| 1. |
- Antonopoulou, Io, 1989-
(författare)
-
Development of biocatalytic processes for selective antioxidant production
- 2018
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Feruloyl esterases (FAEs, EC 3.1.1.73) represent a subclass of carboxylic acid esterases that under normal conditions catalyze the hydrolysis of the ester bond between hydroxycinnamic acids (ferulic acid, sinapic acid, caffeic acid, p-coumaric acid) and sugar residues in plant cell walls. Based on their specificity towards monoferulates and diferulates, substitutions on the phenolic ring and on their amino acid sequence identity, they have been classified into four types (A-D) while phylogenetic analysis has resulted in classification into thirteen subfamilies (SF1-13). Under low water content, these enzymes are able to catalyze the esterification of hydroxycinnamic acids or the transesterification of their esters (donor) with alcohols or sugars (acceptor) resulting in compounds with modified lipophilicity, having a great potential for use in the tailor-made modification of natural antioxidants for cosmetic, cosmeceutical and pharmaceutical industries. The work described in this thesis focused on the selection,characterization and application of FAEs for the synthesis of bioactive esters with antioxidant activity in non-conventional media. The basis of the current classification systems was investigated in relation with the enzymes’ synthetic and hydrolytic abilities while the developed processes were evaluated for their efficiency and sustainability.Paper I was dedicated to the screening and evaluation of the synthetic abilities of 28 fungal FAEs using acceptors of different lipophilicity at fixed conditions in detergentless microemulsions. It was revealed that FAEs classified in phylogenetic subfamilies related to acetyl xylan esterases (SF5 and 6) showed increased transesterification rates and selectivity. In general, FAEs showed preference on more hydrophilic alcohol acceptors and in descending order to glycerol > 1-butanol > prenol. Homology modeling and small molecule docking simulations were employed as tools for the identification of a potential relationship between the predicted surface and active site properties of selected FAEs and the transesterification selectivity.Papers II- IV focused on the characterization of eight promising FAEs and the optimization of reaction conditions for the synthesis of two bioactive esters (prenyl ferulate and L-arabinose ferulate) in detergentless microemulsions. The effect of the medium composition, the donor and acceptor concentration, the enzyme load, the pH, the temperature and the agitation on the transesterification yield and selectivity were investigated. It was observed that the acceptor concentration and enzyme load were crucial parameters for selectivity. Fae125 (Type A, SF5) iiexhibited highest prenyl ferulate yield (81.1%) and selectivity (4.685) converting 98.5% of VFA to products after optimization at 60 mM VFA, 1.5 M prenol, 0.04 mg FAE mL-1, 40oC, 24 h, 53.4:43.4:3.2 v/v/v n-hexane: t-butanol: 100 mM MOPS-NaOH pH 8.0. On the other hand, FaeA1 (Type A, SF5) showed highest L-arabinose ferulate yield (52.2 %) and selectivity (1.120) at 80 mM VFA, 55 mM L-arabinose, 0.02 mg FAE mL-1, 50oC, 8 h, 19.8: 74.7: 5.5 v/v/v n-hexane: t-butanol: 100 mM MOPS-NaOH pH 8.0.In paper V, the effect of reaction media on the enzyme stability and transesterification yield and selectivity was studied in different solvents for the synthesis of two bioactive esters: prenyl ferulate and L-arabinose ferulate. The best performing enzyme (Fae125) was used in the optimization of reaction conditions in the best solvent (n-hexane) via response surface methodology. Both bioconversions were best described by a two-factor interaction model while optimal conditions were determined as the ones resulting in highest yield and selectivity.Highest prenyl ferulate yield (87.5%) and selectivity (7.616) were observed at 18.56 mM prenol mM-1VFA, 0.04 mg FAE mL-1, 24.5 oC, 24.5 h, 91.8: 8.2 v/v n-hexane: 100 mM sodium acetate pH 4.7. Highest L-arabinose ferulate yield (56.2%) and selectivity (1.284) were observed at 2.96 mM L-arabinose mM-1VFA, 0.02 mg FAE mL-1, 38.9 oC, 12 h, 90.5: 5.0: 4.5 v/v/v n-hexane: dimethyl sulfoxide: 100 mM sodium acetate pH 4.7. The enzyme could be reused for six consecutive reaction cycles maintaining 66.6% of its initial synthetic activity. The developed bioconversions showed exceptional biocatalyst productivities (> 300 g product g-1FAE) and the waste production was within the range of pharmaceutical processes.Paper VI focused on the investigation of the basis of the type A classification of a well-studied FAE from Aspergillus niger(AnFaeA) by comparing its activity towards methyl and arabinose hydroxycinnamic acid esters. For this purpose, L-arabinose ferulateand caffeate were synthesized enzymatically. kcat/Kmratios revealed that AnFaeA hydrolyzed arabinose ferulate 1600 times and arabinose caffeate 6.5 times more efficiently than methyl esters. This study demonstrated that short alkyl chain hydroxycinnamate esters which are used nowadays for FAE classification can lead to activity misclassification, while L-arabinose esters could potentially substitute synthetic esters in classification describing more adequately the enzyme specificitiesin the natural environment.
|
|
| 2. |
- Zerva, Anastasia, et al.
(författare)
-
Optimization of Transesterification Reactions with CLEA-Immobilized Feruloyl Esterases from Thermothelomyces thermophila and Talaromyces wortmannii
- 2018
-
Ingår i: Molecules. - : MDPI. - 1431-5157 .- 1420-3049. ; 23:9
-
Tidskriftsartikel (refereegranskat)abstract
- Feruloyl esterases (FAEs, E.C. 3.1.1.73) are biotechnologically important enzymes with several applications in ferulic acid production from biomass, but also in synthesis of hydroxycinnamic acid derivatives. The use of such biocatalysts in commercial processes can become feasible by their immobilization, providing the advantages of isolation and recycling. In this work, eight feruloyl esterases, immobilized in cross-linked enzyme aggregates (CLEAs) were tested in regard to their transesterification performance, towards the production of prenyl ferulate (PFA) and arabinose ferulate (AFA). After solvent screening, comparison with the activity of respective soluble enzymes, and operational stability tests, FAE125 was selected as the most promising biocatalyst. A central composite design revealed the optimum conditions for each transesterification product, in terms of water content, time, and substrate ratio for both products, and temperature and enzyme load additionally for prenyl ferulate. The optimum product yields obtained were 83.7% for PFA and 58.1% for AFA. FAE125 CLEAs are stable in the optimum conditions of transesterification reactions, maintaining 70% residual activity after five consecutive reactions. Overall, FAE125 CLEAs seem to be able to perform as a robust biocatalyst, offering satisfactory yields and stability, and thus showing significant potential for industrial applications.
|
|
| 3. |
- Matsakas, Leonidas, et al.
(författare)
-
Biological Production of 3-Hydroxypropionic Acid : An Update on the Current Status
- 2018
-
Ingår i: Fermentation. - : MDPI. - 2311-5637. ; 4:1
-
Tidskriftsartikel (refereegranskat)abstract
- The production of high added-value chemicals from renewable resources is a necessity inour attempts to switch to a more sustainable society. 3-Hydroxypropionic acid (3HP) is a promisingmolecule that can be used for the production of an important array of high added-value chemicals,such as 1,3-propanediol, acrylic acid, acrylamide, and bioplastics. Biological production of 3HP hasbeen studied extensively, mainly from glycerol and glucose, which are both renewable resources.To enable conversion of these carbon sources to 3HP, extensive work has been performed to identifyappropriate biochemical pathways and the enzymes that are involved in them. Novel enzymeshave also been identified and expressed in host microorganisms to improve the production yieldsof 3HP. Various process configurations have also been proposed, resulting in improved conversionyields. The intense research efforts have resulted in the production of as much as 83.8 g/L 3HP fromrenewable carbon resources, and a system whereby 3-hydroxypropionitrile was converted to 3HPthrough whole-cell catalysis which resulted in 184.7 g/L 3HP. Although there are still challengesand difficulties that need to be addressed, the research results from the past four years have been animportant step towards biological production of 3HP at the industrial level.
|
|
| 4. |
- Nair, Madhu Muraleedharan
(författare)
-
Lytic Polysaccharide MonoOxygenases; their role for lignocellulose depolymerization and production of (functional) biobased compounds
- 2021
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Increased environmental concerns over petroleum-based products triggered the quest to find a sustainable alternative for fuels, chemicals etc. Lignocellulose biomass, due to its abundance, is considered as one of the most promising sustainable sources for the production of fuels and chemicals, while replacing the traditional petroleum resources. In a biorefinery, by choosing a greener biochemical conversion process with cellulolytic enzymes, cellulose from biomass is depolymerized into monomeric sugars and residual fibers; which can be later converted into a spectra of value added products.Lytic polysaccharide monooxygenases (LPMOs) are one of the essential groups of enzymes in the bioconversion of lignocellulose. They are copper active enzymes that are produced by different polysaccharide degrading organisms in nature, such as lignocellulolytic fungi. In lignocellulose degradation, they are different from the traditional hydrolytic cellulolytic enzymes with their unique way of oxidative breakage of cellulose, in the presence of a co-substrate such as oxygen, and a reducing agent like lignin in the biomass. Their ability to enhance the action of traditional cellulases in cellulose depolymerization make them an integral part of today’s commercial cellulosic cocktails.Primary goals of biorefinery research include efficient liquefaction of lignocellulose in order to increase the release of monomeric sugars towards the production of various chemicals and fuels, together with the potential use of residual fibers for the production of value-added products; all by minimizing the release of undesired by-products and the environmental impact of the process. LPMOs, along with other cellulases, have been shown to be very much beneficial in this.This thesis comprises the study of LPMOs from different fungal origin, in their depolymerization ability on various substrates, including both model substrates and natural biomass samples. The evaluation was done based on their ability to release neutral and oxidized sugars, as well as their capability to promote liquefaction. Effect of various pretreatment methods of lignocellulose on the action of LPMOs was studied, together with their capability to use lignin present in the wood as a reducing agent, which gives a better understanding about their function in nature. Lastly, their role in producing value added materials such as nanocellulose, the prebiotic disaccharide cellobiose, from lignocellulose was also evaluated.
|
|
| 5. |
- Patel, Alok, Dr. 1989-, et al.
(författare)
-
Volatile Fatty Acids (VFAs) Generated by Anaerobic Digestion Serve as Feedstock for Freshwater and Marine Oleaginous Microorganisms to Produce Biodiesel and Added-Value Compounds
- 2021
-
Ingår i: Frontiers in Microbiology. - : Frontiers Media S.A.. - 1664-302X. ; 12
-
Tidskriftsartikel (refereegranskat)abstract
- Given an increasing focus on environmental sustainability, microbial oils have been suggested as an alternative to petroleum-based products. However, microbial oil production relies on the use of costly sugar-based feedstocks. Substrate limitation, elevated costs, and risk of contamination have sparked the search for alternatives to sugar-based platforms. Volatile fatty acids are generated during anaerobic digestion of organic waste and are considered a promising substrate for microbial oil production. In the present study, two freshwater and one marine microalga along with two thraustochytrids were evaluated for their potential to produce lipids when cultivated on volatile fatty acids generated from food waste via anaerobic digestion using a membrane bioreactor. Freshwater microalgae Auxenochlorella protothecoides and Chlorella sorokiniana synthesized lipids rich in palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), and linoleic acid (C18:2). This composition corresponds to that of soybean and jatropha oils, which are used as biodiesel feedstock. Production of added-value polyunsaturated fatty acids (PUFA) mainly omega-3 fatty acids was examined in three different marine strains: Aurantiochytrium sp. T66, Schizochytrium limacinum SR21, and Crypthecodinium cohnii. Only Aurantiochytrium sp. T66 seemed promising, generating 43.19% docosahexaenoic acid (DHA) and 13.56% docosapentaenoic acid (DPA) in total lipids. In summary, we show that A. protothecoides, C. sorokiniana, and Aurantiochytrium sp. T66 can be used for microbial oil production from food waste material.
|
|
| 6. |
- Karnaouri, Anthi C, et al.
(författare)
-
Recombinant expression of thermostable processive MtEG5 endoglucanase and its synergism with MtLPMO from Myceliophthora thermophila during the hydrolysis of lignocellulosic substrates
- 2017
-
Ingår i: Biotechnology for Biofuels. - : BioMed Central (BMC). - 1754-6834. ; 10:1
-
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.
|
|
| 7. |
- Lage, Sandra, et al.
(författare)
-
Microalgae Cultivation for the Biotransformation of Birch Wood Hydrolysate and Dairy Effluent
- 2019
-
Ingår i: Catalysts. - : MDPI. - 2073-4344. ; 9:2
-
Tidskriftsartikel (refereegranskat)abstract
- In order to investigate environmentally sustainable sources of organic carbon and nutrients, four Nordic green microalgal strains, Chlorella sorokiniana, Chlorella saccharophila, Chlorella vulgaris, and Coelastrella sp., were grown on a wood (Silver birch, Betula pendula) hydrolysate and dairy effluent mixture. The biomass and lipid production were analysed under mixotrophic, as well as two-stage mixotrophic/heterotrophic regimes. Of all of the species, Coelastrella sp. produced the most total lipids per dry weight (~40%) in the mixture of birch hydrolysate and dairy effluent without requiring nutrient (nitrogen, phosphorus, and potassium-NPK) supplementation. Overall, in the absence of NPK, the two-stage mixotrophic/heterotrophic cultivation enhanced the lipid concentration, but reduced the amount of biomass. Culturing microalgae in integrated waste streams under mixotrophic growth regimes is a promising approach for sustainable biofuel production, especially in regions with large seasonal variation in daylight, like northern Sweden. To the best of our knowledge, this is the first report of using a mixture of wood hydrolysate and dairy effluent for the growth and lipid production of microalgae in the literature.
|
|
| 8. |
- Matsakas, Leonidas, et al.
(författare)
-
A novel hybrid organosolv: Steam explosion method for the efficient fractionation and pretreatment of birch biomass
- 2018
-
Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 11:1
-
Tidskriftsartikel (refereegranskat)abstract
- Background: The main role of pretreatment is to reduce the natural biomass recalcitrance and thus enhance saccharification yield. A further prerequisite for efficient utilization of all biomass components is their efficient fractionation into well-defined process streams. Currently available pretreatment methods only partially fulfill these criteria. Steam explosion, for example, excels as a pretreatment method but has limited potential for fractionation, whereas organosolv is excellent for delignification but offers poor biomass deconstruction. Results: In this article, a hybrid method combining the cooking and fractionation of conventional organosolv pretreatment with the implementation of an explosive discharge of the cooking mixture at the end of pretreatment was developed. The effects of various pretreatment parameters (ethanol content, duration, and addition of sulfuric acid) were evaluated. Pretreatment of birch at 200 °C with 60% v/v ethanol and 1% w/wbiomassH2SO4was proven to be the most efficient pretreatment condition yielding pretreated solids with 77.9% w/w cellulose, 8.9% w/w hemicellulose, and 7.0 w/w lignin content. Under these conditions, high delignification of 86.2% was demonstrated. The recovered lignin was of high purity, with cellulose and hemicellulose contents not exceeding 0.31 and 3.25% w/w, respectively, and ash to be < 0.17% w/w in all cases, making it suitable for various applications. The pretreated solids presented high saccharification yields, reaching 68% at low enzyme load (6 FPU/g) and complete saccharification at high enzyme load (22.5 FPU/g). Finally, simultaneous saccharification and fermentation (SSF) at 20% w/w solids yielded an ethanol titer of 80 g/L after 192 h, corresponding to 90% of the theoretical maximum. Conclusions: The novel hybrid method developed in this study allowed for the efficient fractionation of birch biomass and production of pretreated solids with high cellulose and low lignin contents. Moreover, the explosive discharge at the end of pretreatment had a positive effect on enzymatic saccharification, resulting in high hydrolyzability of the pretreated solids and elevated ethanol titers in the following high-gravity SSF. To the best of our knowledge, the ethanol concentration obtained with this method is the highest so far for birch biomass.
|
|
| 9. |
- Matsakas, Leonidas, et al.
(författare)
-
A novel hybrid organosolv-steam explosion pretreatment and fractionation method delivers solids with superior thermophilic digestibility to methane
- 2020
-
Ingår i: Bioresource Technology. - : Elsevier. - 0960-8524 .- 1873-2976. ; 316
-
Tidskriftsartikel (refereegranskat)abstract
- Rising environmental concerns and the imminent depletion of fossil resources have sparked a strong interest towards the production of renewable energy such as biomethane. Inclusion of alternative feedstock’s such as lignocellulosic biomass could further expand the production of biomethane. The present study evaluated the potential of a novel hybrid organosolv-steam explosion fractionation for delivering highly digestible pretreated solids from birch and spruce woodchips. The highest methane production yield was 176.5 mLCH4 gVS−1 for spruce and 327.2 mL CH4 gVS−1 for birch. High methane production rates of 1.0–6.3 mL min−1 (spruce) and 6.0–35.5 mL min−1 (birch) were obtained, leading to a rapid digestion, with 92% of total methane from spruce being generated in 80 h and 95% of that from birch in 120 h. These results demonstrate the elevated potential of the novel method to fractionate spruce and birch biomass and deliver cellulose-rich pretreated solids with superior digestibility.
|
|
| 10. |
- Matsakas, Leonidas, et al.
(författare)
-
High-Titer Methane from Organosolv-Pretreated Spruce and Birch
- 2017
-
Ingår i: Energies. - : MDPI. - 1996-1073. ; 10:3
-
Tidskriftsartikel (refereegranskat)abstract
- The negative impact of fossil fuels and the increased demand for renewable energy sources has led to the use of novel raw material sources. Lignocellulosic biomass could serve as a possible raw material for anaerobic digestion and production of biogas. This work is aimed at using forest biomass, both softwood (spruce) and hardwood (birch), as a raw material for anaerobic digestion. We examined the effect of different operational conditions for the organosolv pretreatment (ethanol content, duration of treatment, and addition of acid catalyst) on the methane yield. In addition, we investigated the effect of addition of cellulolytic enzymes during the digestion. We found that inclusion of an acid catalyst during organosolv pretreatment improved the yields from spruce, but it did not affect the yields from birch. Shorter duration of treatment was advantageous with both materials. Methane yields from spruce were higher with lower ethanol content whereas higher ethanol content was more beneficial for birch. The highest yields obtained were 185 mL CH4/g VS from spruce and 259.9 mL CH4/g VS from birch. Addition of cellulolytic enzymes improved these yields to 266.6 mL CH4/g VS and 284.2 mL CH4/g VS, respectively.
|
|
