerance against inhibitors is desirable in a consolidated bio-process in which the fermenting microorganism also contributes by producing enzymes.

Place, publisher, year, edition, pages
Umeå: Umeå universitet , 2013. , 80 p.
National Category
Biocatalysis and Enzyme Technology
Identifiers
URN: urn:nbn:se:umu:diva-82486ISBN: 978-91-7459-759-2 (print)OAI: oai:DiVA.org:umu-82486DiVA: diva2:661377
Public defence
2013-11-29, KBC-huset, KB3A9, Umeå universitet, Umeå, 10:15 (English)
Opponent
Supervisors
Available from: 2013-11-08 Created: 2013-11-03 Last updated: 2013-11-08Bibliographically approved
List of papers
1. Improving the fermentability of enzymatic hydrolysates of lignocellulose through chemical in-situ detoxification with reducing agents
Open this publication in new window or tab >>Improving the fermentability of enzymatic hydrolysates of lignocellulose through chemical in-situ detoxification with reducing agents
2011 (English)In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 102, no 2, 1254-1263 p.Article in journal (Refereed) Published
Abstract [en]

Inhibitory lignocellulose hydrolysates were treated with the reducing agents dithionite and sulfite to achieve improved fermentability. Addition of these reducing agents (in the concentration range 5.0-17.5mM) to enzymatic hydrolysates of spruce wood or sugarcane bagasse improved processes based on both SHF (simultaneous hydrolysis and fermentation) and SSF (simultaneous saccharification and fermentation). The approach was exemplified in ethanolic fermentations with Saccharomyces cerevisiae and by using hydrolysates with sugar concentrations >100g/L (for SHF) and with 10% dry-matter content (for SSF). In the SHF experiments, treatments with dithionite raised the ethanol productivities of the spruce hydrolysate from 0.2 to 2.5g×L(-1)×h(-1) and of the bagasse hydrolysate from 0.9 to 3.9g×L(-1)×h(-1), values even higher than those of fermentations with reference sugar solutions without inhibitors. Benefits of the approach include that the addition of the reducing agent can be made in-situ directly in the fermentation vessel, that the treatment can be performed at a temperature and pH suitable for fermentation, and that the treatment results in dramatically improved fermentability without degradation of fermentable sugars. The many benefits and the simplicity of the approach offer a new way to achieve more efficient manufacture of fermentation products from lignocellulose hydrolysates.

Place, publisher, year, edition, pages
Elsevier Ltd, 2011
Keyword
Lignocellulose hydrolysates, Cellulosic ethanol, Fermentation inhibitors, In-situ detoxification, Reducing agents
Identifiers
urn:nbn:se:umu:diva-38851 (URN)10.1016/j.biortech.2010.08.037 (DOI)20822900 (PubMedID)
Available from: 2011-01-04 Created: 2011-01-04 Last updated: 2017-12-11Bibliographically approved
2. Effect of sulfur oxyanions on lignocellulose-derived fermentation inhibitors
Open this publication in new window or tab >>Effect of sulfur oxyanions on lignocellulose-derived fermentation inhibitors
2011 (English)In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 108, no 11, 2592-2599 p.Article in journal (Refereed) Published
Abstract [en]

Recent results show that treatments with reducing agents, including the sulfur oxyanions dithionite and hydrogen sulfite, efficiently improve the fermentability of inhibitory lignocellulose hydrolysates, and that the treatments are effective when the reducing agents are added in situ into the fermentation vessel at low temperature. In the present investigation, dithionite was added to medium with model inhibitors (coniferyl aldehyde, furfural, 5-hydroxymethylfurfural, or acetic acid) and the effects on the fermentability with yeast were studied. Addition of 10 mM dithionite to medium containing 2.5 mM coniferyl aldehyde resulted in a nine-fold increase in the glucose consumption rate and a three-fold increase in the ethanol yield. To investigate the mechanism behind the positive effects of adding sulfur oxyanions, mixtures containing 2.5 mM of a model inhibitor (an aromatic compound, a furan aldehyde, or an aliphatic acid) and 15 mM dithionite or hydrogen sulfite were analyzed using mass spectrometry (MS). The results of the analyses, which were performed by using UHPLC-ESI-TOF-MS and UHPLC-LTQ/Orbitrap-MS/MS, indicate that the positive effects of sulfur oxyanions are primarily due to their capability to react with and sulfonate inhibitory aromatic compounds and furan aldehydes at low temperature and slightly acidic pH (such as 25°C and pH 5.5).

Place, publisher, year, edition, pages
Wiley, 2011
Keyword
inhibitors, lignocellulose hydrolysates, liquid biofuels, detoxification, reducing agents, sulfur oxyanions
National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-45419 (URN)10.1002/bit.23244 (DOI)21702027 (PubMedID)
Available from: 2011-07-04 Created: 2011-07-04 Last updated: 2017-12-11Bibliographically approved
3. Detoxification of lignocellulosic hydrolysates using sodium borohydride
Open this publication in new window or tab >>Detoxification of lignocellulosic hydrolysates using sodium borohydride
2013 (English)In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 136, 368-376 p.Article in journal (Refereed) Published
Abstract [en]

Addition of sodium borohydride to a lignocellulose hydrolysate of Norway spruce affected the fermentability when cellulosic ethanol was produced using Saccharomyces cerevisiae. Treatment of the hydrolysate with borohydride improved the ethanol yield on consumed sugar from 0.09 to 0.31 g/g, the balanced ethanol yield from 0.02 to 0.30 g/g, and the ethanol productivity from 0.05 to 0.57 g/(L×h). Treatment of a sugarcane bagasse hydrolysate gave similar results, and the experiments indicate that sodium borohydride is suitable for chemical in-situ detoxification. The model inhibitors coniferyl aldehyde, p-benzoquinone, 2,6-dimethoxybenzoquinone, and furfural were efficiently reduced by treatment with sodium borohydride, even under mild reaction conditions (20°C and pH 6.0). While addition of sodium dithionite to pretreatment liquid from spruce improved enzymatic hydrolysis of cellulose, addition of sodium borohydride did not. This result indicates that the strong hydrophilicity resulting from sulfonation of inhibitors by dithionite treatment was particularly important for alleviating enzyme inhibition.

Place, publisher, year, edition, pages
Elsevier, 2013
Keyword
Lignocellulose, Sodium borohydride, Detoxification, Cellulosic ethanol, Inhibitors
National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-67828 (URN)10.1016/j.biortech.2013.03.014 (DOI)000319309400050 ()
Available from: 2013-04-03 Created: 2013-04-03 Last updated: 2017-12-06Bibliographically approved
4. Biorefining of wood: combined production of ethanol and xylanase from waste fiber sludge
Open this publication in new window or tab >>Biorefining of wood: combined production of ethanol and xylanase from waste fiber sludge
Show others...
2011 (English)In: Journal of Industrial Microbiology & Biotechnology, ISSN 1367-5435, E-ISSN 1476-5535, Vol. 38, no 8, 891-899 p.Article in journal (Refereed) Published
Abstract [en]

The possibility to utilize fiber sludge, waste fibers from pulp mills and lignocellulose-based biorefineries, for combined production of liquid biofuel and biocatalysts was investigated. Without pretreatment, fiber sludge was hydrolyzed enzymatically to monosaccharides, mainly glucose and xylose. In the first of two sequential fermentation steps, the fiber sludge hydrolysate was fermented to cellulosic ethanol with the yeast Saccharomyces cerevisiae. Although the final ethanol yields were similar, the ethanol productivity after 9.5 h was 3.3 g/l/h for the fiber sludge hydrolysate compared with only 2.2 g/l/h for a reference fermentation with similar sugar content. In the second fermentation step, the spent fiber sludge hydrolysate (the stillage obtained after distillation) was used as growth medium for recombinant Aspergillus niger expressing the xylanase-encoding Trichoderma reesei (Hypocrea jecorina) xyn2 gene. The xylanase activity obtained with the spent fiber sludge hydrolysate (8,500 nkat/ml) was higher than that obtained in a standard medium with similar monosaccharide content (1,400 nkat/ml). Analyses based on deglycosylation with N-glycosidase F suggest that the main part of the recombinant xylanase was unglycosylated and had molecular mass of 20.7 kDa, while a minor part had N-linked glycosylation and molecular mass of 23.6 kDa. Chemical analyses of the growth medium showed that important carbon sources in the spent fiber sludge hydrolysate included xylose, small aliphatic acids, and oligosaccharides. The results show the potential of converting waste fiber sludge to liquid biofuel and enzymes as coproducts in lignocellulose-based biorefineries.

Place, publisher, year, edition, pages
Springer, 2011
Keyword
Biorefinery, Fiber sludge, Cellulosic ethanol, Enzymes, Xylanase
Identifiers
urn:nbn:se:umu:diva-38767 (URN)10.1007/s10295-010-0856-9 (DOI)20824487 (PubMedID)
Note
E-pub ahead of print. This article is based on a presentation at the 32nd Symposium on Biotechnology for Fuels and Chemicals. Available from: 2011-01-28 Created: 2010-12-28 Last updated: 2017-12-11Bibliographically approved
5. Production of cellulosic ethanol and enzyme from waste fiber sludge using SSF, recycling of hydrolytic enzymes and yeast, and recombinant cellulase-producing Aspergillus niger
Open this publication in new window or tab >>Production of cellulosic ethanol and enzyme from waste fiber sludge using SSF, recycling of hydrolytic enzymes and yeast, and recombinant cellulase-producing Aspergillus niger
Show others...
2014 (English)In: Journal of Industrial Microbiology & Biotechnology, ISSN 1367-5435, E-ISSN 1476-5535, Vol. 41, no 8, 1191-1200 p.Article in journal (Refereed) Published
Abstract [en]

Bioethanol and enzymes were produced from fiber sludges through sequential microbial cultivations. After a first simultaneous saccharification and fermentation (SSF) with yeast, the bioethanol concentrations of sulfate and sulfite fiber sludges were 45.6 and 64.7 g/L, respectively. The second SSF, which included fresh fiber sludges and recycled yeast and enzymes from the first SSF, resulted in ethanol concentrations of 38.3 g/L for sulfate fiber sludge and 24.4 g/L for sulfite fiber sludge. Aspergillus niger carrying the endoglucanase-encoding Cel7B gene of Trichoderma reesei was grown in the spent fiber sludge hydrolysates. The cellulase activities obtained with spent hydrolysates of sulfate and sulfite fiber sludges were 2,700 and 2,900 nkat/mL, respectively. The high cellulase activities produced by using stillage and the significant ethanol concentrations produced in the second SSF suggest that onsite enzyme production and recycling of enzyme are realistic concepts that warrant further attention.

Place, publisher, year, edition, pages
Springer Berlin/Heidelberg, 2014
Keyword
biorefinery, fiber sludge, cellulosic ethanol, enzymes, cellulase
National Category
Biocatalysis and Enzyme Technology Microbiology
Identifiers
urn:nbn:se:umu:diva-82484 (URN)10.1007/s10295-014-1457-9 (DOI)000339385400002 ()
Note

Included in thesis in manuscript form.

Available from: 2013-11-03 Created: 2013-11-03 Last updated: 2017-12-06Bibliographically approved
6. Comparison of the growth of filamentous fungi and yeasts in lignocellulose-derived media
Open this publication in new window or tab >>Comparison of the growth of filamentous fungi and yeasts in lignocellulose-derived media
(English)Manuscript (preprint) (Other academic)
National Category
Biocatalysis and Enzyme Technology
Identifiers
urn:nbn:se:umu:diva-82485 (URN)
Available from: 2013-11-03 Created: 2013-11-03 Last updated: 2013-11-04

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