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  • 1.
    Chalima, Angelina
    et al.
    National Technical University of Athens, Athens, Greece.
    Hatzidaki, Angeliki
    National Technical University of Athens, Athens, Greece.
    Karnaouri, Anthi
    National Technical University of Athens, Athens, Greece.
    Topakas, Evangelos
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering. National Technical University of Athens, Athens, Greece.
    Integration of a dark fermentation effluent in a microalgal-based biorefinery for the production of high-added value omega-3 fatty acids2019In: Applied Energy, ISSN 0306-2619, E-ISSN 1872-9118, Vol. 241, p. 130-138Article in journal (Refereed)
    Abstract [en]

    Dark fermentation is an anaerobic digestion process of biowaste, used to produce hydrogen- for generation of energy- that however releases high amounts of polluting volatile fatty acids, such as acetic acid, in the environment. In order for this biohydrogen production process to become more competitive, the volatile fatty acids stream can be utilized through conversion to high added-value metabolites, such as omega-3 fatty acids. The docosahexaenoic acid is one of the two most known omega-3 fatty acids and has been found to be necessary for a healthy brain and proper cardiovascular function. The main source is currently fish, which obtain the fatty acid from the primary producers, microalgae, through the food chain. Crypthecodinium cohnii, a heterotrophic marine microalga, is known for accumulating high amounts of docosahexaenoic acid, while offering the advantage of assimilating various carbon sources, such as glucose, ethanol, glycerol and acetic acid. The purpose of this work was to examine the ability of a C. cohnii strain to grow on different volatile fatty acids, as well as, on a pretreated dark fermentation effluent and accumulate omega-3. The strain was found to grow well on relatively high concentrations of acetic, butyric or propionic acid as main carbon source in a fed-batch pH-auxostat. Most importantly, C. cohnii totally depleted the organic acid content of an ultra-filtrated dark fermentation effluent after 60 h of fed-batch cultivation, therefore offering a bioprocess not only able to mitigate environmental pollutants, but also to provide a solution for a sustainable energy production process. The accumulated docosahexaenoic acid content was as high as 29.8% (w/w) of total fatty acids. 

  • 2.
    Chalima, Angelina
    et al.
    IndBioCat Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, Zografou Campus, 5 Iroon Polytechniou Str., Athens, Greece.
    Oliver, Laura
    Food and Health Group, Health Division, Tecnalia Research and Innovation, Parque Tecnológico de Álava, Leonardo Da Vinci, 11, Miñano-Álava, Spain.
    De Castro, Laura Fernández
    Food and Health Group, Health Division, Tecnalia Research and Innovation, Parque Tecnológico de Álava, Leonardo Da Vinci, 11, Miñano-Álava, Spain.
    Karnaouri, Anthi
    IndBioCat Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, Zografou Campus, 5 Iroon Polytechniou Str., Athens, Greece.
    Dietrich, Thomas
    Food and Health Group, Health Division, Tecnalia Research and Innovation, Parque Tecnológico de Álava, Leonardo Da Vinci, 11, Miñano-Álava, Spain.
    Topakas, Evangelos
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering. IndBioCat Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, Zografou Campus, 5 Iroon Polytechniou Str., Athens, Greece.
    Utilization of volatile fatty acids from microalgae for the production of high added value compounds2017In: Fermentation, E-ISSN 2311-5637, Vol. 3, no 4, article id 3040054Article, review/survey (Refereed)
    Abstract [en]

    Volatile Fatty Acids (VFA) are small organic compounds that have attracted much attention lately, due to their use as a carbon source for microorganisms involved in the production of bioactive compounds, biodegradable materials and energy. Low cost production of VFA from different types of waste streams can occur via dark fermentation, offering a promising approach for the production of biofuels and biochemicals with simultaneous reduction of waste volume. VFA can be subsequently utilized in fermentation processes and efficiently transformed into bioactive compounds that can be used in the food and nutraceutical industry for the development of functional foods with scientifically sustained claims. Microalgae are oleaginous microorganisms that are able to grow in heterotrophic cultures supported by VFA as a carbon source and accumulate high amounts of valuable products, such as omega-3 fatty acids and exopolysaccharides. This article reviews the different types of waste streams in concert with their potential to produce VFA, the possible factors that affect the VFA production process and the utilization of the resulting VFA in microalgae fermentation processes. The biology of VFA utilization, the potential products and the downstream processes are discussed in detail.

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  • 3.
    Chalima, Angelina
    et al.
    Industrial Biotechnology & Biocatalysis Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, Zografou Campus, Athens, Greece.
    Taxeidis, George
    Industrial Biotechnology & Biocatalysis Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, Zografou Campus, Athens, Greece.
    Topakas, Evangelos
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering. Industrial Biotechnology & Biocatalysis Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, Zografou Campus, Athens, Greece.
    Optimization of the production of docosahexaenoic fatty acid by the heterotrophic microalga Crypthecodinium cohnii utilizing a dark fermentation effluent2020In: Renewable energy, ISSN 0960-1481, E-ISSN 1879-0682, Vol. 152, p. 102-109Article in journal (Refereed)
    Abstract [en]

    Dark fermentation is an anaerobic digestion process of biowaste, used to produce hydrogen as a fuel, which however releases high amounts of polluting volatile fatty acids in the environment. In order for the process to become more competitive, the acids stream can be utilized through conversion to high added-value docosahexaenoic acid by the microalga Crypthecodinium cohnii. Docosahexaenoic acid is one of the two main omega-3 fatty acids, necessary for human nutrition. The purpose of this work was to optimize the production of omega-3 fatty acids by the cells, utilizing the organic content of a dark fermentation effluent. For that purpose, the effect of different fermentation conditions was examined, such as incubation temperature, nitrogen source and concentration, the addition of chemical modulators, as well as the feeding composition. The volatile fatty acid content of the effluent was totally depleted in a fed-batch culture of the microalga, while the cells accumulated DHA in a percentage of 35.6% of total lipids, when fed with yeast extract or 34.2% when fed with ammonium sulfate. Taking into consideration the economic feasibility of the culture conditions proposed it was concluded that the use of yeast extract could be substituted by the much economic ammonium sulfate.

  • 4.
    Christakopoulos, Paul
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Sjöblom, Magnus
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Topakas, Evangelos
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Project: BIOcatalytic Carbon Capture and Conversion of steel flue gas to liquid hydrocarbons (FORMAS)2016Other (Other (popular science, discussion, etc.))
  • 5.
    Dedes, Grigorios
    et al.
    Industrial Biotechnology & Biocatalysis Group, School of Chemical Engineering, National Technical University of Athens, Zografou Campus, 9 Iroon Polytechniou Str, 15780, Athens, Greece.
    Karnaouri, Anthi C.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering. Industrial Biotechnology & Biocatalysis Group, School of Chemical Engineering, National Technical University of Athens, Zografou Campus, 9 Iroon Polytechniou Str, 15780, Athens, Greece.
    Marianou, Asimina A.
    Center for Research and Technology Hellas, Chemical Process and Energy Resources Institute, 57001, Thessaloniki, Greece.
    Kalogiannis, Konstantinos G.
    Center for Research and Technology Hellas, Chemical Process and Energy Resources Institute, 57001, Thessaloniki, Greece.
    Michailof, Chrysoula M.
    Center for Research and Technology Hellas, Chemical Process and Energy Resources Institute, 57001, Thessaloniki, Greece.
    Lappas, Angelos A.
    Center for Research and Technology Hellas, Chemical Process and Energy Resources Institute, 57001, Thessaloniki, Greece.
    Topakas, Evangelos
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering. Industrial Biotechnology & Biocatalysis Group, School of Chemical Engineering, National Technical University of Athens, Zografou Campus, 9 Iroon Polytechniou Str, 15780, Athens, Greece.
    Conversion of organosolv pretreated hardwood biomass into 5-hydroxymethylfurfural (HMF) by combining enzymatic hydrolysis and isomerization with homogeneous catalysis2021In: Biotechnology for Biofuels, ISSN 1754-6834, E-ISSN 1754-6834, Vol. 14, article id 172Article in journal (Refereed)
    Abstract [en]

    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.

  • 6.
    Kalogiannis, Konstantinos G.
    et al.
    Chemical Process and Energy Resources Institute (CPERI), CERTH, 6th Km Harilaou-Thermi Road, 57001 Thermi, Thessaloniki, Greece.
    Karnaouri, Anthi
    Chemical Process and Energy Resources Institute (CPERI), CERTH, 6th Km Harilaou-Thermi Road, 57001 Thermi, Thessaloniki, Greece. Industrial Biotechnology & Biocatalysis Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 9 Iroon Polytechniou Str., Zografou Campus, Athens 15780, Greece.
    Michailof, Chrysoula
    Chemical Process and Energy Resources Institute (CPERI), CERTH, 6th Km Harilaou-Thermi Road, 57001 Thermi, Thessaloniki, Greece.
    Tzika, Anna Maria
    Chemical Process and Energy Resources Institute (CPERI), CERTH, 6th Km Harilaou-Thermi Road, 57001 Thermi, Thessaloniki, Greece.
    Asimakopoulou, Georgia
    Industrial Biotechnology & Biocatalysis Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 9 Iroon Polytechniou Str., Zografou Campus, Athens 15780, Greece.
    Topakas, Evangelos
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering. Industrial Biotechnology & Biocatalysis Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 9 Iroon Polytechniou Str., Zografou Campus, Athens 15780, Greece.
    Lappas, Angelos A.
    Chemical Process and Energy Resources Institute (CPERI), CERTH, 6th Km Harilaou-Thermi Road, 57001 Thermi, Thessaloniki, Greece.
    OxiOrganosolv: A novel acid free oxidative organosolv fractionation for lignocellulose fine sugar streams2020In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 313, article id 123599Article in journal (Refereed)
    Abstract [en]

    The valorization of lignocellulosic biomass towards the production of value-added products requires an efficient pretreatment/fractionation step. In this work we present a novel, acid-free, mildly oxidative organosolv delignification process -OxiOrganosolv- which employs oxygen gas to depolymerize and remove lignin. The results demonstrate that the OxiOrganosolv process achieved lignin removal as high as 97% in a single stage, with a variety of solvents; it was also efficient in delignifying both beechwood (hardwood) and pine (softwood), a task in which organosolv pretreatments have failed in the past. Minimal amounts of sugar degradation products were detected, while cellulose recovery was ~100% in the solid pulp. Enzymatic hydrolysis of pulps showed >80 wt% cellulose conversion to glucose. Overall, the OxiOrganosolv pretreatment has significant advantages, including high delignification efficiency of hardwood and softwood biomass, absence of acid homogeneous catalysis and all corresponding challenges involved, and close to zero losses of sugars to degradation products.

  • 7.
    Kanelli, Maria
    et al.
    IndBioCat Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Mandic, Mina
    Institute of Molecular Genetics and Genetic Engineering, University of Belgrade.
    Kalakona, Margarita
    IndBioCat Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athen.
    Vasilakos, Sozon
    Materials Industrial Research and Technology Center S.A., Athens.
    Kekos, Dimitris
    IndBioCat Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Nikodinovic-Runic, Jasmina
    Institute of Molecular Genetics and Genetic Engineering, University of Belgrade.
    Topakas, Evangelos
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering. IndBioCat Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Microbial Production of Violacein and Process Optimization for Dyeing Polyamide Fabrics With Acquired Antimicrobial Properties2018In: Frontiers in Microbiology, E-ISSN 1664-302X, Vol. 9, article id 1495Article in journal (Refereed)
    Abstract [en]

    In the present study, crude bacterial extract containing violacein is investigated for the preparation of antimicrobial polyamide fabrics. The optimal culture conditions of Janthinobacterium lividum (JL) for maximum biomass and violacein production were found to be 25°C, pH 7.0, while the addition of ampicillin of 0.2 mg mL-1 in the small scale increased violacein production 1.3-fold. In scale-up trials, the addition of 1% (v/v) glycerol in a fed-batch bioreactor, resulted in fivefold extracted crude violacein increase with final concentration of 1.828 g L-1. Polyamide 6.6 fabrics were dyed following three different processes; through simultaneous fermentation and dyeing (SFD), by incubating the fabric in the sonicated bacterial culture after fermentation and by using cell-free extract containing violacein. Maximum color change (ΔE) and color strength (K/S) obtained for SFD fabrics were 74.81 and 22.01, respectively, while no alteration of fastness and staining of dye at acid and alkaline perspiration or at water was indicated. The dyed fabrics presented significant antifungal activity against Candida albicans, C. parapsilosis, and C. krusei, as well as antibacterial properties against Escherichia coli, Staphylococcus aureus, and the S. aureus MRSA. We have shown that J. lividum cultures can be successfully used for violacein production and for simultaneous dying of fabrics resulting in dyed fabrics with antimicrobial properties without utilization of organic solvents.

  • 8.
    Kanelli, Maria
    et al.
    National Technical University of Athens.
    Vasilakos, Sozon
    MIRTEC, Materials Industrial Research & Technology Center S.A.
    Nikolaivits, Efstratios
    Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Ladas, Spyridon
    Surface Science Laboratory, Department of Chemical Engineering, University of Patras.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Topakas, Evangelos
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Surface modification of poly(ethylene terephthalate) (PET) fibers by a cutinase from Fusarium oxysporum2015In: Process Biochemistry, ISSN 1359-5113, E-ISSN 1873-3298, Vol. 50, no 11, p. 1885-1892Article in journal (Refereed)
    Abstract [en]

    Synthetic polyester fabrics occupy a great part of the textile industry production satisfying variable ordinary needs. Nonetheless, their high hydrophobicity constitutes an important weakness that impedes process manufacture, as well as permeability and evaporation of sweat when used in clothing industry. The enzymatic treatment of these materials is a modern and eco-friendly procedure that aims at the increase of the hydrophilicity through superficial modification. In this study, the enzymatic surface hydrolysis of poly(ethylene terephthalate) (PET) fabric is succeeded using a recombinant cutinase from Fusarium oxysporum. The effect of various parameters is studied for the enzymatic modification of PET, such as temperature, pH, enzyme loading and reaction time. The optimal parameters are found to be 40 °C, pH 8, and 1.92 mg enzyme loading per gram of fabric. The controlled enzymatic hydrolysis of PET textile is further confirmed and characterized using various spectroscopic and analytical methods, including Fourier Transform Infrared (FT-IR) in the Attenuated Total Reflectance mode (ATR) and X-ray photoelectron spectroscopy (XPS). Tensile test and dyeability analyses were also employed achieving a K/S increase up to 150%, confirming the successful surface modification without degrading the quality of the starting material.

  • 9.
    Karnaouri, Anthi
    et al.
    Industrial Biotechnology & Biocatalysis Group, School of Chemical Engineering, National Technical University of Athens, 9 Iroon Polytechniou Str., Zografou Campus, 15780, Athens, Greece.
    Asimakopoulou, Georgia
    Industrial Biotechnology & Biocatalysis Group, School of Chemical Engineering, National Technical University of Athens, 9 Iroon Polytechniou Str., Zografou Campus, 15780, Athens, Greece.
    Kalogiannis, Konstaninos G.
    Chemical Process and Energy Resources Institute (CPERI), CERTH, 6th km Harilaou-Thermi Road, Thermi, 57001, Thessaloniki, Greece.
    Lappas, Angelos
    Chemical Process and Energy Resources Institute (CPERI), CERTH, 6th km Harilaou-Thermi Road, Thermi, 57001, Thessaloniki, Greece.
    Topakas, Evangelos
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering. Industrial Biotechnology & Biocatalysis Group, School of Chemical Engineering, National Technical University of Athens, 9 Iroon Polytechniou Str., Zografou Campus, 15780, Athens, Greece.
    Efficient d-lactic acid production by Lactobacillus delbrueckii subsp. bulgaricus through conversion of organosolv pretreated lignocellulosic biomass2020In: Biomass and Bioenergy, ISSN 0961-9534, E-ISSN 1873-2909, Vol. 140, article id 105672Article in journal (Refereed)
    Abstract [en]

    Lactic acid bioconversion processes have numerous advantages over the chemical synthesis route, not only due to the high-titer yield of the final product with great optical purity, but also due to the possibility of utilizing lignocellulosic biomass feedstocks as carbon source in an economic and environmentally friendly way. In the present study, beechwood and pine were pretreated with a novel mild oxidative organosolv process to produce cellulose-rich solid fractions, which were tested for their ability to support the growth and high lactic acid productivity of Lactobacillus delbrueckii subsp. bulgaricus (ATCC® 11842). We employed a simultaneous saccharification and fermentation (SSF) strategy in batch cultures with 9% w v−1 solids loading. The results for beechwood showed the highest production of 62 g L−1 lactic acid after 72 h of incubation, corresponding to a yield of 0.69 g g−1 of biomass (82.7% of the theoretical maximum yield) and a productivity of 0.86 g L−1 h−1. In the case of pine, the productivity was lower at 0.51 g L−1 h−1, leading to accumulation of 36.4 g L−1 lactic acid, corresponding to a yield of 0.40 g g−1 of biomass (41.4% of the theoretical maximum yield). Our study suggests that L. delbrueckii subsp. bulgaricus is an efficient lactic acid bacterial strain for the production of optically pure d-lactic acid from non-edible, organosolv pretreated hardwood and softwood biomass for the synthesis of bio-based plastics and other products.

  • 10.
    Karnaouri, Anthi C.
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Antonopoulou, Io
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Zerva, Anastasia
    Biotechnology Laboratory, Department of Synthesis and Development of Industrial Processes, School of Chemical Engineering, National Technical University of Athens, Athens, Greece.
    Dimarogona, Maria
    Section of Process and Environmental Engineering, Department of Chemical Engineering, University of Patras, Patras, Greece.
    Topakas, Evangelos
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering. Biotechnology Laboratory, Department of Synthesis and Development of Industrial Processes, School of Chemical Engineering, National Technical University of Athens, Athens, Greece.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Thermophilic enzyme systems for efficient conversion of lignocellulose to valuable products: Structural insights and future perspectives for esterases and oxidative catalysts2019In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 279, p. 362-372Article in journal (Refereed)
    Abstract [en]

    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.

  • 11.
    Karnaouri, Anthi C
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Muraleedharan, Madhu Nair
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Dimarogona, Maria
    Biotechnology Laboratory, Department of Synthesis and Development of Industrial Processes, School of Chemical Engineering, National Technical University of Athens, Athens, Greece.
    Topakas, Evangelos
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering. Biotechnology Laboratory, Department of Synthesis and Development of Industrial Processes, School of Chemical Engineering, National Technical University of Athens, Athens, Greece.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Sandgren, Mats
    Department of Chemistry and Biotechnology, Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Recombinant expression of thermostable processive MtEG5 endoglucanase and its synergism with MtLPMO from Myceliophthora thermophila during the hydrolysis of lignocellulosic substrates2017In: Biotechnology for Biofuels, E-ISSN 1754-6834, Vol. 10, no 1, article id 126Article in journal (Refereed)
    Abstract [en]

    Background

    Filamentous 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.

    Results

    In 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.

    Conclusions

    The 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.

  • 12.
    Karnaouri, Anthi C
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Topakas, Evangelos
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering. School of Chemical Engineering, National Technical University of Athens.
    Matsakas, Leonidas
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Fine-tuned enzymatic hydrolysis of organosolv pretreated forest materials for the efficient production of cellobiose2018In: Frontiers in Chemistry, E-ISSN 2296-2646, Vol. 6, article id 128Article in journal (Refereed)
    Abstract [en]

    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.

  • 13.
    Karnaouri, Anthi
    et al.
    Industrial Biotechnology & Biocatalysis Group, School of Chemical Engineering, National Technical University of Athens.
    Chalima, Angelina
    Industrial Biotechnology & Biocatalysis Group, School of Chemical Engineering, National Technical University of Athens.
    Kalogiannis, Konstantinos G.
    Chemical Process and Energy Resources Institute (CPERI), CERTH.
    Varamogianni-Mamatsi, Despoina
    Industrial Biotechnology & Biocatalysis Group, School of Chemical Engineering, National Technical University of Athens.
    Lappas, Angelos
    Chemical Process and Energy Resources Institute (CPERI), CERTH.
    Topakas, Evangelos
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering. Industrial Biotechnology & Biocatalysis Group, School of Chemical Engineering, National Technical University of Athens.
    Utilization of lignocellulosic biomass towards the production of omega-3 fatty acids by the heterotrophic marine microalga Crypthecodinium cohnii2020In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 303, article id 122899Article in journal (Refereed)
    Abstract [en]

    Omega-3 fatty acids have become a commodity of high nutritional and commercial value; intensive fishing and its environmental and social cost has led researchers to seeking alternative more sustainable ways of producing them. Heterotrophic microalgae such as Crypthecodinium cohnii, a marine dinoflagellate, have the ability to utilize various substrates and accumulate high amounts of docosahexaenoic acid (DHA). In this work, a mild oxidative organosolv pretreatment of beechwood pulps was employed that allowed up to 95% of lignin removal in a single stage, thus yielding a cellulose-rich solid fraction. The enzymatic hydrolysates were evaluated for their ability to support the growth and lipid accumulation of C. cohnii in batch and fed-batch cultures; the results verified the successful microalgae growth, while DHA reached up to 43.5% of the cell’s total lipids. The proposed bioprocess demonstrated the utilization of non-edible biomass towards high added value food supplements in a sustainable and efficient manner.

  • 14.
    Karnaouri, Anthi
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Matsakas, Leonidas
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Topakas, Evangelos
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering. Department of Chemical Engineering, Biotechnology Laboratory, National Technical University of Athens, Greece.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Development of Thermophilic Tailor-Made Enzyme Mixtures for the Bioconversion of Agricultural and Forest Residues2016In: Frontiers in Microbiology, E-ISSN 1664-302X, Vol. 7, article id 177Article in journal (Refereed)
    Abstract [en]

    Even though the main components of all lignocellulosic feedstocks include cellulose, hemicellulose, as well as the protective lignin matrix, there are some differences in structure, such as in hardwoods and softwoods, which may influence the degradability of the materials. Under this view, various types of biomass might require a minimal set of enzymes that has to be tailor-made. Partially defined complex mixtures that are currently commercially used are not adapted to efficiently degrade different materials, so novel enzyme mixtures have to be customized. Development of these cocktails requires better knowledge about the specific activities involved, in order to optimize hydrolysis. The role of filamentous fungus Myceliophthora thermophila and its complete enzymatic repertoire for the bioconversion of complex carbohydrates has been widely proven. In this study, four core cellulases (MtCBH7, MtCBH6, MtEG5, and MtEG7), in the presence of other four “accessory” enzymes (mannanase, lytic polyssacharide monooxygenase MtGH61, xylanase, MtFae1a) and β-glucosidase MtBGL3, were tested as a nine-component cocktail against one model substrate (phosphoric acid swollen cellulose) and four hydrothermally pretreated natural substrates (wheat straw as an agricultural waste, birch, and spruce biomass, as forest residues). Synergistic interactions among different enzymes were determined using a suitable design of experiments methodology. The results suggest that for the hydrolysis of the pure substrate (PASC), high proportions of MtEG7 are needed for efficient yields. MtCBH7 and MtEG7 are enzymes of major importance during the hydrolysis of pretreated wheat straw, while MtCBH7 plays a crucial role in case of spruce. Cellobiohydrolases MtCBH6 and MtCBH7 act in combination and are key enzymes for the hydrolysis of the hardwood (birch). Optimum combinations were predicted from suitable statistical models which were able to further increase hydrolysis yields, suggesting that tailor-made enzyme mixtures targeted toward a particular residual biomass can help maximize hydrolysis yields. The present work demonstrates the change from “one cocktail for all” to “tailor-made cocktails” that are needed for the efficient saccharification of targeted feed stocks prior to the production of biobased products through the biorefinery concept.

  • 15.
    Karnaouri, Anthi
    et al.
    Industrial Biotechnology and Biocatalysis Group, Biotechnology Laboratory, Department of Synthesis and Development of Industrial Processes, School of Chemical Engineering, National Technical University of Athens, Athens, Greece.
    Zerva, Anastasia
    Industrial Biotechnology and Biocatalysis Group, Biotechnology Laboratory, Department of Synthesis and Development of Industrial Processes, School of Chemical Engineering, National Technical University of Athens, Athens, Greece.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Topakas, Evangelos
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering. Industrial Biotechnology and Biocatalysis Group, Biotechnology Laboratory, Department of Synthesis and Development of Industrial Processes, School of Chemical Engineering, National Technical University of Athens, Athens, Greece.
    Screening of Recombinant Lignocellulolytic Enzymes Through Rapid Plate Assays2021In: Protein Downstream Processing: Design, Development, and Application of High and Low-Resolution Methods / [ed] Nikolaos E. Labrou, Springer Nature, 2021, 2, p. 479-503Chapter in book (Other academic)
    Abstract [en]

    In the search for novel biomass-degrading enzymes through mining microbial genomes, it is necessary to apply functional tests during high-throughput screenings, which are capable of detecting enzymatic activities directly by way of plate assay. Using the most efficient expression systems of Escherichia coli and Pichia pastoris, the production of a high amount of His-tagged recombinant proteins could be thrived, allowing the one-step isolation by affinity chromatography. Here, we describe simple and efficient assay techniques for the detection of various biomass-degrading enzymatic activities on agar plates, such as cellulolytic, hemicellulolytic, and ligninolytic activities and their isolation using immobilized-metal affinity chromatography.

  • 16.
    Katsimpouras, Constantinos
    et al.
    Industrial Biotechnology & Biocatalysis Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Dedes, Grigorios
    Industrial Biotechnology & Biocatalysis Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Bistis, Perrakis
    Industrial Biotechnology & Biocatalysis Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Kekos, Dimitrios
    Industrial Biotechnology & Biocatalysis Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Kalogiannis, Konstantinos G.
    Chemical Process and Energy Resources Institute (CPERI) .
    Topakas, Evangelos
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering. Industrial Biotechnology & Biocatalysis Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Acetone/water oxidation of corn stover for the production of bioethanol and prebiotic oligosaccharides2018In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 270, p. 208-215Article in journal (Refereed)
    Abstract [en]

    Ethanol production at high-gravity promise to achieve concentrations over the threshold for an economical distillation process and concurrently reduce water consumption. However, a persisting limitation is the poor mass transfer conditions resulting in low ethanol yields and concentrations. Hereby, the combination of an acetone/water oxidation pretreatment process (AWO) with a liquefaction/saccharification step, using a free-fall mixer, before simultaneous saccharification and fermentation (SSF) can realize ethanol concentrations of up to ca. 74 g/L at a solids content of 20 wt.%. The free-fall mixer achieved a biomass slurry’s viscosity reduction by 87 % after only 2 h of enzymatic saccharification, indicating the efficiency of the mixing system. Furthermore, the direct enzymatic treatment of AWO pretreated corn stover (CS) by a GH11 recombinant xylanase, led to the production of xylooligosaccharides (XOS) with prebiotic potential and the removal of insoluble fibers of hemicellulose improved the glucose release of AWOCS by 22 %.

  • 17.
    Katsimpouras, Constantinos
    et al.
    Industrial Biotechnology & Biocatalysis Group, School of Chemical Engineering, National Technical University of Athens, 9 Iroon Polytechniou Str., Zografou Campus, Athens, Greece.
    Dedes, Grigorios
    Industrial Biotechnology & Biocatalysis Group, School of Chemical Engineering, National Technical University of Athens, 9 Iroon Polytechniou Str., Zografou Campus, Athens, Greece.
    Thomaidis, Nikolaos S.
    Laboratory of Analytical Chemistry, Department of Chemistry, National and Kapodistrian University of Athens, Panepistimioupolis Zografou, Athens, Greece.
    Topakas, Evangelos
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering. Industrial Biotechnology & Biocatalysis Group, School of Chemical Engineering, National Technical University of Athens, 9 Iroon Polytechniou Str., Zografou Campus, Athens, Greece.
    A novel fungal GH30 xylanase with xylobiohydrolase auxiliary activity2019In: Biotechnology for Biofuels, ISSN 1754-6834, E-ISSN 1754-6834, Vol. 12, article id 120Article in journal (Refereed)
    Abstract [en]

    Background:

    The main representatives of hemicellulose are xylans, usually decorated β-1,4-linked d-xylose polymers, which are hydrolyzed by xylanases. The efficient utilization and complete hydrolysis of xylans necessitate the understanding of the mode of action of xylan degrading enzymes. The glycoside hydrolase family 30 (GH30) xylanases comprise a less studied group of such enzymes, and differences regarding the substrate recognition have been reported between fungal and bacterial GH30 xylanases. Besides their role in the utilization of lignocellulosic biomass for bioenergy, such enzymes could be used for the tailored production of prebiotic xylooligosaccharides (XOS) due to their substrate specificity.

    Results:

    The expression of a putative GH30_7 xylanase from the fungus Thermothelomyces thermophila (synonyms Myceliophthora thermophila, Sporotrichum thermophile) in Pichia pastoris resulted in the production and isolation of a novel xylanase with unique catalytic properties. The novel enzyme designated TtXyn30A, exhibited an endo- mode of action similar to that of bacterial GH30 xylanases that require 4-O-methyl-d-glucuronic acid (MeGlcA) decorations, in contrast to most characterized fungal ones. However, TtXyn30A also exhibited an exo-acting catalytic behavior by releasing the disaccharide xylobiose from the non-reducing end of XOS. The hydrolysis products from beechwood glucuronoxylan were MeGlcA substituted XOS, and xylobiose. The major uronic XOS (UXOS) were the aldotriuronic and aldotetrauronic acid after longer incubation indicating the ability of TtXyn30A to cleave linear parts of xylan and UXOS as well.

    Conclusions:

    Hereby, we reported the heterologous production and biochemical characterization of a novel fungal GH30 xylanase exhibiting endo- and exo-xylanase activity. To date, considering its novel catalytic properties, TtXyn30A shows differences with most characterized fungal and bacterial GH30 xylanases. The discovered xylobiohydrolase mode of action offers new insights into fungal enzymatic systems that are employed for the utilization of lignocellulosic biomass. The recombinant xylanase could be used for the production of X2 and UXOS from glucuronoxylan, which in turn would be utilized as prebiotics carrying manifold health benefits.

  • 18.
    Katsimpouras, Constantinos
    et al.
    Industrial Biotechnology & Biocatalysis Group, School of Chemical Engineering, National Technical University of Athens.
    Zacharopoulou, Maria
    Industrial Biotechnology & Biocatalysis Group, School of Chemical Engineering, National Technical University of Athens.
    Matsakas, Leonidas
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Topakas, Evangelos
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering. Industrial Biotechnology & Biocatalysis Group, School of Chemical Engineering, National Technical University of Athens.
    Sequential high gravity ethanol fermentation and anaerobic digestion of steam explosion and organosolv pretreated corn stover2017In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 244:1, p. 1129-1136Article in journal (Refereed)
    Abstract [en]

    The present work investigates the suitability of pretreated corn stover (CS) to serve as feedstock for high gravity (HG) ethanol production at solids-content of 24 wt%. Steam explosion, with and without the addition of H2SO4, and organosolv pretreated CS samples underwent a liquefaction/saccharification step followed by simultaneous saccharification and fermentation (SSF). Maximum ethanol concentration of ca. 76 g/L (78.3% ethanol yield) was obtained from steam exploded CS (SECS) with 0.2% H2SO4. Organosolv pretreated CS (OCS) also resulted in high ethanol concentration of ca. 65 g/L (62.3% ethanol yield). Moreover, methane production through anaerobic digestion (AD) was conducted from fermentation residues and resulted in maximum methane yields of ca. 120 and 69 mL/g volatile solids (VS) for SECS and OCS samples, respectively. The results indicated that the implementation of a liquefaction/saccharification step before SSF employing a liquefaction reactor seemed to handle HG conditions adequately.

  • 19.
    Mandic, Mina
    et al.
    Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Belgrade, Serbia.
    Djokic, Lidija
    Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Belgrade, Serbia.
    Nikolaivits, Efstratios
    School of Chemical Engineering, National Technical University of Athens, Athens, Greece.
    Prodanovic, Radivoje
    Faculty of Chemistry, University of Belgrade, Belgrade, Serbia.
    O’Connor, Kevin
    BEACON SFI Bioeconomy Research Centre and School of Biomolecular and Biomedical Science, University College Dublin, Belfield, Dublin, Ireland.
    Jeremic, Sanja
    Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Belgrade, Serbia.
    Topakas, Evangelos
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering. School of Chemical Engineering, National Technical University of Athens, Athens, Greece.
    Nikodinovic-Runic, Jasmina
    Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Belgrade, Serbia.
    Identification and Characterization of New Laccase Biocatalysts from Pseudomonas Species Suitable for Degradation of Synthetic Textile Dyes2019In: Catalysts, E-ISSN 2073-4344, Vol. 9, no 7, article id 629Article in journal (Refereed)
    Abstract [en]

    Laccases are multicopper-oxidases with variety of biotechnological applications. While predominantly used, fungal laccases have limitations such as narrow pH and temperature range and their production via heterologous protein expression is more complex due to posttranslational modifications. In comparison, bacterial enzymes, including laccases, usually possess higher thermal and pH stability, and are more suitable for expression and genetic manipulations in bacterial expression hosts. Therefore, the aim of this study was to identify, recombinantly express, and characterize novel laccases from Pseudomonas spp. A combination of approaches including DNA sequence analysis, N-terminal protein sequencing, and genome sequencing data analysis for laccase amplification, cloning, and overexpression have been used. Four active recombinant laccases were obtained, one each from P. putida KT2440 and P. putida CA-3, and two from P. putida F6. The new laccases exhibited broad temperature and pH range and high thermal stability, as well as the potential to degrade selection of synthetic textile dyes. The best performing laccase was CopA from P. putida F6 which degraded five out of seven tested dyes, including Amido Black 10B, Brom Cresol Purple, Evans Blue, Reactive Black 5, and Remazol Brilliant Blue. This work highlighted species of Pseudomonas genus as still being good sources of biocatalytically relevant enzymes.

  • 20.
    Muraleedharan, Madhu Nair
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Zouraris, Dimitrios
    Laboratory of Physical Chemistry and Applied Electrochemistry, School of Chemical Engineering, National Technical University of Athens, Athens, Greece.
    Karantonis, Antonis
    Laboratory of Physical Chemistry and Applied Electrochemistry, School of Chemical Engineering, National Technical University of Athens, Athens, Greece.
    Topakas, Evangelos
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering. Biotechnology Laboratory, Department of Synthesis and Development of Industrial Processes, School of Chemical Engineering, National Technical University of Athens, Athens, Greece.
    Sandgren, Mats
    Department of Chemistry and Biotechnology, Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Karnaouri, Anthi C.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Effect of lignin fractions isolated from different biomass sources on cellulose oxidation by fungal lytic polysaccharide monooxygenases2018In: Biotechnology for Biofuels, ISSN 1754-6834, E-ISSN 1754-6834, Vol. 11, no 1, article id 296Article in journal (Refereed)
    Abstract [en]

    Background

    Lytic 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.

    Results

    The 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.

    Conclusions

    Lignins 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.

  • 21.
    Nikolaivits, Efstratios
    et al.
    Industrial Biotechnology & Biocatalysis Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 15780 Athens, Greece.
    Agrafiotis, Andreas
    Industrial Biotechnology & Biocatalysis Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 15780 Athens, Greece.
    Baira, Eirini
    Division of Pharmacognosy and Chemistry of Natural Products, Department of Pharmacy, National and Kapodistrian University of Athens, 15771 Athens, Greece.
    Le Goff, Géraldine
    Institut de Chimie des Substances Naturelles, ICSN, CNRS, 91198 Gif sur Yvette, France.
    Tsafantakis, Nikolaos
    Division of Pharmacognosy and Chemistry of Natural Products, Department of Pharmacy, National and Kapodistrian University of Athens, 15771 Athens, Greece.
    Chavanich, Suchana A.
    Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
    Benayahu, Yehuda
    School of Zoology, Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
    Ouazzani, Jamal
    Institut de Chimie des Substances Naturelles, ICSN, CNRS, 91198 Gif sur Yvette, France.
    Fokialakis, Nikolas
    Division of Pharmacognosy and Chemistry of Natural Products, Department of Pharmacy, National and Kapodistrian University of Athens, 15771 Athens, Greece.
    Topakas, Evangelos
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering. Industrial Biotechnology & Biocatalysis Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 15780 Athens, Greece.
    Degradation Mechanism of 2,4-Dichlorophenol by Fungi Isolated from Marine Invertebrates2020In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 21, no 9, article id 3317Article in journal (Refereed)
    Abstract [en]

    2,4-Dichlorophenol (2,4-DCP) is a ubiquitous environmental pollutant categorized as a priority pollutant by the United States (US) Environmental Protection Agency, posing adverse health effects on humans and wildlife. Bioremediation is proposed as an eco-friendly, cost-effective alternative to traditional physicochemical remediation techniques. In the present study, fungal strains were isolated from marine invertebrates and tested for their ability to biotransform 2,4-DCP at a concentration of 1 mM. The most competent strains were studied further for the expression of catechol dioxygenase activities and the produced metabolites. One strain, identified as Tritirachium sp., expressed high levels of extracellular catechol 1,2-dioxygenase activity. The same strain also produced a dechlorinated cleavage product of the starting compound, indicating the assimilation of the xenobiotic by the fungus. This work also enriches the knowledge about the mechanisms employed by marine-derived fungi in order to defend themselves against chlorinated xenobiotics.

  • 22.
    Nikolaivits, Efstratios
    et al.
    Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, Athens, Greece.
    Dimarogona, Maria
    Department of Chemical Engineering, University of Patras, Patras, Greece.
    Karagiannaki, Ioanna
    Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, Athens, Greece.
    Chalima, Angelina
    Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, Athens, Greece.
    Fishman, Ayelet
    Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa, Israel.
    Topakas, Evangelos
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering. Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, Athens, Greece.
    Versatile fungal polyphenol oxidase with chlorophenol bioremediation potential: Characterization and protein engineering2018In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 84, no 23, article id e01628-18Article in journal (Refereed)
    Abstract [en]

    Polyphenol oxidases (PPOs) have been mostly associated with the undesirable post-harvest browning in fruits and vegetables and with implications in human melanogenesis. Nonetheless, they are considered useful biocatalysts in the food, pharmaceutical and cosmetic industries. The aim of the present work was to characterize a novel PPO and explore its potential as a bioremediation agent. A gene encoding an extracellular tyrosinase-like enzyme was amplified from the genome of Thermothelomyces thermophila and expressed in Pichia pastoris. The recombinant enzyme (TtPPO) was purified and biochemically characterized. Its production reached 40 mg/L and it appeared to be a glycosylated and N-terminally processed protein. TtPPO showed broad substrate specificity as it could oxidize 28/30 compounds tested, including polyphenols, substituted phenols, catechols and methoxyphenols. Its optimum temperature was 65 °C with a half-life of 18.3 h at 50 °C, while its optimum pH was 7.5. The homology model of TtPPO was constructed and site-directed mutagenesis was performed in order to increase its activity on mono- and di-chlorophenols (CPs). TtPPO variant G292N/Y296V had a 5.3-fold increased activity on 3,5-diCP compared to wild-type.

    Importance A novel fungal PPO was heterologously expressed and biochemically characterized. Construction of single and double mutants led to the generation of variants with altered specificity against CPs. Through this work, knowledge is gained regarding the effect of mutations on the substrate specificity of PPOs. This work also demonstrates that more potent biocatalysts for the bioremediation of harmful CPs can be developed by applying site-directed mutagenesis.

  • 23.
    Papaspyridi, Lefki Maria
    et al.
    Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Zerva, Anastasia
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Topakas, Evangelos
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering. Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Biocatalytic synthesis of fungal β-glucans2018In: Catalysts, E-ISSN 2073-4344, Vol. 8, no 7, article id 274Article in journal (Refereed)
    Abstract [en]

    Glucans are the dominant polysaccharide constituents of fungal cell walls. Remarkably, these major bioactive polysaccharides account for the beneficial effects that have been observed by many mushrooms of medicinal interest. Accordingly, the prevailing tendency is the use of bioactive mushroom β-glucans mainly in pharmaceutical industries or as food additives, since it seems that they can be involved in meeting the overall growing demand for food in the future, but also in medical and material sectors. β-(1,3)-Glucan synthase (GLS) is the responsible enzyme for the synthesis of these important polysaccharides, which is a member of the glycosyl transferase (GT) family. For optimizing the production of such natural polymers of great interest, the comprehension of the fungal synthetic mechanism, as well as the biochemical and molecular characteristics of the key enzyme GLS and its expression seem to be crucial. Overall, in this review article, the fungal β-glucans biosynthesis by GLS is summarized, while the in vitro synthesis of major polysaccharides is also discussed, catalyzed by glycoside hydrolases (GHs) and GTs. Possible future prospects of GLS in medicine and in developing other potential artificial composite materials with industrial applications are also summarized

  • 24.
    Zerva, Anastasia
    et al.
    Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, Athens, Greece.
    Koutroufini, Efthymia
    Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, Athens, Greece.
    Kostopoulou, Ioanna
    Laboratory of Organic Chemistry, School of Chemical Engineering, National Technical University of Athens, Athens, Greece.
    Detsi, Anastasia
    Laboratory of Organic Chemistry, School of Chemical Engineering, National Technical University of Athens, Athens, Greece.
    Topakas, Evangelos
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    A novel thermophilic laccase-like multicopper oxidase from Thermothelomyces thermophila and its application in the oxidative cyclization of 2′,3,4-trihydroxychalcone2019In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 49, p. 10-18Article in journal (Refereed)
    Abstract [en]

    Laccase-like multicopper oxidases (LMCOs) are a heterogeneous group of oxidases, acting mainly on phenolic compounds and which are widespread among many microorganisms, including Basidiomycetes and Ascomycetes. Here, we report the cloning, heterologous expression, purification and characterization of a novel LMCO from the thermophilic fungus Thermothelomyces thermophila. The 1953 bp lmco gene sequence comprises of 3 exons interrupted by 2 introns and according to the LccED database the translated sequence belongs to superfamily 6 of multicopper oxidases. After removal of the introns, the gene was transformed into Pichia pastoris, under the control of the alcohol oxidase (AOX1) promoter. The heterologous enzyme was purified with an apparent molecular weight of 80 kDa. TtLMCO1 displayed optimum activity at pH 4 and 50 °C and appeared thermostable up to 50 °C. A variety of phenolic compounds were oxidized by TtLMCO1, including standard laccase substrates such as ABTS and 2,6 dimethoxyphenol. The UV/Vis spectrum of purified TtLMCO1 indicates that it belongs to yellow laccase-like oxidases. The enzyme was used for the bioconversion of 2′,3,4-trihydroxychalcone to 3′,4′-dihydroxy-aurone, a bioactive aurone recently shown to possess inhibitory activity against several isoforms of the histone deacetylase complex (HDAC). Overall, the thermophilic yellow LMCO TtLMCO1 presents a number of superior properties with potential use in industrial biocatalysis.

  • 25.
    Zerva, Anastasia
    et al.
    Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Manos, Nikolaos
    Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Vouyiouka, Stamatina
    National Technical University of Athens, Laboratory of Polymer Technology, School of Chemical Engineering, National Technical University of Athens.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Topakas, Evangelos
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Bioconversion of Biomass-Derived Phenols Catalyzed by Myceliophthora thermophila Laccase2016In: Molecules, ISSN 1431-5157, E-ISSN 1420-3049, Vol. 21, no 5, article id 550Article in journal (Refereed)
    Abstract [en]

    Biomass-derived phenols have recently arisen as an attractive alternative for building blocks to be used in synthetic applications, due to their widespread availability as an abundant renewable resource. In the present paper, commercial laccase from the thermophilic fungus Myceliophthora thermophila was used to bioconvert phenol monomers, namely catechol, pyrogallol and gallic acid in water. The resulting products from catechol and gallic acid were polymers that were partially characterized in respect to their optical and thermal properties, and their average molecular weight was estimated via solution viscosity measurements and GPC. FT-IR and 1H-NMR data suggest that phenol monomers are connected with ether or C–C bonds depending on the starting monomer, while the achieved molecular weight of polycatechol is found higher than the corresponding poly(gallic acid). On the other hand, under the same condition, pyrogallol was dimerized in a pure red crystalline compound and its structure was confirmed by 1H-NMR as purpurogallin. The herein studied green synthesis of enzymatically synthesized phenol polymers or biological active compounds could be exploited as an alternative synthetic route targeting a variety of applications.

  • 26.
    Zerva, Anastasia
    et al.
    Industrial Biotechnology & Biocatalysis Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, Athens, Greece.
    Pentari, Christina
    Industrial Biotechnology & Biocatalysis Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, Athens, Greece.
    Ferousi, Christina
    Industrial Biotechnology & Biocatalysis Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, Athens, Greece.
    Nikolaivits, Efstratios
    Industrial Biotechnology & Biocatalysis Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, Athens, Greece.
    Karnaouri, Anthi
    Industrial Biotechnology & Biocatalysis Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, Athens, Greece.
    Topakas, Evangelos
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering. Industrial Biotechnology & Biocatalysis Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, Athens, Greece.
    Recent advances on key enzymatic activities for the utilisation of lignocellulosic biomass2021In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 342, article id 126058Article, review/survey (Refereed)
    Abstract [en]

    The field of enzymatic degradation of lignocellulose is actively growing and the recent updates of the last few years indicate that there is still much to learn. The growing number of protein sequences with unknown function in microbial genomes indicates that there is still much to learn on the mechanisms of lignocellulose degradation. In this review, a summary of the progress in the field is presented, including recent discoveries on the nature of the structural polysaccharides, new technologies for the discovery and functional annotation of gene sequences including omics technologies, and the novel lignocellulose-acting enzymes described. Novel enzymatic activities and enzyme families as well as on accessory enzymes and their synergistic relationships regarding biomass breakdown are described. Moreover, it is shown that all the valuable knowledge of the enzymatic decomposition of plant biomass polymers can be employed towards the decomposition and upgrading of synthetic polymers, such as plastics.

  • 27.
    Zerva, Anastasia
    et al.
    InduBioCat Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 5 Iroon Polytechniou Str., Zografou Campus, 15780 Athens, Greece.
    Pentari, Christina
    InduBioCat Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 5 Iroon Polytechniou Str., Zografou Campus, 15780 Athens, Greece.
    Topakas, Evangelos
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering. InduBioCat Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 5 Iroon Polytechniou Str., Zografou Campus, 15780 Athens, Greece.
    Crosslinked Enzyme Aggregates (CLEAs) of Laccases from Pleurotus citrinopileatus Induced in Olive Oil Mill Wastewater (OOMW)2020In: Molecules, ISSN 1431-5157, E-ISSN 1420-3049, Vol. 25, no 9, article id 2221Article in journal (Refereed)
    Abstract [en]

    The enzymatic factory of ligninolytic fungi has proven to be a powerful tool in applications regarding the degradation of various types of pollutants. The degradative potential of fungi is mainly due to the production of different types of oxidases, of which laccases is one of the most prominent enzymatic activities. In the present work, crude laccases from the supernatant of Pleurotus citrinopileatus cultures grown in olive oil mill wastewater (OOMW) were immobilized in crosslinked enzyme aggregates (CLEAs), aiming at the development of biocatalysts suitable for the enzymatic treatment of OOMW. The preparation of laccase CLEAs was optimized, resulting in a maximum of 72% residual activity. The resulting CLEAs were shown to be more stable in the presence of solvents and at elevated temperatures compared to the soluble laccase preparation. The removal of the phenolic component of OOMW catalyzed by laccase-CLEAs exceeded 35%, while they were found to retain their activity for at least three cycles of repetitive use. The described CLEAs can be applied for the pretreatment of OOMW, prior to its use for valorization processes, and thus, facilitate its complete biodegradation towards a consolidated process in the context of circular economy.

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