| 1. |
- Dedes, Grigorios, et al.
(författare)
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Conversion of organosolv pretreated hardwood biomass into 5-hydroxymethylfurfural (HMF) by combining enzymatic hydrolysis and isomerization with homogeneous catalysis
- 2021
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Ingår i: Biotechnology for Biofuels. - : Springer Nature. - 1754-6834. ; 14
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Tidskriftsartikel (refereegranskat)abstract
- Background: Over the last few years, valorization of lignocellulosic biomass has been expanded beyond the production of second-generation biofuels to the synthesis of numerous platform chemicals to be used instead of their fossil-based counterparts. One such well-researched example is 5-hydroxymethylfurfural (HMF), which is preferably produced by the dehydration of fructose. Fructose is obtained by the isomerization of glucose, which in turn is derived by the hydrolysis of cellulose. However, to avoid harsh reaction conditions with high environmental impact, an isomerization step towards fructose is necessary, as fructose can be directly dehydrated to HMF under mild conditions. This work presents an optimized process to produce fructose from beechwood biomass hydrolysate and subsequently convert it to HMF by employing homogeneous catalysis.Results: The optimal saccharification conditions were identified at 10% wt. solids loading and 15 mg enzyme/gsolids, as determined from preliminary trials on pure cellulose (Avicel® PH-101). Furthermore, since high rate glucose isomerization to fructose requires the addition of sodium tetraborate, the optimum borate to glucose molar ratio was determined to 0.28 and was used in all experiments. Among 20 beechwood solid pulps obtained from different organosolv pretreatment conditions tested, the highest fructose production was obtained with acetone (160 °C, 120 min), reaching 56.8 g/100 g pretreated biomass. A scale-up hydrolysis in high solids (25% wt.) was then conducted. The hydrolysate was subjected to isomerization eventually leading to a high-fructose solution (104.5 g/L). Dehydration of fructose to HMF was tested with 5 different catalysts (HCl, H3PO4, formic acid, maleic acid and H-mordenite). Formic acid was found to be the best one displaying 79.9% sugars conversion with an HMF yield and selectivity of 44.6% and 55.8%, respectively.Conclusions: Overall, this work shows the feasibility of coupling bio- and chemo-catalytic processes to produce HMF from lignocellulose in an environmentally friendly manner. Further work for the deployment of biocatalysts for the oxidation of HMF to its derivatives could pave the way for the emergence of an integrated process to effectively produce biobased monomers from lignocellulose.
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| 2. |
- Karnaouri, Anthi C, et al.
(författare)
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Chemoenzymatic Fractionation and Characterization of Pretreated Birch Outer Bark
- 2016
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Ingår i: ACS Sustainable Chemistry and Engineering. - : American Chemical Society (ACS). - 2168-0485. ; 4:10, s. 5289-5302
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Tidskriftsartikel (refereegranskat)abstract
- In this study, the application of different chemical and enzymatic treatment methods for the fractionation of the birch outer bark components was evaluated. More specifically, untreated and steam exploded, hydrothermally and organosolv treated bark samples were incubated with enzyme mixtures that consisted of cellulases, hemicellulases and esterases, and the effect of enzymes was analyzed with 31P NMR and {13C-1H} HSQC. The biocatalysts performed the cleavage of ester bonds resulting in reduction of methoxy and aliphatic groups in the remaining solid fraction, whereas the aromatic fraction remained intact. Moreover, the suberin and lignin fraction were isolated chemically and their properties were characterized by gas chromatography (GC-MS), 31P NMR, {13C-1H} HSQC and gel permeation chromatography (GPC). It was demonstrated that the lignin fraction was enriched in guaiacyl phenolics but still contained some associated aliphatic acids and carbohydrates, whereas the suberin fraction presented a polymodal pattern of structures with different molecular weight distributions. This work will help in getting a deeper fundamental knowledge of the bark structure, the intermolecular connection between lignin and suberin fractions, as well as the potential use of enzymes in order to degrade the recalcitrant bark structure toward its valorization.
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| 3. |
- Karnaouri, Anthi C., et al.
(författare)
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Cloning, expression, and characterization of a thermostable GH7 endoglucanase from Myceliophthora thermophila capable of high-consistency enzymatic liquefaction
- 2014
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 98:1, s. 231-242
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Tidskriftsartikel (refereegranskat)abstract
- An endoglucanase gene from the thermophilic fungus Myceliophthora thermophila, belonging to the glycoside hydrolase family 7, was functionally expressed in methylotrophic yeast Pichia pastoris. The putative endoglucanase from the genomic DNA was successfully cloned in P. pastoris X-33 and the recombinant enzyme was purified to its homogeneity (65 kDa) and subsequently characterized. Substrate specificity analysis revealed that the enzyme exhibits high activity on substrates containing β-1,4-glycosidic bonds such as carboxymethyl cellulose, barley β-glucan, and cello-oligosaccharides, as well as activity on xylan-containing substrates, including arabinoxylan and oat spelt xylan. MtEG7a was proved to liquefy rapidly and efficiently pretreated wheat straw, indicating its key role to the initial step of hydrolysis of high-solids lignocellulose substrates. High thermostability of the endoglucanase reflects potential commercial significance of the enzyme
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| 4. |
- Karnaouri, Anthi C., et al.
(författare)
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Cloning, expression and characterization of an ethanol tolerant GH3 β-glucosidase from Myceliophthora thermophile
- 2013
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Ingår i: PeerJ. - : PeerJ. - 2167-8359.
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Tidskriftsartikel (refereegranskat)abstract
- The β-glucosidase gene bgl3a from Myceliophthora thermophila, member of the fungal glycosyl hydrolase (GH) family 3, was cloned and expressed in Pichia pastoris. The mature β-glucosidase gene, which results after the excision of one intron and the secreting signal peptide, was placed under the control of the strong alcohol oxidase promoter (AOX1) in the plasmid pPICZαC. The recombinant enzyme (90 kDa) was purified and characterized in order to evaluate its biotechnological potential. Recombinant P. pastoris efficiently secreted β-glucosidase into the medium and produced high level of enzymatic activity (41 U/ml) after 192 h of growth, under methanol induction. MtBgl3a was able to hydrolyze low molecular weight substrates and polysaccharides containing β-glucosidic residues. The Km was found to be 0.39 mM on p-β-NPG and 2.64 mM on cellobiose. Optimal pH and temperature for the p-β-NPG hydrolysis were 5.0 and 70 °C. The β-glucosidase exhibits a half life of 143 min at 60 °C. Kinetic parameters of inhibition were determined for D-glucose, D-xylose and D-gluconic acid, indicating tolerance of the enzyme for these sugars and oxidized products. The recombinant enzyme was stimulated by short chain alcohols and has been shown to efficiently synthesize methyl-D-glucoside in the presence of methanol due to its transglycosylation activity. The stability of MtBgl3a in ethanol was prominent, and it retained most of its original activity after we exposed it to 50% ethanol for 6 h. The high catalytic performance, good thermal stability and tolerance to elevated concentrations of ethanol, D-xylose and D-glucose qualify this enzyme for use in the hydrolysis of lignocellulosic biomass for biofuel production, as part of an efficient complete multi-enzyme cocktail.
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| 5. |
- Karnaouri, Anthi C, et al.
(författare)
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Effect of Different Pretreatment Methods on Birch Outer Bark : New Biorefinery Routes
- 2016
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Ingår i: Molecules. - : MDPI AG. - 1431-5157 .- 1420-3049. ; 21:4
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Tidskriftsartikel (refereegranskat)abstract
- A comparative study among different pretreatment methods used for the fractionation of the birch outer bark components, including steam explosion, hydrothermal and organosolv treatments based on the use of ethanol/water media, is reported. The residual solid fractions have been characterized by ATR-FTIR, 13C-solid-state NMR and morphological alterations afterpretreatment were detected by scanning electron microscopy. The general chemical composition of the untreated and treated bark including determination of extractives, suberin, lignin and monosaccharides was also studied. Composition of the residual solid fraction and relative proportions of different components, as a function of the processing conditions, could be established. Organosolv treatment produces a suberin-rich solid fraction, while duringhydrothermal and steam explosion treatment cleavage of polysaccharide bonds occurs. This work will provide a deeper fundamental knowledge of the bark chemical composition, thus increasing the utilization efficiency of birch outer bark and may create possibilities to up-scale the fractionation processes.
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| 6. |
- Karnaouri, Anthi C, et al.
(författare)
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Fine-tuned enzymatic hydrolysis of organosolv pretreated forest materials for the efficient production of cellobiose
- 2018
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Ingår i: Frontiers in Chemistry. - : Frontiers Media S.A.. - 2296-2646. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Non-digestible oligosaccharides (NDOs) are likely prebiotic candidates that have been related to the prevention of intestinal infections and other disorders for both humans and animals. Lignocellulosic biomass is the largest carbon source in the biosphere, therefore cello-oligosacharides (COS), especially cellobiose, are potentially the most widely available choice of NDOs. Production of COS and cellobiose with enzymes offers numerous benefits over acid-catalyzed processes, as it is milder, environmentally friendly and produces fewer by-products. Cellobiohydrolases (CBHs) and a class of endoglucanases (EGs), namely processive EGs, are key enzymes for the production of COS, as they have higher preference toward glycosidic bonds near the end of cellulose chains and are able to release soluble products. In this work, we describe the heterologous expression and characterization of two CBHs from the filamentous fungus Thermothelomyces thermophila, as well as their synergism with proccessive EGs for cellobiose release from organosolv pretreated spruce and birch. The properties, inhibition kinetics and substrate specific activities for each enzyme are described in detail. The results show that a combination of EGs belonging to Glycosyl hydrolase families 5, 6 and 9, with a CBHI and CBHII in appropriate proportions, can enhance the production of COS from forest materials, underpinning the potential of these biocatalysts in the production of NDOs.
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| 7. |
- Karnaouri, Anthi C., et al.
(författare)
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Lytic Polysaccharide Monooxygenase-Assisted Preparation of Oxidized-Cellulose Nanocrystals with a High Carboxyl Content from the Tunic of Marine Invertebrate Ciona intestinalis
- 2020
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Ingår i: ACS Sustainable Chemistry and Engineering. - : American Chemical Society (ACS). - 2168-0485. ; 8:50, s. 18400-18412
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Tidskriftsartikel (refereegranskat)abstract
- The tunicate species Ciona intestinalis is a fast-growing marine invertebrate animal that contains cellulose in its outer part - the tunic. The high crystallinity and microfibril aspect ratio of tunicate cellulose make it an excellent starting material for the isolation of nanocellulose. In the present work, tunic from C. intestinalis was subjected to organosolv pretreatment followed by bleaching and acid-hydrolysis steps for the isolation of nanocrystals. Applying an intermediate enzymatic treatment step with a lytic polysaccharide monooxygenase (LPMO) from the thermophilic fungus Thermothelomyces thermophila was proved to facilitate the isolation of nanocellulose and to improve the overall process yield, even when the bleaching step was omitted. LPMOs are able to oxidatively cleave the glycosidic bonds of a polysaccharide substrate, either at the C1 and/or C4 position, with the former leading to introduction of carboxylate moieties. X-ray photoelectron spectroscopy analysis showed a significant increase in the atomic percentage of the Câ•O/O-C-O and O-Câ•O bonds upon the addition of LPMO, while the obtained nanocrystals exhibited higher thermal stability compared to the untreated ones. Moreover, an enzymatic post-treatment with LPMOs was performed to additionally functionalize the cellulose nanocrystals. Our results demonstrate that LPMOs are promising candidates for the enzymatic modification of cellulose fibers, including the preparation of oxidized-nanocellulose, and offer great perspectives for the production of novel biobased nanomaterials. ©
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| 8. |
- Karnaouri, Anthi C, et al.
(författare)
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Recombinant expression of thermostable processive MtEG5 endoglucanase and its synergism with MtLPMO from Myceliophthora thermophila during the hydrolysis of lignocellulosic substrates
- 2017
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Ingår i: Biotechnology for Biofuels. - : BioMed Central (BMC). - 1754-6834. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundFilamentous fungi are among the most powerful cellulolytic organisms in terrestrial ecosystems. To perform the degradation of lignocellulosic substrates, these microorganisms employ both hydrolytic and oxidative mechanisms that involve the secretion and synergism of a wide variety of enzymes. Interactions between these enzymes occur on the level of saccharification, i.e., the release of neutral and oxidized products, but sometimes also reflected in the substrate liquefaction. Although the synergism regarding the yield of neutral sugars has been extensively studied, further studies should focus on the oxidized sugars, as well as the effect of enzyme combinations on the viscosity properties of the substrates.ResultsIn the present study, the heterologous expression of an endoglucanase (EG) and its combined activity together with a lytic polysaccharide monooxygenase (LPMO), both from the thermophilic fungus Myceliophthora thermophila, are described. The EG gene, belonging to the glycoside hydrolase family 5, was functionally expressed in the methylotrophic yeast Pichia pastoris. The produced MtEG5A (75 kDa) featured remarkable thermal stability and showed high specific activity on microcrystalline cellulose compared to CMC, which is indicative of its processivity properties. The enzyme was capable of releasing high amounts of cellobiose from wheat straw, birch, and spruce biomass. Addition of MtLPMO9 together with MtEG5A showed enhanced enzymatic hydrolysis yields against regenerated amorphous cellulose (PASC) by improving the release not only of the neutral but also of the oxidized sugars. Assessment of activity of MtEG5A on the reduction of viscosity of PASC and pretreated wheat straw using dynamic viscosity measurements revealed that the enzyme is able to perform liquefaction of the model substrate and the natural lignocellulosic material, while when added together with MtLPMO9, no further synergistic effect was observed.ConclusionsThe endoglucanase MtEG5A from the thermophilic fungus M. thermophila exhibited excellent properties that render it a suitable candidate for use in biotechnological applications. Its strong synergism with LPMO was reflected in sugars release, but not in substrate viscosity reduction. Based on the level of oxidative sugar formation, this is the first indication of synergy between LPMO and EG reported.
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| 9. |
- Karnaouri, Anthi C., et al.
(författare)
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Tailoring Celluclast (R) Cocktail's Performance towards the Production of Prebiotic Cello-Oligosaccharides from Waste Forest Biomass
- 2019
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Ingår i: Catalysts. - : MDPI. - 2073-4344. ; 9:11
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Tidskriftsartikel (refereegranskat)abstract
- The main objective of this study focused on the sustainable production of cellobiose and other cellulose-derived oligosaccharides from non-edible sources, more specifically, from forest residues. For this purpose, a fine-tuning of the performance of the commercially available enzyme mixture Celluclast® was conducted towards the optimization of cellobiose production. By enzyme reaction engineering (pH, multi-stage hydrolysis with buffer exchange, addition of β-glucosidase inhibitor), a cellobiose-rich product with a high cellobiose to glucose ratio (37.4) was achieved by utilizing organosolv-pretreated birch biomass. In this way, controlled enzymatic hydrolysis combined with efficient downstream processing, including product recovery and purification through ultrafiltration and nanofiltration, can potentially support the sustainable production of food-grade oligosaccharides from forest biomass. The potential of the hydrolysis product to support the growth of two Lactobacilli probiotic strains as a sole carbon source was also demonstrated
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| 10. |
- Karnaouri, Anthi C., et al.
(författare)
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Thermophilic enzyme systems for efficient conversion of lignocellulose to valuable products : Structural insights and future perspectives for esterases and oxidative catalysts
- 2019
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Ingår i: Bioresource Technology. - : Elsevier. - 0960-8524 .- 1873-2976. ; 279, s. 362-372
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Tidskriftsartikel (refereegranskat)abstract
- Thermophilic enzyme systems are of major importance nowadays in all industrial processes due to their great performance at elevated temperatures. In the present review, an overview of the current knowledge on the properties of thermophilic and thermotolerant carbohydrate esterases and oxidative enzymes with great thermostability is provided, with respect to their potential use in biotechnological applications. A special focus is given to the lytic polysaccharide monooxygenases that are able to oxidatively cleave lignocellulose through the use of oxygen or hydrogen peroxide as co-substrate and a reducing agent as electron donor. Structural characteristics of the enzymes, including active site conformation and surface properties are discussed and correlated with their substrate specificity and thermostability properties.
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| 11. |
- Karnaouri, Anthi C., et al.
(författare)
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Valorization of waste forest biomass toward the production of cello-oligosaccharides with potential prebiotic activity by utilizing customized enzyme cocktails
- 2019
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Ingår i: Biotechnology for Biofuels. - : BioMed Central (BMC). - 1754-6834. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundProduction of value-added materials from lignocellulosic biomass residues is an emerging sector that has attracted much attention as it offers numerous benefits from an environmental and economical point of view. Non-digestible oligosaccharides represent a group of carbohydrates that are resistant to gastrointestinal digestion, and therefore, they are considered as potential prebiotic candidates. Such oligosaccharides can derive from the biomass cellulose fraction through a controlled enzymatic hydrolysis that eliminates the yield of monomers.ResultsIn the present study, hydrolysis of organosolv-pretreated forest residues (birch and spruce) was tested in the presence of four cellulases (EG5, CBH7, CBH6, EG7) and one accessory enzyme (LPMO). The optimal enzyme combinations were comprised of 20% EG5, 43% CBH7, 22% TtLPMO, 10% PaCbh6a and 5% EG7 in the case of birch and 35% EG5, 45% CBH7, 10% TtLPMO, 10% PaCbh6a and 5% EG7 in the case of spruce, leading to 22.3% and 19.1 wt% cellulose conversion into cellobiose, respectively. Enzymatic hydrolysis was applied on scale-up reactions, and the produced oligosaccharides (consisted of > 90% cellobiose) were recovered and separated from glucose through nanofiltration at optimized temperature (50 °C) and pressure (10 bar) conditions, yielding a final product with cellobiose-to-glucose ratio of 21.1 (birch) and 20.2 (spruce). Cellobiose-rich hydrolysates were tested as fermentative substrates for different lactic acid bacteria. It was shown that they can efficiently stimulate the growth of two Lactobacilli strains.ConclusionsControlled enzymatic hydrolysis with processive cellulases, combined with product recovery and purification, as well as enzyme recycling can potentially support the sustainable production of food-grade oligosaccharides from forest biomass.
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| 12. |
- Matsakas, Leonidas, et al.
(författare)
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Formation of Lignin Nanoparticles by Combining Organosolv Pretreatment of Birch Biomass and Homogenization Processes
- 2018
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Ingår i: Molecules. - : MDPI. - 1431-5157 .- 1420-3049. ; 23:7
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Tidskriftsartikel (refereegranskat)abstract
- Valorization of lignocellulosic biomass into a biorefinery scheme requires the use of all biomass components; in this, the lignin fraction is often underutilized. Conversion of lignin to nanoparticles is an attractive solution. Here, we investigated the effect of different lignin isolation processes and a post-treatment homogenization step on particle formation. Lignin was isolated from birch chips by using two organosolv processes, traditional organosolv (OS) and hybrid organosolv-steam explosion (HOS-SE) at various ethanol contents. For post-treatment, lignin was homogenized at 500 bar using different ethanol:water ratios. Isolation of lignin with OS resulted in unshaped lignin particles, whereas after HOS-SE, lignin micro-particles were formed directly. Addition of an acidic catalyst during HOS-SE had a negative impact on the particle formation, and the optimal ethanol content was 50⁻60% v/v. Homogenization had a positive effect as it transformed initially unshaped lignin into spherical nanoparticles and reduced the size of the micro-particles isolated by HOS-SE. Ethanol content during homogenization affected the size of the particles, with the optimal results obtained at 75% v/v. We demonstrate that organosolv lignin can be used as an excellent starting material for nanoparticle preparation, with a simple method without the need for extensive chemical modification. It was also demonstrated that tuning of the operational parameters results in nanoparticles of smaller size and with better size homogeneity.
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| 13. |
- Muraleedharan, Madhu Nair, et al.
(författare)
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Effect of lignin fractions isolated from different biomass sources on cellulose oxidation by fungal lytic polysaccharide monooxygenases
- 2018
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Ingår i: Biotechnology for Biofuels. - London : BMC. - 1754-6834. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundLytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that oxidatively cleave recalcitrant lignocellulose in the presence of oxygen or hydrogen peroxide as co-substrate and a reducing agent as electron donor. One of the possible systems that provide electrons to the LPMOs active site and promote the polysaccharide degradation involves the mediation of phenolic agents, such as lignin, low-molecular-weight lignin-derived compounds and other plant phenols. In the present work, the interaction of the bulk insoluble lignin fraction extracted from pretreated biomass with LPMOs and the ability to provide electrons to the active site of the enzymes is studied.ResultsThe catalytic efficiency of three LPMOs, namely MtLPMO9 with C1/C4 regioselectivity, PcLPMO9D which is a C1 active LPMO and NcLPMO9C which is a C4 LPMO, was evaluated in the presence of different lignins. It was correlated with the physicochemical and structural properties of lignins, such as the molecular weight and the composition of aromatic and aliphatic hydroxyl groups. Moreover, the redox potential of lignins was determined with the use of large amplitude Fourier Transform alternating current cyclic voltammetry method and compared to the formal potential of the Cu (II) center in the active site of the LPMOs, providing more information about the lignin-LPMO interaction. The results demonstrated the existence of low-molecular weight lignin-derived compounds that are diffused in the reaction medium, which are able to reduce the enzyme active site and subsequently utilize additional electrons from the insoluble lignin fraction to promote the LPMO oxidative activity. Regarding the bulk lignin fractions, those isolated from the organosolv pretreated materials served as the best candidates in supplying electrons to the soluble compounds and, finally, to the enzymes. This difference, based on biomass pretreatment, was also demonstrated by the activity of LPMOs on natural substrates in the presence and absence of ascorbic acid as additional reducing agent.ConclusionsLignins can support the action of LPMOs and serve indirectly as electron donors through low-molecular-weight soluble compounds. This ability depends on their physicochemical and structural properties and is related to the biomass source and pretreatment method.
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| 14. |
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| 15. |
- Nitsos, Christos, et al.
(författare)
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Isolation and Characterization of Organosolv and Alkaline Lignins from Hardwood and Softwood Biomass
- 2016
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Ingår i: ACS Sustainable Chemistry and Engineering. - : American Chemical Society (ACS). - 2168-0485. ; 4:10, s. 5181-5193
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Tidskriftsartikel (refereegranskat)abstract
- Isolation of lignins from hardwood and softwood biomass samples, containing 26.1% and 28.1% lignin, respectively, has been performed with the use of alkaline and organosolv pretreatment methods. The effect of catalyst loading, ethanol content, particle size, and pretreatment time on the yields and properties of the isolated lignins were investigated. Alkaline lignins had higher carbohydrate content - up to 30% - and exhibited higher molecular weights in the range of 3000 Da, with a maximum phenolic hydroxyl content of 1 mmol g-1 for birch and 2 mmol g-1 for spruce. Organosolv lignins, on the other hand, showed high purity - 93% or higher - despite the more extensive biomass dissolution into the pretreatment medium; they also exhibited a lower range of molecular weights between 600 and 1600 Da depending on the source and pretreatment conditions. Due to the lower molecular weight, phenolic hydroxyl content was also increased, reaching as high as 4 mmol g-1 with a simultaneous decrease in aliphatic hydroxyl content as low as 0.6 mmol g-1. Efficient lignin dissolution of 62% for spruce and 69% for birch, achieved at optimal pretreatment conditions, was combined with extensive hemicellulose removal
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