Enzyme-magnetic nanoparticle reactor for the advanced oxidation of micropollutants in wastewaters

TESIS DOCTORAL Enzyme-magnetic nanoparticle reactor for the advanced oxidation of micropollutants in wastewaters Yolanda Moldes Diz ESCUELA DE DOCTORADO INTERNACIONAL PROGRAMA DE DOCTORADO EN INGENIERIA QUÍMICA Y AMBIENTAL SANTIAGO DE COMPOSTELA 2019 DECLARACIÓN DEL AUTOR DE LA TESIS Enzyme-Magnetic Nanoparticle Reactor for the advanced oxidation of micropollutants in wastewaters D./Dña. Yolanda Moldes Diz Presento mi tesis, siguiendo el procedimiento adecuado al Reglamento, y declaro que: 1) La tesis abarca los resultados de la elaboración de mi trabajo. 2) En su caso, en la tesis se hace referencia a las colaboraciones que tuvo este trabajo. 3) La tesis es la versión definitiva presentada para su defensa y coincide con la versión enviada en formato electrónico. 4) Confirmo que la tesis no incurre en ningún tipo de plagio de otros autores ni de trabajos presentados por mí para la obtención de otros títulos. En Santiago de Compostela, 11 de Octubre de 2019 Fdo. Yolanda Moldes Diz AUTORIZACIÓN DEL DIRECTOR / TUTOR DE LA TESIS Enzyme-Magnetic Nanoparticle Reactor for the advanced oxidation of micropollutants in wastewaters Prof. María Teresa Moreira Vilar Prof. Gumersindo Feijoo Costa INFORMA/N: Que la presente tesis, corresponde con el trabajo realizado por D/Dña. Yolanda Moldes Diz, bajo nuestra dirección, y autorizamos su presentación, considerando que reúne los requisitos exigidos en el Reglamento de Estudios de Doctorado de la USC, y que como directores de ésta no incurre en las causas de abstención establecidas en Ley 40/2015. En Santiago de Compostela, 11 de Octubre de 2019 Fdo. María Teresa Moreira Vilar Fdo. Gumersindo Feijoo Costa Resumen El continuo aumento de la población humana y el crecimiento económico están llevando a una demanda continua de agua. La contaminación química de las aguas naturales se ha convertido ya en una preocupación pública de primer orden en todo el mundo, ya que sus efectos a largo plazo sobre la vida acuática y la salud humana son en gran medida desconocidos. Por lo tanto, la protección de los recursos hídricos contra la explotación del agua y la contaminación antropogénica con productos químicos y patógenos es indispensable para preservar la salud ecológica y humana. La sociedad moderna se beneficia del elevado número de productos químicos disponibles para diversas aplicaciones en la industria, la agricultura, la atención sanitaria y el saneamiento, así como para fines domésticos. En la Unión Europea hay más de 100.000 sustancias químicas registradas, de las cuales entre 30.000 y 70.000 son de uso diario. La mayoría de estos productos químicos llegan a las aguas naturales principalmente a través de las corrientes de aguas residuales municipales e industriales, los derrames de productos químicos y las emisiones difusas de las actividades agrícolas. En la actualidad, la aparición nuevos métodos analíticos con límites de detección más bajos han permitido prevenir de la presencia de contaminantes emergentes en efluentes de aguas potables, superficiales y residuales de todo el mundo. Este tipo de compuestos y su eliminación suponen un desafío científico-tecnológico, sobre todo si se tiene en cuenta el hecho de que las plantas de tratamiento y depuración de aguas se diseñaron sin considerar la presencia de estos contaminantes. Por ello, el desarrollo de procesos que permitan eliminar este tipo de compuestos se sitúa entre las líneas de investigación prioritarias de los principales organismos dedicados a la protección de la salud pública y medioambiental, tales como la Organización Mundial de la Salud (OMS), la Agencia de Estados Unidos para la Protección del Medio Ambiente (US EPA) o la Agencia Europea de Medio Ambiente (EEA). La creciente demanda de aguas más limpias por parte de los ciudadanos y las organizaciones medioambientales ha impulsado a la Comisión Europea a definir una lista de sustancias prioritarias con un riesgo significativo para el medio acuático. La lista actualizada incluye microcontaminantes de diferentes categorías: disruptores endocrinos, como el bisfenol, y productos farmacéuticos, en particular From the outcomes of this research, the target transformation reactions will be later developed on the basis of the selected nanobiocatalyst. CHAPTER 3: Superparamagnetic nanobiocatalyst to biotransform micropollutants from wastewater A nanobiocatalyst developed in the previous chapter will be evaluated in terms of stability against different pH, temperature and inhibiting compounds, and benchmarked with the values obtained for free laccase. In addition, its reusability will be demonstrated in consecutive oxidation cycles of Bisphenol A, estradiol and methyl green, which were selected as model compounds present in wastewater. It is also interesting to assess the toxicity associated to the degradation products from the enzymatic treatment that will be compared with the parent molecules. CHAPTER 4: An eco-friendly nanobiocatalyst: Pycnoporus sanguineus laccase for environmental applications This chapter focuses on the interest of using agro-industrial waste to produce laccase from Pycnoporus sanguineus as sources of natural carbon. The produced enzyme will be immobilized on the superparamagnetic silica-coated nanoparticles to evaluate the immobilization yields and stability. The nanobiocatalyst will be used in the biotransformation of Bisphenol A and methyl green in consecutive cycles. Finally, the potential toxicity of the nanobiocatalyst and the resulting medium will be checked. CHAPTER 5: Superparamagnetic nanobiocatalyst in oxidation technologies for added value bio-based products In this chapter two oxidoreductases, galactose oxidase and aryl alcohol oxidase, will be immobilized on the superparamagnetic silica-coated nanoparticles and used to perform cascade reactions to produce high valueadded products from 5-Hydroxymethylfurfural (HMF). In addition, the genotoxicity of the nanobiocatalyst will be studied to demonstrate the potential DNA damage, condition suggesting that the nanobiocatalyst is safe or not to be used in different biotechnological applications. CHAPTER 6: Development of an enzymatic reactor with internal magnetic separation for biotechnological applications A new sequential batch reactor will be designed, coupled to an internal magnetic separation system. The separator consists of a series of axially magnetized toroidal magnets, distributed along a non-magnetic steel bar and with alternate polarity, which generates an external magnetic field of up to 1.2 T. The proof of concept of the reactor will be conducted by evaluating the transformation of methyl green and HMF, with complete recovery of the nanobiocatalyst. Finally, the reactor will be scaled to a volume of 100-L to estimate costs and environmental impact. CHAPTER 7: Comparative life cycle assessment of different synthesis routes of magnetic nanoparticles In this chapter the environmental assessment of the different routes of synthesis of magnetic nanoparticles used as supports for immobilization will be carried out using the perspective of Life Cycle Analysis (LCA). The complexity of the production process for the nanoparticles suggests that a compromise must be adopted between efficiency and associated environmental impact. Index RESUMEN OBJECTIVES CHAPTER 1 1.1. OXIDOREDUCTASES ......................................................................................... 4 1.2. COPPER-CONTAINING OXIDASES: LACCASES .................................................. 4 1.3. FLAVIN-CONTAINING OXIDASES ...................................................................... 6 1.4. BASIDIOMYCETE PEROXYGENASE: UNSPECIFIC PEROXIGENASE .................... 6 1.5. INDUSTRIAL APPLICATIONS ............................................................................. 6 1.5.1. CONCERN ON THE EFFECT OF EMERGING CONTAMINANTS...................... 7 1.6. ENZYMATIC IMMOBILIZATION ........................................................................ 8 1.6.1. METHODS OF IMMOBILIZATION ................................................................ 9 1.7. NANOPARTICLES AS A SUPPORT FOR IMMOBILIZATION ................................ 11 1.8. OPENING THE SCOPE: BIOTECHNOLOGICAL APPLICATIONS OF ENZYMES ..... 13 1.9. ENZYMATIC REACTORS .................................................................................... 15 1.10. ENZYME-AIDED PROCESSES AS A WAY OF REDUCING THE ENVIRONMENTAL IMPACTS .................................................................................. 16 1.11. REFERENCES .................................................................................................. 16 CHAPTER 2 2.1. INTRODUCTION ............................................................................................... 26 2.2. MATERIALS AND METHODS ............................................................................ 27 2.2.1. CHEMICALS FOR ENZYME IMMOBILIZATION ............................................. 27 2.2.2. ENZYMES ..................................................................................................... 28 2.2.3. SUPPORTS FOR IMMOBILIZATION .............................................................. 29 2.2.4. DETERMINATION OF ENZYMATIC ACTIVITY ............................................... 30 2.2.5. SELF-IMMOBILIZATION OF ENZYMES BY CROSS-LINKING AGGREGATES (CLEAS) ............................................................................................ 32 2.2.6. IMMOBILIZATION OF ENZYMES ON EPOXY AND AGAROSE-GLIOXYL BASED SUPPORTS ................................................................................................... 33 2.2.7. IMMOBILIZATION OF ENZYMES ON MAGNETIC NANOPARTICLES ............. 33 2.3. RESULTS ........................................................................................................... 35 Index 2.3.1. IMMOBILIZATION OF ENZYMES BY CLEAS ................................................. 35 2.3.2. IMMOBILIZATION OF ENZYMES ON SUPPORTS ......................................... 38 2.4. CONCLUSIONS ................................................................................................ 42 2.5. REFERENCES .................................................................................................... 42 CHAPTER 3 3.1. INTRODUCTION .............................................................................................. 50 3.2. MATERIALS AND METHODS ............................................................................ 52 3.2.1. CHEMICALS FOR SYNTHESIS OF NANOPARTICLES AND ENZYME IMMOBILIZATION .................................................................................................. 52 3.2.2. PREPARATION AND CHARACTERIZATION OF MAGNETIC NANOPARTICLES .................................................................................................... 53 3.2.3. CHARACTERIZATION OF MAGNETIC NANOPARTICLES 53 3.2.4. IMMOBILIZATION OF LACCASE ONTO SILICA-COATED MAGNETIC NANOPARTICLES .................................................................................................... 54 3.2.5. INFLUENCE OF PH, T, AND INACTIVATING COMPOUNDS ON THE RELATIVE ACTIVITY AND STABILITY OF FREE AND IMMOBILIZED LACCASE .......... 54 3.2.6. REGENERATION OF THE SUPPORT ............................................................. 55 3.2.7. BIOTRANSFORMATION OF THE TARGET POLLUTANTS .............................. 56 3.2.8. BIOTRANSFORMATION OF THE TARGET POLLUTANTS IN SEQUENTIAL BATCH REACTORS .................................................................................................. 56 3.2.9. EVALUATION OF TOXICITY ......................................................................... 57 3.3. RESULTS AND DISCUSSION ............................................................................. 58 3.3.1. CHARACTERIZATION OF SILICA-COATED IRON OXIDE NANOPARTICLES ... 58 3.3.2. IMMOBILIZATION OF LACCASE ONTO SILICA-COATED MAGNETIC NANOPARTICLES .................................................................................................... 60 3.3.3. INFLUENCE OF PH, T AND INACTIVATING COMPOUNDS ON THE RELATIVE ACTIVITY AND STABILITY OF FREE AND IMMOBILIZED LACCASE .......... 61 3.3.4. REGENERATION OF THE SUPPORT ............................................................. 65 3.3.5. BIOTRANSFORMATION OF THE TARGET POLLUTANTS .............................. 65 3.3.6. BIOTRANSFORMATION OF BPA, E2 AND MG BY LACCASE IMMOBILIZED ON MAGNETIC NANOSUPPORT IN SEQUENTIAL BATCH REACTION ..................... 69 3.3.7. EVALUATION OF TOXICITY ......................................................................... 70 Index 3.4. CONCLUSIONS ................................................................................................. 71 3.5. REFERENCES .................................................................................................... 71 CHAPTER 4 4.1. INTRODUCTION ............................................................................................... 82 4.2. MATERIALS AND METHODS ............................................................................ 83 4.2.1. CHEMICALS AND NANOPARTICLES ............................................................. 83 4.2.2. MICROORGANISM AND CULTURE CONDITIONS . 83 4.2.3. LACCASE PURIFICA TION .............................................................................. 83 4.2.4. NANOBIOCATALYST CHARACTERIZATION .................................................. 84 4.2.5. BIOTRANSFORMATION OF BISPHENOL A AND METHYL GREEN BY FREE AND IMMOBILIZED ENZYME .................................................................................. 84 4.2.6. REUSE OF THE NANOBIOCATALYST IN A SEQUENTIAL BATCH REACTOR ... 84 4.2.7. BPA AND MG ANALYSIS .............................................................................. 85 4.2.8. TOXICITY EVALUATION ............................................................................... 85 4.3. RESULTS AND DISCUSSION .............................................................................. 86 4.3.1. HIGH PRODUCTION RATES OF PYCNOPORUS SANGUINEUS LACCASE ....... 86 4.3.2. STABLE AND ROBUST NANOBIOCATALYST BY EFFICIENTLY IMMOBILIZING OF PYCNOPORUS SANGUINEUS ONTO SMNP ............................... 87 4.3.3. MULTIPOINT COVALENT IMMOBILIZATION AND ENZYME SHELL DISTRIBUTION ON SMNP WERE CONFIRMED ........................................................ 89 4.3.4. THE NANOBIOCATALYST WAS ABLE TO BIOTRANSFORM INDUSTRIAL WASTEWATERS AS RESIN INTERMEDIATE BPA OR TEXTILE DYE MG ..................... 91 4.3.5. REUSABLE AND NO TOXIC SUPERPARAMAGNETIC NANOBIOCATALYST FOR INDUSTRIAL WASTEWATER APPLICATIONS .................................................... 93 4.3.6. SUCCESSFUL DETOXIFICATION OF THE TREATED WASTEWATER ............... 94 4.4. CONCLUSIONS ................................................................................................. 95 4.5. REFERENCES .................................................................................................... 95 CHAPTER 5 5.1. INTRODUCTION ............................................................................................... 104 5.2. MATERIALS AND METHODS ............................................................................ 105 5.2.1. CHEMICALS, NANOPARTICLES AND ENZYMES ........................................... 105 Index 5.2.2. IMMOBILIZATION OF GAO AND AAO ON SUPERPARAMAGNETIC SILICA COATED NANOPARTICLES...................................................................................... 105 5.2.3. HMF BIOTRANSFORMATION WITH GAO AND AAO ................................... 105 5.2.4. CONSECUTIVE CYCLES OF BATCH BIOTRANSFORMATION OF 5-HMF BY AAO IMMOBILIZED ON SMNP ............................................................................... 106 5.2.5. ANALYSIS OF 5-HMF TRANSFORMATION PRODUCTS ............................... 106 5.2.6. GENOTOXICITY OF THE NANOBIOCATALYST ............................................. 106 5.3. RESULTS AND DISCUSSION ............................................................................. 107 5.3.1. HMF BIOTRANSFORMATION TRANSFORMATION BY GAO AND AAO ....... 107 5.3.2. BIOTRANSFORMATION OF 5-HMF BY AAO IMMOBILIZED ON SILICA COATED SUPERPARAMAGNETIC NANOPARTICLES IN SEQUENTIAL BATCH REACTION .............................................................................................................. 109 5.3.3. EVALUATION OF GENOTOXICITY ............................................................... 109 5.4. CONCLUSIONS ................................................................................................ 110 5.5. REFERENCES .................................................................................................... 111 CHAPTER 6 6.1. INTRODUCTION .............................................................................................. 116 6.2. MATERIALS AND METHODS ............................................................................ 117 6.2.1. CHEMICALS, ENZYMES AND NANOPARTICLES ........................................... 117 6.2.2. DECOLORIZATION OF MG BY LACCASE IMMOBILIZED ONTO SILICA-COATED MNPS ................................................................................. 117 6.2.3. DEVELOPMENT AND MODELLING OF THE MAGNETIC SEQUENTIAL BATCH REACTOR (SBR) .......................................................................................... 118 6.2.4. BIOTRANSFORMATION OF MG AND HMF IN THE MAGNETIC SBR BY LACCASE IMMOBILIZED ONTO SILICA-COATED MAGNETIC NANOPARTICLES ...... 120 6.2.5. ENVISIONING THE BIOTRANSFORMATION OF MG PRESENT IN A TEXTILE EFFLUENT .............................................................................................................. 120 6.2.6. IDENTIFICATION OF LACCASE-CATALYZED REACTION PRODUCTS FROM MG DECOLORIZATION ........................................................................................... 121 6.2.7. TOXICITY AND BIODEGRADABILITY ASSAYS ............................................... 121 6.2.8. ENVIRONMENTAL PERFORMANCE AND COST ANALYSIS .......................... 122 6.3. RESULTS AND DISCUSSION ............................................................................. 123 Index 6.3.1. CHARACTERIZATION OF THE MAGNETIC REACTOR AND MODELLING OF THE MAGNETIC FIELD ............................................................................................. 123 6.3.2. DECOLORIZATION OF MG BY LACCASE IMMOBILIZED ONTO SILICACOATED MAGNETIC NANOPARTICLES .................................................................... 125 6.3.3. SEQUENTIAL BATCH REACTOR FOR THE BIOTRANSFORMATION OF METHYL GREEN AND HMF BY LACCASE IMMOBILIZED ONTO MAGNETIC NANOPARTICLES ..................................................................................................... 127 6.3.4. ENVISIONING THE BIOTRANSFORMATION OF MG PRESENT IN TEXTILE EFFLUENT ............................................................................................ 129 6.3.5. EVALUATION OF MG BIOTRANSFORMATION PRODUCTS .......................... 130 6.3.6. TOXICITY AND BIODEGRADABILITY OF THE BIOTRANSFORMATION PRODUCTS .............................................................................................................. 135 6.3.7. ENVIRONMENTAL PERFORMANCE AND COST ANALYSIS ........................... 137 6.4. CONCLUSIONS ................................................................................................. 140 6.5. REFERENCES .................................................................................................... 140 CHAPTER 7 ...................................................................................................... 145 7.1. INTRODUCTION ............................................................................................... 148 7.2. MATERIALS AND METHODS ............................................................................ 149 7.2.1. GOAL AND SCOPE DEFINITION ................................................................... 149 7.2.2. DESCRIPTION OF THE MNPS PRODUCTION SCENARIOS............................. 151 7.2.3. INVENTORY DATA ....................................................................................... 155 7.2.4. IMPACT ASSESSMENT METHODOLOGY ..................................................... 157 7.3. ENVIRONMENTAL RESULTS ............................................................................. 157 7.4. CONCLUSIONS ................................................................................................. 175 7.5. REFERENCES .................................................................................................... 176 GENERAL CONCLUSIONS ................................................................................. 183 PUBLICATIONS ................................................................................................ 189 ACKNOWLEDGEMENTS ................................................................................... 195 Chapter 1 Introduction Oxidative biocatalysts by oxidoreductases arises as a promising alternative from the development of advanced oxidation processes for transformation of emerging contaminants to transformation of compounds into value added bio-based products. For technical and economic reasons, in most enzyme catalysed processes it is necessary to reuse the biocatalyst. In this context, enzyme immobilization can be defined as a technique that allows reuse or continued use of the biocatalyst. Envisaging the application of the different biocatalysts in wastewater treatment plants or other biotechnological applications it is important to design an enzymatic reactor in which the recovery and reuse of the enzyme is fulfilled. Oxidoreductases Wastewater treatments Immobilization? Chapter 1 8 production of safe drinking water and the environmentally responsible release of wastewater (Bolong et al. 2009). The presence of metals, bacteria, hydrocarbons or other ions like nitrates, ammonia in water are described for several decades and their impact on human health and the environment are known; these contaminants are subject to regulation and control. But the occurrence and effects of phthalates, pharmaceuticals compounds and endocrine disrupting compounds (such as bisphenol A) is often not available. They are originated from industry or from the discharge in wastewaters. The problem of emerging pollutants is the lack of knowledge of their impact in the middle or long-term effect on human health, the environment and aquatic environments. The degradation of organic pollutants implies an important challenge as they may have complex structure and/or low bioavailability. Moreover, conventional WWTPs are not primarily designed to remove them (Deblonde et al. 2011). Treatment options which are typically considered for the removal of emerging contaminants from drinking water as well as wastewater include adsorption, advanced oxidation processes (AOPs), nanofiltration (NF), and reverse osmosis (RO) membranes (Grassi et al. 2012). Advanced treatment technologies such as ozonization, UV irradiation and activated carbon adsorption have been suggested to increase the removal of such compounds (Liu et al. 2009) but a full-scale implementation would require significant investment and increased operation costs. In this sense, the costs need to be weighed against the possible benefits of introducing new technologies. As a result, there are still need to develop and further examines new alternatives to efficiently remove these compounds from wastewater. 1.6. Enzymatic immobilization For technical and economic reasons, in most enzyme catalysed processes it is necessary to reuse or continuously use the biocatalyst for a prolonged period of time (Katchalski-Katzir et al., 2000). In this context, enzyme immobilization can be defined as a technique that allows reuse or continued use of the biocatalyst. In addition, immobilization allows for greater stability and longer enzyme storage time (Lloret et al., 2011). General introduction 9 1.6.1. Methods of immobilization There are a large number of immobilization methods and since the method used greatly influences the properties of the resulting biocatalyst, the selection of the immobilization strategy determines the process specifications for the catalyst (Durán et al., 2002). A classification of the main methods may be the following (Illanes et al., 2008):  Enzyme entrapment by physical retention in the inner cavities of a solid matrix such as polyacrylamide, collagen or alginate  Encapsulation of the enzyme by physical retention in synthetic membranes  Immobilization of the enzyme by cross-linking with bifunctional reagents  Immobilization in supports by physical, ionic or covalent bonds Entrapment Entrapment is defined as the physical retention of enzymes in a solid porous matrix, such as polyacrylamide, collagen, alginate, or gelatin. The enzyme is suspended in a monomer solution and a subsequent polymerization process keeps the enzyme trapped, avoiding direct contact with the environment. It is the simplest method of immobilization and there are no alterations in the enzymatic structure. However, this methodology has limitations in terms of mass transfer and also has a low enzyme load (Fernández-Fernandez et al., 2013). Encapsulation The enzyme is retained by a membrane that allows the passage of substrates and reaction products (Illanes et al., 2008). Retention can be achieved by microencapsulation in semi-permeable membranes or by adsorption of the enzyme onto a membrane that will form the reactor. As in the previous case, the enzyme is protected from the environment and the mass transfer represents an important limitation (Fernández-Fernández et al., 2013). Cross-linking The use of solid supports for enzymatic immobilization can reduce the activity of the biocatalyst. Cross-linking is an alternative in which immobilization is carried out without support, achieved through the use of a bifunctional cross-linker agent. Chapter 1 10 Cross-linkers include dialdehydes, diiminosteres, diisocyanates and diamines activated by carbodiimide (Fernández-Fernández et al., 2013). Despite achieving greater activity and remaining stable in extreme conditions, they present deficiencies in mechanical properties and a high cost in protein purification (Illanes et al., 2008). Immobilization on supports Different types of materials have been used as supports for the immobilization of enzymes, both organic and inorganic compounds. The main properties of the substrates are: large surface area, high bonding capacity with the enzyme, compatibility and insolubility in the reaction medium, mechanical and chemical stability, recovery after use and conformational flexibility. There is not a support that fulfills all the characteristics reason why this one is chosen depending on the requirements of the process (Illanes et al., 2008). In the present work, immobilization will be considered according to two methods of immobilization: adsorption and covalent bond (Fernández-Fernández et al., 2013). Both methods will be explained in more detail below. Adsorption The adsorption of enzymes on a substrate is based on ionic bonds and other weak attraction forces. Adsorption is a relatively simple and low-cost method of immobilization, making it potentially more competitive than other methodologies. Variables such as pH, ionic strength of the medium and hydrophobicity of the support must be taken into account in the immobilization process (FernándezFernández et al., 2013). In spite of obtaining a high immobilization yield, in this method the enzyme is easily desorbed, especially in aqueous solutions (Illanes et al., 2008). Covalent bond Immobilization by covalent bond is the most interesting method in industrial applications. Therefore, covalent bonding has become the most widely used method of immobilization. In this technique, the chemical groups on the surface of the support are activated and react with the nucleophilic groups of the protein. Different supports have been studied for the immobilization of laccase by covalent bond, from silica supports, epoxy resins, gold, silver and magnetic supports, among others (Fernández-Fernández et al., 2013). In the present work, magnetic General introduction 11 nanoparticles will be used as supports both in the case of covalent bonding and in adsorption. 1.7. Nanoparticles as a support for immobilization The rapid development of nanotechnology has provided a basis for innovation in a wide range of fields, such as health, agriculture, food, transport, environment, electronics and communications, resulting in a significant increase in research into new nanomaterials (Figura 1.3) (Grillo et al., 2015). Figure 1.3. Number of publications on nanoparticles over time (ISI Web of Knowledge) According to the European Committee for Standardisation (CEN), nanomaterials are defined as materials with any external dimension at the nanoscale, or having internal or surface nanoscale structures. The nanoscale size range is between 1 and 100 nm (ISO/TS 27687:2008) (JRC, 2010). Nanoparticles (NPs), the most studied field within nanomaterials, have external dimensions of the order of 100 nm or less (JRC, 2010). NPs may be spherical, tubular, or irregularly shaped, and may exist as aggregates, fused or agglomerated (Nowack et al., 2007). There are two main types: engineered nanoparticles (ENP) and non-engineered nanoparticles. Non-engineered NPs are present in the environment in natural phenomena such as dust storms, erosion, volcanic eruptions and forest fires (Nowack et al., 2007; Cupaioli et al., 2014). ENPs are intentionally produced by humans as metals (including Ag, Zn, Au, Ni, Fe, and Chapter 1 12 Cu) (Kaegi et al., 2013), metal oxides (TiO2, Fe3O4, SiO2, CeO2 and Al2O3) (BozonVerduraz et al., 2009), non-metallic (silica and quantum dots) (Probst et al., 2013), carbon nanotubes and fullerene) (Isaacson et al., 2009; Ma et al., 2010), polymers (alginate, chitosan and polyhydroxyalkanoates) (Rao et al., 2011), and lipids (Faraji et al., 2009). Nanomaterials can be used as support in enzymatic immobilization thanks to their properties such as surface area, resistance to mass transfer and others (Feng et al., 2011). Table 1.2 summarizes the advantages and disadvantages of the use of nanomaterials for enzymatic immobilization. Table 1.2. Advantages and disadvantages of nanoinmobilisation (Cipolatti et al., 2014) Advantages Disadvantages Mass transfer resistance Cost of the process High enzyme load Large scale application High surface area High mechanical resistance Minimization of diffusion problems Separation of the reaction medium In recent decades, superparamagnetic particles of nanometric size (maghemite, γ-Fe3O4 or magnetite, Fe3O4) have been widely studied both in biology and medicine in areas such as magnetic resonance, DNA and RNA purification and immobilization of proteins and enzymes (Cipolatti et al., 2014). Immobilization in nanomaterials offers numerous advantages in terms of separation as it is possible to separate them by physical methods, such as filtration or sedimentation (centrifugation) and they can be reused. Magnetic nanoparticles as media provide a simple and fast recovery of the biocatalyst by applying an external magnetic field and, compared to centrifugation or sedimentation, are subjected to much less mechanical stress. In addition, the use of magnetic nanoparticles as media has the following advantages: greater specific surface area to achieve greater enzyme load, lower resistance to mass transfer, less fouling, less diffusion problems and reduced operational cost (Kalkan et al., 2011). Based on the above, it is interesting to evaluate the immobilization of oxidorreductases in magnetic nanoparticles for the elimination of endocrine General introduction 13 disrupting compounds and the production of high value-added compounds in order to gain experience for the development of a possible future process on an industrial scale. 1.8. Opening the scope: Biotechnological applications of enzymes Beyond the perspective of degradation of target contaminants, the use of an enzyme emerges as a biocatalyst in conversion/transformation processes. Accordingly, the oxidation process based on the use of oxidases and peroxidases may be of special interest in the case that the objective pursued is the transformation of substrates based on synthesis reactions. Several industries and research/academic groups have demonstrated the feasibility of using new or improved (i.e. genetically modified) oxidative enzymes in the production of value-added compounds from molecules derived from biomass (such as sugars and lipids) to replace others of petrochemical origin and/or to develop more ecological and specific transformations. Of special interest in this field are several biochemical reactions of oxidation and oxyfunctionalization by fungal oxidases (Dijkman et al., 2013; Hernández-Ortega et al., 2012) and peroxidases, including new hemetiolate biocatalysts with monooxygenase activity (peroxygenases) (Bormann et al., 2015; Hofrichter and Ullrich, 2014). These peroxygenases are excellent biocatalysts for the regal and stereoscopic oxyfunctionalization of a variety of organic compounds. These enzymes also have the advantage of being stable (due to their secreted nature) and self-sufficient (Hofrichter and Ullrich, 2006), in the sense that they do not require the activation of a reducing power source and/or an auxiliary flavoenzyme/domain as in the case of other monooxygenases, such as cytochromes P450 (P450s) and flavinoxygenases (Kim and Oh, 2013). In this context, this thesis evaluates the exploratory biochemical conversion of 5-hydroxymethylfurfural (HMF) into diformylfurane, a platform chemical, and 2,5furandicarboxylic acid (FDCA), a basic plastic component. Oxidases and peroxygenases will be used to perform three-stage oxidation of HMF to FDCA in a single pot conversion without substrate and without by-products. FDCA (and HMF) occupied one of the first positions in the ranking of most interesting bio-based chemical building blocks published by the US Department of Energy in 2004 (Werpy Chapter 1 14 and Petersen, 2004), that they still maintain in similar studies published several years after (Bozell and Petersen, 2010; IAR, 2015a; 2015b). Concerning FDCA production, the possibility to contribute to substitution of non-renewable PET by biomass-based PEF by using an enzymatic technology is a desirable goal, making the process more efficient and competitive. PEF itself has several advantages with respect to PET, including 50% better CO2 footprint, several folds better O2 and CO2 permeability, higher density, and higher field strength, among others. The main advantage of the bio-chemical technology (using oxidases and/or peroxygenases) for PEF production is, in addition to the cost reduction due to milder operation conditions (high pressure and temperature is required when using Co/Mn/Br catalysts), the absence of the undesirable by-products formed due to the low selectivity of the current chemical technology for FDCA production (de Diego et al., 2011). Such by-products include monocarboxylic acids that cause chain termination reactions in the intended polymer applications, and require greater energy and capital expenditure for product purification (van Putten et al., 2013). Once optimized, the enzymatic conversion will circumvent the above problem due to its high selectivity. Biotechnological production of FDCA by selected Pseudomonas strains has been proposed as an alternative to the chemical conversion in several studies of Corbion (a leader company in the lactic acid sector, which is also interested in PEF production) (Wierckx et al., 2015). However, due to different issues -including the need of a carbon and nitrogen source in the microbial transformation and the toxicity of furfural products for microorganisms - the biochemical process based on oxidative enzymes would be undoubtedly superior once optimized. This thesis aims to provide innovative answers to develop a new generation of biochemical technologies for future biorefineries, allowing the transformation of molecules of plant origin with exquisite regio-selectivity and stereoselectivity that cannot be achieved by classical chemical technologies. Due to the above characteristics, the biochemical technologies to be developed will represent a real advance in lignocellulose biorefineries, improving competitiveness and allowing the development of new production routes. These oxidation/oxyfunctionalization reactions by oxidases and peroxygenases will allow a more integral and efficient use of the different types of feedstocks. General introduction 15 1.9. Enzymatic reactors Envisaging the application of the different magnetic nanobiocatalysts in wastewater treatment plants or other biotechnological applications it is important to design an enzymatic reactor in which the recovery and reuse of the enzyme is fulfilled. In previous works, different systems based on the use of free enzyme or immobilized systems were proposed. The first approach relies on the use of an enzymatic membrane reactor (EMR), which comprises a CSTR coupled with a semipermeable ultrafiltration membrane to ensure the retention of free biocatalyst. This system has been successfully applied for the enzymatic decolorization of dyes (López et al.2011) and also for the removal of endocrine disrupting compounds from filtered-secondary wastewater effluent. Laccase was highly stable during 100 h of operation until the reactor was discontinued. Alternatively, to the use of free enzyme, the use of immobilized laccase to facilitate its retention allowing the separation of the enzyme from the reaction medium, so that the biocatalyst can be applied in continuous systems and it can be beneficial for enzyme stability and prolonged storage (Lloret et al. 2011). Although laccase showed high stability during the operation of the reactor and the pollutants were efficiently removed, the main constraints are related to the complexity of the operation and the difficulty to add fresh enzyme. Regarding magnetic reactors, the available literature on configurations is very limited. Some alternatives are provided below. In recent investigations, magnetic enzyme reactors have been used, such as the case of Wang et al. (2012), which designed a magnetically stabilized fluidized bed reactor, or Duan et al. (2014) in which reactor had a system of electromagnets where the reaction took place, both for the transformation of phenol in wastewater. However, these two configurations need an external power supply which implies drawbacks as the cost of the electricity or the high temperatures that reaches, among others. Ardao et al. (2013), also presented a configuration of a continuous reactor for eliminating micropollutants in which the separation system was a magnet in the outlet stream that collects the nanoparticles by means of a system of valves that reversed the flow, bringing the nanoparticles back to the reactor. Chapter 1 16 1.10. Enzyme-aided processes as a way of reducing the environmental impacts The application of oxidative enzymes combined with nanoparticles as a highperformance nanobiocatalysts for environmental and bio-chemical processes is directly linked to the concept of Green Chemistry as a carrier for effective environmental sustainability. It is widely accepted that biocatalytic processes offer extensive advantages over traditional chemistry in terms of environmental impact as a result of milder reactions conditions (physiological pH and temperature), biodegradable enzymatic catalysts that reduce waste disposal requirements, and substitution of organic/toxic solvents by water (Sheldon, 2007). In addition, the combination of higher regioand stereo-selectivities also enables shorter processes that generate less waste. It has been shown that enzyme-catalyzed oxidations result in: i) Reduction of toxic reagents, solvents and metal catalysts; ii) Reduced consumption of energy and water; and iii) Reduction of waste and GHG emissions. Besides its intrinsic, underlying sustainable character, this thesis aims at quantifying these indicators, especially those related to the production of the nanoparticles and the operation of the enzymatic reactor. This work will specifically address the evaluation of environmental impact reductions through process scaleup of selected case studies, which will support life cycle assessment of the processes. The broader use of optimized oxidoreductase based biocatalysts will help to develop enzyme-aided processes for environmental and biotechnological processes in a sustainable process that helps in their successful implementation. 1.11. References Ardao, I.D., P. Demarche, R. Nair, S.N. Agathos. 2013.Micropollutants clean-up by bioinspired entrapped laccases in a continuous reactor with magnetic retention, Proceedings of the 2nd European Symposium of Water Technology and Management. (2013) 141-146. General introduction 17 Barreca A, Fabrini, M., Galli, C., Gentili, P., Ljunggren, S. 2003. Laccase mediated oxidation of a lignin model for improved delignification procedures. Journal of Molecular Catalysis B: Enzymatic, 26: 105-110. Bolong, N Ismail, A.F. Salim, M.R. M.R. Matsuura, M.R.2009. A review of the effects of emerging contaminants in wastewater and options for their removal, Desalination,229 (1-3). 229-246 Bormann,S, Baraibar,AG, Ni,Y, Holtmann,D, Hollmann,F. 2015. Specific oxyfunctionalisations catalysed by peroxygenases: opportunities, challenges and solutions. Catal. Sci. Technol. 5:2038-2052. Bozell,JJ, Petersen,GR. 2010. Technology development for the production of biobased products from biorefinery carbohydrates-the US Department of Energy's "Top 10" revisited. Green Chem. 12:539-55 Bozon-Verduraz F, Fernand-Fivet, J., Piquemal, R., Brayer, K, El, K., Yaghoub, S. 2009. Nanoparticles of metal oxides: some peculiar synthesis methods, size and shape control, application to catalysts preparation. Journal of Physics, 39: 134-140 Cañas A, Camarero, S. 2010. Laccases and their natural mediators: Biotechnological tools for sustainable eco-friendly processes. Biotechnology Advances, 28: 694-705 Cupaioli F, Zucca, F.A., Boraschi, D., Zecca, L. 2014. Engineered nanoparticles. How brain friendly is this new guest?. Progress in Neurobiology, 119120: 20-38 Deblonde, T., Cossu-Leguille, C., Hartemann,P. 2011. Emerging pollutants in wastewater: A review of the literature, International Journal of Hygiene and Environmental Health, 214 (6): 442-448, Immobilization of oxidoreductases to formulate robust and suitable biocatalysts 25 OUTLINE CHAPTER 2 INTRODUCTION 26 2.1. MATERIALS AND METHODS 27 2.2. 2.2.1. CHEMICALS FOR ENZYME IMMOBILIZATION 27 2.2.2. ENZYMES 28 2.2.3. SUPPORTS FOR IMMOBILIZATION 29 2.2.4. DETERMINATION OF ENZYMATIC ACTIVITY 30 2.2.5. SELF-IMMOBILIZATION OF ENZYMES BY CROSS-LINKING AGGREGATES (CLEAS) 32 2.2.6. IMMOBILIZATION OF ENZYMES ON EPOXY AND AGAROSE-GLIOXYL BASED SUPPORTS 33 2.2.7. IMMOBILIZATION OF ENZYMES ON MAGNETIC NANOPARTICLES 33 RESULTS 35 2.3. 2.3.1. IMMOBILIZATION OF ENZYMES BY CLEAS35 2.3.2. IMMOBILIZATION OF ENZYMES ON SUPPORTS 38 CONCLUSIONS 42 2.4. REFERENCES 42 2.5. Chapter 2 26 Introduction 2.1. In this research, the use of enzymes is proposed as an alternative to perform the oxidation of pollutants of emerging concern and the production of building blocks. Although enzymes constitute promising biocatalysts, the use of enzymes for large-scale applications needs to bear in mid not only the catalytic activity and specificity but also its stability and reusability in a continuous operation. When free enzymes are used, their recovery can be only be performed by means of ultrafiltration membranes coupled to an enzymatic reactor. In contrast, the use of enzymes in an immobilized or insolubilized form favours their easy recovery and retention in the reaction system (Datta et al. 2013; DiCosimo et al. 2013). Conceptually, there are two basic methods for enzyme immobilization, as the immobilization can occur by physical or chemical interactions. Physical coupling methods include the entrapment of the enzyme within a tridimensional matrix, its encapsulation in an organic or inorganic polymer, and its absorption to the support surface by ionic exchange, whereas covalent bonding assures the irreversible binding of enzyme by self-immobilization using bifunctional cross-linkers or the binding to a support matrix (Fernández-Fernández et al. 2013). In this research, attention was paid to two different immobilization methods: self-immobilization using crosslinking agents and covalent immobilization of enzymes on commercial supports and superparamagnetic nanoparticles. Self-immobilization of enzymes as cross-linked aggregates (CLEAs) is a simple technique to increase stability and reusability of biocatalyst (Torres et al. 2013). The procedure to prepare CLEAs usually involves an enzyme precipitation step and followed by protein cross-linking via the reaction of glutaraldehyde with reactive amine residues of the protein (Schoevaart et al. 2004). As aggregation and precipitation are commonly used for enzyme purification, the production of CLEAs combines purification and immobilization into a single operation (Sheldon et al. 2011). The method is extremely simple and amenable to swift optimization. However, CLEAs may suffer drawbacks as softness or poor mechanical stability, and often leaching of enzyme in the reaction medium during the biocatalysis may occur (Wilson et al. 2006). On the other hand, enzyme immobilization by covalent binding onto a support has the advantage of multipoint covalent attachment which typically rigidifies the Oxidoreductases immobilization to formulate robust and suitable biocatalysts 27 enzyme structure, preventing it from undergoing inactivation deformations, which translates in an increased stability, activity and selectivity (Torres et al. 2013). The application of covalent immobilization techniques considers two different possibilities: either the use of inert supports which can be properly activated or the use of commercially available active supports. Among the latter, glyoxyl and epoxyactivated supports are considered very promising and have been reported to be effective supports for enzyme immobilization (Mateo et al. 2007; Lloret et al. 2012). However, to avoid enzyme leaching, it is convenient to disperse the enzyme on high specific surface areas, an intrinsic characteristic of nanomaterials. In the development of nanotechnology, a number of different materials may be used for this purpose, such as nanoparticles, nanosheets, nanotubes, nanofibers, and nanocomposites (Johnson et al. 2011; Tran et al. 2012; Ma et al. 2012; Plessis et al. 2013). Among the different nanomaterials, magnetic nanoparticles (mNP) offer an additional advantage over others because the retention of the biocatalyst is possible by means of a magnetic field, as an alternative to centrifugation or filtration stages (Kalkan et al. 2012). The main goal of the work presented in this chapter was to investigate the immobilization of different oxidoreductases: Trametes versicolor and Myceliophtora thermophile laccases, Galactose oxidase, Aryl alcohol oxidase, 5Hydroxymethyl furfural oxidase and Unspecific peroxygenase, to select the best biocatalysts for the different applications. With this aim, enzymes were immobilized as CLEAs as well as on different supports from commercial to superparamagnetic nanoparticles and both procedures were optimized. Materials and methods 2.2. 2.2.1. Chemicals for enzyme immobilization 3-(aminopropyl)triethoxysilane (APTES) (≥98%), 2-2´-azinobis(3ethylbenzothiazoline-6-sulfonic acid) (ABTS) (≥98%), glutaraldehyde (25%), 3- (Ethyliminomethyleneamino)-N,N-dimethylpropan-1-amine (EDC) (≥98%), Nhydroxysuccinimide (NHS), vanillyl alcohol (≥98%) and polyethylene glycol (PEG) 6000 and 3350 were purchased from Sigma-Aldrich. Ammonium sulfate (98%) and tert-butyl alcohol (98%) were obtained from Panreac. Veratryl alcohol (97%) was Chapter 2 28 purchased in Fluka. Ethanol (≥96%) and acetone (≥99.6%) were purchased from Merck and Labkem, respectively. 2.2.2. Enzymes Commercial laccase from Myceliopthora thermophile (Mt) (85 kDa) and galactose oxidase (GAO) (68 kDa) were provided by Novozymes. These enzymes were produced by submerged fermentation of genetically modified Aspergillus oryzae. Trametes versicolor (Tv) laccase (70 kDa) was purchased by Sigma-Aldrich. Recombinant aryl alcohol oxidase (AAO) (67 kDa) from Pleurotus ostreatus was obtained by expressing in Escherichia coli and was kindly provided by the Environmental Biotechnology Laboratory (IHI Zittau, Technical Dresden University, Germany). The recombinant unspecific peroxygenase (UPO) (45 kDa) from isolate S358 (ascomycete) was obtained by expressing in Pichia pastoris and provided by Jena Bios GmbH. 5-Hydroxymethyl furfural oxidase (HMFO) (70 kDa) from Pseudomonas nitroreducens was provided by the Biological Research Center (CIB, Spanish National Research Council, Spain). Three-dimensional structures of the enzymes can be found in literature and are shown in Figure 3.1. Figure 2.1. Mt (A), Tv (B), GAO (C), AAO (D), HMFO (E) and UPO (F). Figures generated using Pymol (pdb accession numbers 5LOI, 1GYC, 2EIB, 3FIM, 4UDP and 2YOR) (B) (C) (D) (E) (F) (A) Oxidoreductases immobilization to formulate robust and suitable biocatalysts 29 2.2.3. Supports for immobilization Epoxy and agarose-glioxyl based supports 2.2.3.1. Epoxy-activated methacrylate polymers, Lifetech® ECR8204/F and ECR8215/F were purchased in Purolite and Relizyme® EP 403/M was purchased in Resindion. Agarose-glioxyl support was prepared from Agarose BCL-6 as described by other authors (Vieira et al. 2011) and was provided by the Institute of Catalysis and Petrochemistry (ICP, Spanish National Research Council, Spain). Detailed characteristics of these supports are presented in Table 2.1. Table 2.1. Characteristics of the different supports Support Size (µm) Porosity (Å) Epoxy functional group Lifetech® ECR8204/F 150-300 300-600 Lifetech® ECR8215/F 150-300 1200-1800 Relizyme® EP 403/M 200-500 400-600 A garose-glioxyl functional group Agarose-glioxyl 100 Superparamagnetic nanoparticles 2.2.3.2. Non-coated magnetite nanoparticles (mNP), single-core silica-coated magnetic nanoparticles (smNP), polyacrylic acid magnetic nanoparticles (PAAmNP), polyethyleneimine-coated magnetic nanoparticles (PEImNP) were supplied by Nanomag (Universidade de Santiago de Compostela, Spain). Detailed characteristics of the nanoparticles evaluated are presented in Table 2.2. Chapter 2 30 Table 2.2. Characteristics of the different magnetic nanoparticles Support Size (nm) Zeta potential (mV) smNP 21.5±2.1 -24.2 PAAmNP 10.1±2.4 -40.1 PEImNP 10±1.2 4.7 mNP 9.9±1.4 -45.2 Magnetized fumed silica nanoparticles 2.2.3.3. Magnetized fumed silica nanoparticles (mfsNP) were prepared according with Safarik et al. (2012). Fumed silica nanoparticles (fsNP) (surface area: 390±40 m 2 g -1 ; aggregates of particles with a size of 7 nm) were purchased from Sigma-Aldrich. The ferrofluid was provided by the Institute of Nanobiology and Structural Biology (GCRC, Czech Republic) and is composed of magnetic iron oxide nanoparticles (1020 nm of diameter) and with a concentration of iron (Fe 2+ , Fe 3+ ) oxide content of 48.11 mg mL -1 . The magnetized support from fumed silica nanoparticles is obtained after incubation with ferrofluid and methanol (1:7) under gentle agitation for 1 h. Afterwards, the nanoparticles were washed and re-suspended in phosphate buffer (pH 7) to remove residual methanol. 2.2.4. Determination of enzymatic activity The enzyme activities of the free and immobilized enzymes were performed to assess the efficiency of the immobilization process. Below the analytical procedures that allow the determination of the activity of the different enzymes used in this study are described. The basis for the determination of enzyme activity is based on a common concept: the measurement of the oxidation of a model substrate, specific to each enzyme, which allows the indirect determination of activity by correlating the oxidation rate of the compounds with the reference enzyme units. All spectrophotometric measurements were carried out on a BioTeK PowerWave XS2 microplate spectrophotometer Oxidoreductases immobilization to formulate robust and suitable biocatalysts 31 Myceliophthora thermophila (Mt) and Trametes versicolor (Tv) 2.2.4.1. Laccase activity was determined by monitoring the oxidation rate of 0.267 mM ABTS to its cation radical (ABTS + ) at 420 nm (ε 420 = 36,000 M -1 cm -1 ) in Mcllvaine buffer (80 mM citric acid and 40 mM Na 2 HPO 4 ); pH 3) at 30˚C for 7 min (6-s intervals). One unit (U) of activity was defined as the amount of enzyme forming 1 µmol of ABTS + per min. Galactose oxidase (GAO) 2.2.4.2. The activity of GAO was measured following the oxidation of 0.85 mM of ABTS into the radical cation ABTS + at 30˚C for 2 min (6-s intervals) under the catalysis of 2.55 U mL -1 of Horseradish peroxidase (HRP) and 85 mM of galactose in phosphate buffer (50 mM, pH 6) at 436 nm (ε 436 = 29,300 M -1 cm -1 ). One unit (U) of activity was defined as the amount of enzyme forming 1 µmol of ABTS + per min. Aryl alcohol oxidase (AAO) and Unspecific peroxygenase (UPO) 2.2.4.3. AAO activity was measured following the oxidation of 10 mM of VE into veratraldehyde (as major compound) at 30˚C for 1 min (6-s intervals) in phosphate buffer (50 mM, pH7) at 310 (ε 310 = 9,300 M -1 cm -1 ). UPO activity was measured following the oxidation of 10 mM of VE into veratraldehyde (as major compound) at 30˚C for 1 min (6-s intervals) in presence of 0.5 mM of hydroxgen peroxide in phosphate buffer (50 mM, pH 7) at 310 nm (ε 310 = 9,300 M -1 cm -1 ). One unit (U) of activity was defined as the amount of enzyme forming 1 µmol of veratraldehyde per min. 5-Hydroxymethyl furfural oxidase (HMFO) 2.2.4.4. The activity of HMFO was measured following the oxidation of 35 mM of VA into vanillin at 30˚C for 1 min (6-s intervals) in TRIS buffer (50 mM, pH 7) at 309 nm (ε 309 = 8,332 M -1 cm -1 ). One unit (U) of activity was defined as the amount of enzyme forming 1 µmol of vanillin per min. Chapter 2 32 2.2.5. Self-immobilization of enzymes by cross-linking aggregates (CLEAs) CLEAs are support-free covalently immobilized biocatalysts. First, a precipitant is used to form the enzyme agglomerates, and then a cross-linking agent binds these enzymes together, retaining them in the agglomerated form according to the scheme depicted in Figure 2.2. Figure 2.2. Scheme of CLEAs preparation. As a first step, the enzyme (10,000 U L -1 ) was incubated with the selected precipitants (PEG 3350 and 6000 (20 and 40 g L -1 ), (NH 4 ) 2 SO 4 (50% wt/v), EtOH (50% v/v), Acetone (36% v/v) and t-BuOH (90% v/v)) for 24 h at room temperature and at 100 rpm on a C24 Incubator shaker. The amount of aggregates formed is determined and re-dissolved in phosphate buffer (0.1 M, pH 7) and the activity was measured and compared to the initial activity. Thereafter, the aggregates of enzymes were incubated under agitation (100 rpm) at room temperature for 2, 8 and 24 h with the cross-linking agent glutaraldehyde 25% (v/v). Different concentrations of cross-linking agent (10, 50 and 100 mM) were considered. After each stage, the unreacted chemicals were removed after consecutive cycles of centrifugation/re-suspension in phosphate buffer (100 mN, pH 7). The immobilization efficiency was calculated as the ratio of the initial activity and the leached enzyme on the supernatants. All the experiments were performed in triplicate. Precipitant Crosslinker Oxidoreductases immobilization to formulate robust and suitable biocatalysts 33 2.2.6. Immobilization of enzymes on epoxy and agarose-glioxyl based supports Previous to immobilization process the supports (Lifetech® ECR8204/F, Lifetech® ECR8215/F, Relizyme® EP 403/M and agarose-glioxyl) were washed four times with carbonate buffer (100 mM, pH 10). The subsequent enzyme immobilization was carried out following the method described by Mateo et al. (2007). The schemes of the mechanisms of both immobilization processes are showed in Figure 2.3. Figure 2.3. Mechanisms of enzyme immobilization onto epoxy (A) and agarose-glioxyl (B) based supports. The washed support (100 g L -1 ) was incubated with the enzymes (1.5 U mg -1 support) at 4°C for 24 h and 100 rpm on a C24 Incubator shaker. The noncovalently bound enzyme was washed with phosphate buffer (100 mM, pH 7) in five centrifugation/re-suspension steps for 5 min at 8000 g-force in a EBA-270 centrifuge. The immobilization yield was calculated as the ratio of the enzyme activity theoretically immobilized and the final enzyme loading. 2.2.7. Immobilization of enzymes on magnetic nanoparticles Immobilization of enzymes onto mNP 2.2.7.1. Enzyme immobilization in magnetite nanoparticles (lacking any external coating) was carried out by ionic exchange of the enzyme with magnetite nanoparticles. Enzyme was added (0.55 U mg -1 mNP) to previously washed nanoparticles and incubated at 4°C and 25°C, 100 rpm, and pH 5, 6 and 7 for 4 h. After incubation, the nanobiocatalyst was washed five times in phosphate buffer (100 mM, pH 7) before storage. (A) (B) Table 2.4. Immobilization yield (IY) and enzyme loading (EL) for the different enzymes and supports Supports Tv Mt GAO IY (%) EL (U mg-1)IY (%) EL (U mg-1) IY (%) EL (U mg-1) Agarose glyoxyl based supports Agarose-glyoxil (Ag) 0.22 ± 0.02 <0.01 0.51 ± 0.14 <0.01 1.53 ± 0.01 <0.01 Epoxy based supports ECR8204/F 0.07 ± 0.01 <0.01 0.13 ±0.01 <0.01 1.11 ± 0.01 <0.01 ECR8215/F 0.14 ± 0.02 <0.01 0.36 ± 0.17 <0.01 3.40 ± 3.90 <0.01 Relizyme 0.06 ± 0.01 <0.01 0.62 ± 0.60 <0.01 8.00 ± 5.00 <0.01 Nanoparticles mNP 58.50±0.22 0.69±0.01 31.60±0.57 0.45±0.05 - - smNP 103.50 ± 0.35 2.66 ± 0.65 102.84 ± 12.90 0.65 ± 0.01 51.21 ± 1.68 2.66 ± 0.65 PEI-mNP 80.50 ± 0.21 1.54 ± 0.03 70.32 ± 0.05 <0.01 55.19 ± 1.80 1.54 ± 0.03 mfsmNP 70.30 ± 0.10 1.48 ± 0.01 55.32 ± 0.12 <0.01 42.30 ± 1.99 1.48 ± 0.01 Chapter 2 40 Table 2.5. Immobilization yield (IY) and enzyme loading (EL) for the different enzymes and supports (cont.) Supports AAO HMFO UPO IY (%) EL (U mg-1)IY (%) EL (U mg-1) IY (%) EL (U mg-1) Agarose glioxyl based supports Agarose-glyoxil (Ag) 0.10 ± 0.02 <0.01 2 .00± 0.56 <0.01 15.60 ± 2.05 0.08±0.02 Epoxy based supports ECR8204/F 0.03 ± 0.01 <0.01 0.50 ±0.01 <0.01 0.50 ± 0.03 <0.01 ECR8215/F 0.03 ± 0.02 <0.01 2.00± 0.23 <0.01 1.20 ± 0.67 <0.01 Relizyme 0.04 ± 0.01 <0.01 2.30 ± 0.76 <0.01 3.00 ± 0.35 <0.01 Nanoparticles mNP - - - - - - smNP 46.05 ± 7.71 0.95± 0.01 45.30 ± 11.80 0.21 ± 0.02 115.87 ± 5.95 1.04 ± 0.05 PEI-mNP 16.97 ± 5.58 0.55 ± 0.09 82.20 ± 11.3 0.37 ± 0.03 137.06 ± 0.92 0.92 ± 0.06 mfsmNP 16.68 ± 2.28 0.36 ± 0.01 68.30 ± 12.1 0.16 ± 0.02 128.32 ± 8.90 1.04 ± 0.02 Oxidoreductases immobilization to formulate robust and suitable biocatalysts 41 Oxidoreductases immobilization to formulate robust and suitable biocatalysts 42 Conclusions 2.4. Immobilization of two laccases (Myceliophthora thermophila and Trametes versicolor), three oxidases (Galactose oxidase, Aryl alcohol oxidase and Hydroxymethyl furfural oxidase) and one peroxygenase (Unspecific peroxygenase) was investigated aiming to facilitate its reuse and application in continuous operation such as water detoxification or for the production of building blocks. For this purpose, different methods were evaluated: first, the enzymes were immobilized by self-immobilization by precipitating the enzyme and further crosslinking with glutaraldehyde; also, enzyme immobilization was conducted by covalent and ionic exchange bonding to commercial epoxyand glyloxyl-activated acrylic supports and superparamagnetic nanoparticles with different coatings (silica, polyethyleneimine and polyacrylic acid) by glutaraldehyde-activated and sulfo-NHS/EDC-activated. CLEAs were produced in all cases excepted for aryl alcohol oxidase which was completely inactivated when presence of glutaraldehyde. CLEAs of both laccases even leads to hyperactivation and high immobilization yields. However, the enzyme consumption and the difficulty of handling them are significantly higher compared with immobilization onto supports. Of all supports, superparamagnetic nanoparticles (functionalized with amino groups) showed the highest overall potential for enzyme immobilization with immobilization yields higher than 80%. Moreover, it was observed that the silica-coated superparamagnetic nanoparticles brought out a most stable biocatalyst after three months of storage. For those reasons silica-coated superparamagnetic nanoparticles arise as the most suitable form of immobilization of these enzymes. References 2.5. Arca-Ramos, A. et al. (2016). Assessing the use of nanoimmobilized laccases to remove micropollutants from wastewater. 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Cross-Linked Enzyme Aggregates (CLEAs): Stable and Recyclable Biocatalysts. Biochemical Society Transactions 35 (Pt 6): 1583-87. Sheldon, R. A. et al. (2011). Characteristic features and biotechnological applications of cross-linked enzyme aggregates (CLEAs). Applied Microbiology and Biotechnology 92 (3): 467-77. Chapter 2 46 Torres, M.P. et al. (2013). Cross-Linked Enzyme Aggregates (CLEAs) of Selected Lipases: A Procedure for the Proper Calculation of Their Recovered Activity. AMB Express 3: 25. Tran, D-T. et al. (2012). Immobilization of Burkholderia sp. Lipase on a Ferric Silica Nanocomposite for Biodiesel Production. Journal of Biotechnology 158 (3): 112-19. Vieira, M. et al. (2011). β-Glucosidase immobilized and stabilized on agarose matrix functionalized with distinct reactive groups. Journal of Molecular Catalysis B: Enzymatic 69 (1): 47-53. Kumar, V. et al. (2012). Preparation and characterization of porous cross linked laccase aggregates for the decolorization of triphenyl methane and reactive dyes. Bioresource Technology 119: 28-34. Wilson, L. et al. (2006). CLEAs of lipases and poly-ionic polymers: A simple way of preparing stable biocatalysts with improved properties. Enzyme and Microbial Technology 39 (4): 750-55. 1. Zimmermann, Y-S. et al. (2011). Sorption-Assisted Surface Conjugation: A Way to Stabilize Laccase Enzyme. Applied Microbiology and Biotechnology 92 (1): 169-78. Chapter 3 Superparamagnetic nanobiocatalyst to biotransform micropollutants from wastewater The development of nanotechnology has provided a range of diverse nanoscale carriers that can be potentially applied for enzyme immobilization. Among the different types of support, silica-coated magnetic nanoparticles (smNPs) have been selected and a high redox potential laccase from Trametes versicolor (Tv) was successfully immobilized. Enzyme loads of 2.66±0.07 U mg -1 smNPs were attained for the optimal doses of Tv. In general, the laccase-smNP conjugates showed higher resistance against acidic pH and higher storage stability, especially when incubated in the secondary effluent from a municipal wastewater treatment plant (WWTP). The ability of laccase-smNP to biotransform Bisphenol A (BPA), Phenol, Diclofenac (DCF), Estrone (E1), Estradiol (E2), 17ß-estradiol (EE2) and Methyl green (MG) was assessed in batch experiments. Compared to free laccase, immobilized enzyme led slower biotransformation rates but the superparamagnetic characteristic of the support allowed simple and fast recovery of the nanobiocatalyst. APTES Fe3O4@SiO2Functionalized Fe3O4@SiO2 Laccase-Fe3O4 @ SiO2 1)Laccase 2)Glutaraldehyde Superparamagnetic nanobiocatalyst to biotransform micropollutants from wastewater 49 OUTLINE CHAPTER 3 3.1. INTRODUCTION 50 3.2. MATERIALS AND METHODS 52 C HEMICALS FOR SYNTHESIS OF NANOPARTICLES AND ENZYME 3.2.1. IMMOBILIZATION 52 P REPARATION AND CHARACTERIZATION OF MAGNETIC NANOPARTICLES 52 3.2.2. CHARACTERIZATION OF MAGNETIC NANOPARTICLES 53 3.2.3. IMMOBILIZATION OF LACCASE ONTO SILICA-COATED MAGNETIC NANOPARTICLES 3.2.4. 54 I NFLUENCE OF PH, T, AND INACTIVATING COMPOUNDS ON THE RELATIVE 3.2.5. ACTIVITY AND STABILITY OF FREE AND IMMOBILIZED LACCASE 54 R EGENERATION OF THE SUPPORT 55 3.2.6. BIOTRANSFORMATION OF THE TARGET POLLUTANTS 56 3.2.7. BIOTRANSFORMATION OF THE TARGET POLLUTANTS IN SEQUENTIAL BATCH 3.2.8. REACTORS 56 E VALUATION OF TOXICITY 57 3.2.9. 3.3. RESULTS AND DISCUSSION 58 C HARACTERIZATION OF SILICA-COATED IRON OXIDE NANOPARTICLES 58 3.3.1. IMMOBILIZATION OF LACCASE ONTO SILICA-COATED MAGNETIC NANOPARTICLES 3.3.2. 60 I NFLUENCE OF PH, T AND INACTIVATING COMPOUNDS ON THE RELATIVE 3.3.3. ACTIVITY AND STABILITY OF FREE AND IMMOBILIZED LACCASE 61 R EGENERATION OF THE SUPPORT 65 3.3.4. BIOTRANSFORMATION OF THE TARGET POLLUTANTS 65 3.3.5. BIOTRANSFORMATION OF BPA, E2 AND MG BY LACCASE IMMOBILIZED ON 3.3.6. MAGNETIC NANOSUPPORT IN SEQUENTIAL BATCH REACTION 69 E VALUATION OF TOXICITY 70 3.3.7. 3.4. CONCLUSIONS 71 3.5. REFERENCES 71 Chapter 3 56 Biotransformation of the target pollutants 3.2.7. To determine the capability of the enzymatic system the oxidation toward the target pollutants by free and immobilized enzyme (1000 U L -1 ) was evaluated in batch reactors (30 mL flasks). The conditions of each experiment are detailed in Table 3.3. Table 3.3. Summary of experiment conditions for the different target pollutant Target pollutant Concentration (mg L -1 ) Reaction medium BPA 10 Phosphate buffer (100 mM), pH 6 E1 2.5 E2 2.5 EE2 2.5 Phenol 10 MG 20 DCF 5 Acetate buffer (100 mM), pH 5 BPA, DCF, E1, E2, EE2 and Phenol were quantified by High-Performance Liquid Chromatography (HPLC) at a detection wavelength of 270 nm on a Jasco XLC HPLC (Jasco Analítica, Madrid, Spain). This equipment was coupled with a diode detector 3110 MD, a 4.6 x 150 nm Gemini reversed-phase column (3 µm C 18 110 Å) from Phenomenex (supplied by Jasco Analítica, Madrid, Spain), and an HP ChromNav data processor. A 25-µL-sample volume was injected into the column. The mobile phase contained 50% acetonitrile and 50% water. The flow rate was fixed at 0.4 mL min -1 in isocratic conditions. MG was analyzed by following the decrease in absorbance at 630 nm. Biotransformation of the target pollutants in sequential batch reactors 3.2.8. The biotransformation of Bisphenol A (100 μg L −1 ), E2 (1 μg L −1 ) and MG (20 mg L −1 ) by immobilized laccase (1,000 U L −1 ) incubated in the secondary effluent of a wastewater treatment plant (WWTP) was investigated in a sequential batch stirred reactor of 1 L for 10 cycles. In parallel, a control experiment with Superparamagnetic nanobiocatalyst to biotransform micropollutants from wastewater 57 functionalized mNP lacking laccase under the same conditions was also performed. After 6 h, the reaction medium was withdrawn, and the nanobiocatalyst and functionalized mNP (control) were magnetically separated for a new cycle biotransformation. The extent of the reaction was calculated as a ratio of the final and initial concentration of the compound. All experiments were performed in triplicate. The percentage of BPA and E2 biotransformation was determined by Gas Chromatography-Mass Spectrometry (GC-MS) after a Solid Phase Extraction (SPE). The extraction procedure was carried out using 60 mg OASIS Hydrophilic-LipophilicBalanced (HLB) cartridges (Waters closet, Milford) previously conditioned with 3 mL ethyl acetate, 3 mL methanol, and 3 mL of distilled water acidified with HCl to pH 2. A nitrogen stream was used to dry the cartridges for 45 min and eluted with 200 mL ethyl acetate. The GC-MS analysis was conducted in a MS Saturn 2100T system with Zebron column (ZB-SemiVolatiles 30 m x 0.25 mm x 0.25 µm) (Phenomenex). The carrier gas was He with a flow rate of 1 mL min -1 . The injection was performed in splitless mode (2 min) at 280°C. The oven temperature was programmed to rise from 70 to 150°C at 25°C min -1 , at 3°C min -1 until 200°C and finally, at 8°C min -1 to reach 280°C, that was maintained for 5 min. Electron impact (EI) mass spectra were generated at 70 eV with a scan range of 50–500 amu. Each sample was analyzed in duplicate. MG decolorization was followed by spectrophotometry at 630 nm. Evaluation of toxicity 3.2.9. To investigate the potential toxicity of the transformation products obtained from the enzymatic treatment, a Microtox ® test based on the luminescent marine bacterium Vibrium fischeri was performed in triplicate using a Microtox ® model 500 Analyzer. The results were expressed as EC 50 (15 min) , which corresponds to the concentration of the pollutant that causes a reduction in the light output by 50% after 15 min incubation. All the measurements were performed in triplicate. Chapter 3 58 3.3. Results and discussion Characterization of silica-coated iron oxide nanoparticles 3.3.1. The X-ray diffraction patterns of both oleic acid and silica coated iron oxide nanoparticles are depicted in Figure 3.1. The crystalline phase was identified as magnetite (PDF-2 card number 19-0629) and the main reflections were labelled with the Miller indexes. In the silica-coated magnetite nanoparticles, a broad band between 20-30 º2Θ was observed due to the amorphous silica shell. Figure 3.1. X-ray diffraction patterns of oleic-acid-coated magnetite and silica-coated magnetite nanoparticles Transmission electron micrographs show the nearly spherical shape of both oleic-acid and silica-coated magnetite nanoparticles (Figure 3.2). The average particle size of the oleic-acid coated magnetite nanoparticles was 7.7±3.3 nm, and the silica-coated magnetite nanoparticles was 21.5±1.3 nm. From the TEM micrographs, it can be evidenced that the reverse microemulsion method allowed to develop an excellent silica coating on the magnetite nanoparticle. The samples were magnetically characterized by measuring the variation of the magnetization as a function of the applied magnetic field at 300 K (Figure 3.3). Both samples show superparamagnetic behaviour due to the small particle size (Lu et al., 2007). The oleic-acid-coated magnetite nanoparticles showed a high saturation magnetization Superparamagnetic nanobiocatalyst to biotransform micropollutants from wastewater 59 (measured as the magnetization at 10 kOe), Ms = 61.75 emu g-1, and nearly zero values for both coercivity (Hc = 14Oe) and remanence (Mr = 1.35 emu g-1). The decrease of saturation magnetization in the silica-coated magnetite nanoparticles was due to the non-magnetic silica shell, which did not affect the superparamagnetic behaviour negatively. Figure 3.2. Transmission electron micrographs and corresponding size histograms of the oleic-acid-coated magnetite and silica-coated magnetite nanoparticles. Scale bar (50 nm). Figure 3.3. Magnetization study of the samples: plot of the variation of the magnetization with the applied magnetic field at 300 K Chapter 3 60 Immobilization of laccase onto silica coated magnetic nanoparticles 3.3.2. Silica allows the surface modification of the magnetite nanoparticles. The hydroxyl surface groups can be chemically modified to provide different bioconjugation groups, such as amine. To perform the aminofunctionalization of the nanoparticles, (3-aminopropyl) triethoxysilane (APTES) was added. In a further step, glutaraldehyde was used as cross-linker for the attachment of proteins via the amine groups of the lysine residues. Different concentrations of APTES and glutaraldehyde were evaluated in excess of laccase (3.29 U mg -1 mNP) with the aim of maximizing immobilization yield and enzyme loading while minimizing enzyme loss after washing. As observed in Table 3.4, the optimal concentration of APTES was 0.8 mmol mg -1 mNP (run 2), which was the value selected for the following experiments. Regarding glutaraldehyde, the lowest concentration considered (run 4) was probably not sufficient to immobilize laccase, and almost 60% of the activity was lost after washing. As the concentration increased, the enzyme loading was also increased. However, washing losses markedly increased at the highest concentration (run 7), probably due to laccase inactivation caused by glutaraldehyde (Roy et al., 2006). Consequently, the optimal concentration of glutaraldehyde was found at 8 mmol mg -1 mNP, which led to the highest immobilization yield (83.7%). Finally, the maximum laccase immobilized on the mNP was similar (2.66-2.68 U mg -1 mNP for run 6 and 10, respectively) for the highest enzyme activities: 2.82 and 3.29 U mg -1 mNP, which is attributed to the likely saturation of the binding sites of the support by the enzyme. The enhancement of catalytic activity of the nanobiocatalyst was evidenced by the increase of ABTS oxidation, which rendered into values of immobilization yields above 100% (Table 3.4), revealing the superior affinity of immobilized laccase toward ABTS (Cabana et al., 2011; Arca-Ramos et al., 2016). The immobilization of laccase into amino-modified silica nanoparticles using glutaraldehyde as cross-linker was shown to be dependent on laccase source, and reached values ranging from 0.77 to 4.77 U mg -1 mNP (Zimmermann et al., 2011; Hommes et al., 2012). When comparing the immobilization of laccase from T. versicolor onto fumed silica non-magnetic and magnetic nanoparticles, the maximum enzyme loading was significantly lower, between 2.5 and 1.7 times lower than the value reported here (Ammann et al., 2013; Deng et al., 2015). Superparamagnetic nanobiocatalyst to biotransform micropollutants from wastewater 61 Table 3.4. Immobilization of laccase in silica coated magnetic nanoparticles at different concentrations of APTES, glutaraldehyde and laccase activity Influence of pH, T and inactivating compounds on the relative activity 3.3.3. and stability of free and immobilized laccase While the optimal pH for the measurement of enzymatic activity was similar for both free and immobilized laccase: 3 (Figure 3.4), a significant effect was evident when evaluating the effect of pH on the enzyme stability at different incubation periods (Figure 3.5). It was observed that the immobilized enzyme showed higher activity, markedly enhanced under acidic conditions (pH 3-5), in agreement with other reports (Lloret et al., 2011; Rossi et al., 2004). On the contrary, free laccase was inactive after 24 h of incubation at pH 3.4, and remarkably low (18%) at pH 5.2. Comparatively, for the same incubation period, the activity of the immobilized laccase was similar: 41 and 43% at pH 3.4 and 5.2, respectively. Run Washing loss (%) Immobilization yield (%) Enzyme loading (U mg -1 mNPs) APTES (mmol mg -1 mNPs) 1 0.4 56.4±7.0 50.6±7.3 1.55±0.23 2 0.8 49.1±5.2 65.1±8.3 1.81±0.15 3 1.6 55.2±1.7 52.3±7.9 1.60±0.13 Glutaraldehyde (mmol mg -1 mNPs) 4 1 59.8±2.3 36.0±3.8 0.89±0.07 5 4 22.5±17.8 82.8±11.6 2.07±0.25 6 8 21.1±1.8 83.3±1.1 2.66±0.07 7 12 35.7±12.3 77.0±11.0 2.72±0.45 Laccase (U mg -1 mNPs) 8 1.88 16.4±2.81 103.5±1.3 2.11±0.18 9 2.35 28.4±6.9 103.7±16.0 2.31±0.43 10 2.82 30.7±6.4 64.2±4.6 2.68±0.08 Chapter 3 62 Figure 3.4. Effect of pH on the relative activity of free laccase (○) and laccase immobilized into mNP (●). Figure 3.5. Residual activity (A/A 0 ) of free laccase (●) and immobilized on smNP (●) at different pH after 24 h incubation. Regarding the effect of the temperature on the activity of free and immobilized laccase, the maximum activity was obtained at 60°C in both cases. However, immobilized laccase provided a remarkable broader profile and its relative activity was 4-13% higher than that of free laccase over the range tested (data not shown) (Lloret et al., 2011; D´Annibale et al., 1999; Kunamneni et al., 2008). Short-term stability of immobilized enzyme was also proved to be enhanced in comparison with free laccase, which is especially evident for the immobilized enzyme at 50°C (Figure 3.6). In contrast, the activity of free laccase dropped more rapidly than that of immobilized laccase, which is attributed to the conformational changes of the immobilized enzyme that increase enzyme rigidity, which protects the enzyme against denaturation at high temperature (Lloret et al., 2011; Osma et al., 2010). Superparamagnetic nanobiocatalyst to biotransform micropollutants from wastewater 63 Storage stability is one of the most important parameters to be considered in enzyme immobilization as it affects overall productivity. Free and immobilized laccases were stored at 4°C, with periodical sampling and monitoring. After 4 months, both biocatalysts maintain nearly 99% of its initial activity. When we compared the relative activity of the immobilized laccase by APTES/glutaradehyde with other immobilization methods, higher values of activity were obtained in this work (Lu et al., 2007; Deng et al., 2015; D´Annibale et al., 1999; Wang et al., 2012; Wang et al., 2014; Xu et al., 2013; Zhang et al., 2014). Figure 3.6. Residual activity (A/A 0 ) of free laccase (●) and immobilized on smNP (●) at different temperature after 24 h incubation. Figure 3.7 presents the residual activity of immobilized and free laccase after the incubation of the enzyme in the presence of sodium acetate buffer (pH 5) and inactivating compounds (NaCl, CaCl 2 , methanol, ethanol and acetone) at room temperature for 30 min. The immobilized laccase was observed to present higher stability in presence of the organic solvents and similar to free laccase when salts: NaCl and CaCl 2 are present. Chapter 3 64 Figure 3.7. Residual activity (A/A 0 ) of free laccase (●) and immobilized on smNP (●) at different inactivating compounds after 24 h incubation. The influence of the composition of a secondary effluent on enzyme stability was also evaluated (Figure 3.8). After an initial period, immobilized laccase was more stable than free laccase (about 36% higher activity from day 9 to 22). As a comparison with laccase immobilized on fumed silica nanoparticles, Hommes et al. (2011) observed a residual activity above 80% after 7 days of incubation in the secondary effluent of municipal wastewater, in contrast with free enzyme that retained only 2% of its initial activity. Figure 3.8. Residual activity of free (●) and immobilized laccase (●) during 22 days of incubation in a wastewater effluent collected from the WWTP of Calo (Milladoiro, Spain) Superparamagnetic nanobiocatalyst to biotransform micropollutants from wastewater 65 Regeneration of the support 3.3.4. The reusability of immobilized enzyme is fundamental for its practical application. However, since the loss of activity is a certainty over time, it would be interesting to regenerate the support for its further reuse with fresh enzyme. In this work, protein denaturation took place after ultrasonic incubation, heating and organic solvents addition. Thereafter, the three-dimensional network structure of protein would be broken and the protein chains would stretch up, thus the residual functional groups (e.g., –NH 2 , –COOH, –SH) in protein chains could be used to react with other groups, as for instance, glutaraldehyde. After reactivation with glutaraldehyde (Zhao et al., 2011; Liu et al., 2012), immobilization of fresh enzyme was carried out following the optimal conditions previously described. Under these conditions, the enzyme loading obtained was 1.28±0.38 U mg -1 mNP, the immobilization yield was 61.8±0.5 % and the washing loss was 45.9±0.7%. Despite the reduction of enzyme loading, the immobilization yield was still high. Hence, it has been demonstrated that silica-coated mNP can be regenerated for enzyme repeated immobilization (Table 3.5). Similarly, a sharp drop in the immobilization capacity of the regenerated supports comparing to the original one was reported (Zhao et al., 2011). However, after three regeneration cycles, the immobilization capacity was not negatively affected and the enzyme activity was maintained. Table 3.5 Potential of regeneration of the support in terms of washing loss, immobilization and enzyme loading with fresh enzyme with and without APTES functionalization. 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Laccase immobilized on a pan/adsorbents composite nanofibrous membrane for catechol treatment by a biocatalysis/adsorption process. Molecules. 19, 3376-3388 WWAP, 2017 - Wastewater, The Untapped Resource | United Nations Educational, Scientific and Cultural Organization». http://www.unesco.org/new/en/naturalsciences/environment/water/wwap/wwdr/2017-wastewater-the-untapped-resource/. Xu R, Chi C, Li F, Zhang B, 2013. Laccase–Polyacrylonitrile nanofibrous membrane: Highly immobilized, stable, reusable, and efficacious for 2,4,6Trichlorophenol removal. ACS Appl Mater Interfaces. 5, 12554-12560 Superparamagnetic nanobiocatalyst to biotransform micropollutants from wastewater 77 Zeng, Jun, Qinghe Zhu, Yucheng Wu, y Xiangui Lin. 2016. Oxidation of Polycyclic Aromatic Hydrocarbons using Bacillus Subtilis CotA with High Laccase activity and Copper independence. Chemosphere 148 (4): 1-7. Zhang P, Wang Q, Zhang J, Li G, Wei Q. 2014. Preparation of amidoximemodified polyacrylonitrile nanofibers immobilized with laccase for dye degradation. Fibers Polym. 15, 30-34 Zhao, Guanghui, Jianzhi Wang, Yanfeng Li, Xia Chen, y Yaping Liu. 2011. Enzymes immobilized on superparamagnetic Fe3O4@clays nanocomposites: Preparation, characterization, and a new strategy for the regeneration of supports. The Journal of Physical Chemistry C 115 (14): 6350-59. Zimmermann, Yannick-Serge, Patrick Shahgaldian, Philippe F. X. Corvini, y Gregor Hommes. 2011. Sorption-assisted surface conjugation: a way to stabilize laccase enzyme. Applied Microbiology and Biotechnology 92 (1): 169-78. Chapter 4 An eco-friendly nanobiocatalyst: Pycnoporus sanguineus laccase for environmental applications Agro-industrial wastes, such as sugar cane bagasse, coffee pulp, apple pomace, or tomato juice are abundantly available and it would be interesting to transform this waste into value-added products in order to create a circular economy. The present study was aimed to production of laccase Pycnoporus sanguineus using agro-industrial wastes (tomato juice and soybean oil) as the natural carbon sources. Furthermore, laccase was immobilized onto silica-coated magnetic nanoparticles (smNP) with high enzyme loadings (1.60 U mg -1 smNP) and characterized in terms of physical, chemical and morphological aspects. The nanobiocatalyst was applied for biotransformation of the emerging contaminant BPA and the triphenyl methane dye MG and the enzymatic action toward them was proven in 6 cycles with biotransformations rates higher than 85% in both cases. Finally, the potential toxicity of the nanobiocatalyst and the medium resulting from the enzymatic treatment were evaluated and not toxicity was detected. Pycnoporus sanguineus Laccase-smNP Si Fe Introduction 81 OUTLINE CHAPTER 4 INTRODUCTION 82 4.1. MATERIALS AND METHODS 83 4.2. CHEMICALS AND NANOPARTICLES 83 4.2.1. MICROORGANISM AND CULTURE CONDITIONS 83 4.2.2. LACCASE PURIFICATION 83 4.2.3. NANOBIOCATALYST CHARACTERIZATION 84 4.2.4. BIOTRANSFORMATION OF BISPHENOL A AND METHYL GREEN BY FREE AND 4.2.5. IMMOBILIZED ENZYME 84 R EUSE OF THE NANOBIOCATALYST IN A SEQUENTIAL BATCH REACTOR 84 4.2.6. BPA AND MG ANALYSIS 85 4.2.7. TOXICITY EVALUATION 85 4.2.8. RESULTS AND DISCUSSION 86 4.3. HIGH PRODUCTION RATES OF PYCNOPORUS SANGUINEUS LACCASE 86 4.3.1. STABLE AND ROBUST NANOBIOCATALYST BY EFFICIENTLY IMMOBILIZING OF 4.3.2. PYCNOPORUS SANGUINEUS ONTO SMNP 87 M ULTIPOINT COVALENT IMMOBILIZATION AND ENZYME SHELL DISTRIBUTION ON 4.3.3. SMNP WERE CONFIRMED 89 T HE NANOBIOCATALYST WAS ABLE TO BIOTRANSFORM INDUSTRIAL 4.3.4. WASTEWATERS AS RESIN INTERMEDIATE BPA OR TEXTILE DYE MG 91 R EUSABLE AND NO TOXIC SUPERPARAMAGNETIC NANOBIOCATALYST FOR 4.3.5. INDUSTRIAL WASTEWATER APPLICATIONS 93 S UCCESSFUL DETOXIFICATION OF THE TREATED WASTEWATER 94 4.3.6. CONCLUSIONS 95 4.4. REFERENCES 95 4.5. Chapter 4 88 Figure 4.2. Residual activity (A/A 0 ) of free (green bars) and immobilized laccase (orange bars) at different pH after 24 h of incubation As for the effect of the inactivating agents (methanol, ethanol, acetone, CaCl 2 and NaCl) on the free and immobilized laccase, the immobilized enzyme presented higher stability in the presence on organic solvents and CaCl 2 and similar to free laccase when NaCl was present (Table 4.1). The increase in organic solvents was also notable compared to previous studies, almost 58, 49 and 24 times higher in the presence of acetone, ethanol and methanol, respectively (Moldes-Diz et al. 2018). The stability of the nanobiocatalyst was also studied and after 3 months of storage at 4˚C, almost 99% of its initial activity was maintained. Table 4.1. Residual activity (A/A 0 ) of free and immobilized laccase after 24 h of incubation under different deactivating agents Residual activity (%) Deactivating agent Free enzyme Immobilized enzyme Methanol (25% v/v) - 81.01±7.06 Ehtanol (25% v/v) 1.68±1.57 75.93±0.22 Acetone (25% v/v) 44.37±2.69 80.83±6.85 CaCl 2 (10 µM) 5.11±5.01 70.32±2.27 NaCl (2.5 mM) 56.11±5.11 74.96±13.04 An eco-friendly nanobiocatalyst: Pycnoporus sanguineus laccase for environmental applications 89 Multipoint covalent immobilization and enzyme shell distribution on 4.3.3. sMNP were confirmed The morphology of the nanoparticles was characterized by TEM. The sMNP with core-shell architecture showed good dispersity, and the average size was 21.52.1 nm (Figure 4.3). The EDX elemental mapping showed that the elements Fe and Si existed in the sMNP (Figure 4.3). Figure 4.3. TEM image of Fe 3 O 4 @SiO 2 ; EDX elemental mapping images of Fe (red), and Si (blue) of sMNPs. The functionalization of sMNP and the laccase conjugates was evaluated by ATR-FTIR analysis and compared with sMNP-free samples (Figure 4.4). The existence of SiO 2 layers in the sMNP spectrum can be seen by the Fe-O-Si and Si-OSi stretching vibration at the characteristics absorption bands at 1103 and 1077 cm - 1 , respectively. The sMNP spectrum should show an absorption at around 1390 cm -1 for the Fe-O stretching mode, but cannot be observed due to the existence of SiO 2 layers on the surface (Quy et al., 2013). ATR-FTIR spectra involve successful functionalization of sMNPS with APTES (C 9 H 23 NO 3 Si) using a chemical bonded reaction via Si-O covalent bonds and modified with the amino group. Two peaks associated with the stretching and bending of the amino group (NH 2- ) can be found Chapter 4 90 at 3403 and 1652 cm -1 in the functionalized sMNP. Besides, the peak at 1077 cm -1 is associated with the asymmetric stretch vibration of Si-O bond. It can also be seen from the spectrum that the nanoparticles were successfully functionalized as the CH stretching (3018 cm -1 ) is increased because the hydrocarbon chain length is C 9 for APTES (Zhang et al. 2007). Similar to the confirmation of functionalization, the laccase conjugates obtained by reaction of functionalized sMNP amino groups and glutaraldehyde amino groups were confirmed by ATR-FTIR. The characteristic absorption of 1735 and 1421 cm -1 in laccase-smNP conjugates bands is associated with characteristic absorptions of C=O and C-O bonds resulting from the glutaraldehyde reaction. Moreover, the characteristic absorption of the amino group (3403 and 1652 cm -1 ) and C-H stretching (3018 cm -1 ) increased compared to functionalized sMNPs. The above observations imply that the enzyme is chemically conjugated on the surface of the functionalized sMNP via multipoint covalent immobilization mechanism. Figure 4.4. ATR-FTIR spectra of initial sMNP (green line) amino-functionalized sMNP (blue line) and laccase-sMNP conjugates (orange line) Furthermore, the successful modification of APTES, and enzyme was also confirmed by the zeta-potential measurements with change of the surface charge from negative (-21.24±2.54 mV) to positive values (28.26±5.25 mV) and TGA analysis with a reduction in the masses of the magnetic materials. Elemental An eco-friendly nanobiocatalyst: Pycnoporus sanguineus laccase for environmental applications 91 analysis of the materials (Table 4.2) also indicated that the C, Si and O contents change after modification with APTES and enzyme. Although laccase was immobilized in the nanoparticles, it was unclear the distribution on it. The FM characterization of the laccase-sMNP conjugates verified that most of nanoparticles emitted green fluorescence under excitation and showed core-shell structures, indicating the FITC labeled laccase had been successfully immobilized into the shell of the nanoparticles (Figure 4.5). Table 4.2. Results of elemental analysis for magnetic materials Compound sMNP APTES-sMNP Laccase-sMNP C (%) 85.9 10.1 27.1 Si (%) 1.2 16.6 16.5 O (%) 2.2 20.1 26.9 Figure 4.3. Pycnoporus sanguineus laccase-sMNP conjugates with laccase labelled by fluorescein isothiocyanate The nanobiocatalyst was able to biotransform industrial wastewaters as 4.3.4. resin intermediate BPA or textile dye MG The oxidative potential of the free and immobilized enzyme for environmental purposes was evaluated for target pollutants such as BPA, an endocrine disrupting compound, and MG, as a triphenyl methane dye. When BPA was considered, higher BPA transformation was achieved using free laccase (>96%) after 2 h, whereas BPA conversion was around 88% for immobilized laccase (Figure 4.6). Other authors also observed slower BPA transformation rates for immobilized laccase (ArcaRamos et al. 2016b). This lower reaction rate was related to the accessibility of the substrate to the enzyme active site (Sun et al. 2015). The activity remained constant in both free and immobilized enzymes. When considering the Chapter 4 92 biotransformation of MG by free and immobilized enzyme, the results showed that alike BPA, free enzyme has a high rate (Figure 4.7). However, the biotransformation rate of the nanobiocatalyst is 1.4 times higher than that observed by Kunamneni et al. (2008) when the MG was decolorized by Myceliophtora thermophile laccase immobilized on epoxy-activated carriers. The higher efficiency implies higher flow of pollutants treated and consequently to a reduction in the treatment cost. The immobilized enzyme maintained activity throughout the oxidation experiment, while for the free enzyme there is a slight decay of up to 25% of its initial activity. In both cases, controls with BPA and MG lacking laccase and with functionalized nanoparticles were performed, with no decrease in BPA or MG concentration (Figure 4.6 and 4.7). Figure 4.4. Biotransformation rate of 10 mg L -1 BPA (•), BPA control (o) and enzymatic residual activity (x) by free (green) and laccase-sMNP conjugates (orange) An eco-friendly nanobiocatalyst: Pycnoporus sanguineus laccase for environmental applications 93 Figure 4.5. Biotransformation rate of 20 mg L -1 MG (•), BPA control (o) and enzymatic residual activity (x) by free (green) and laccase-sMNP conjugates (orange) Reusable and no toxic superparamagnetic nanobiocatalyst for industrial 4.3.5. wastewater applications The reuse of the immobilized enzyme is essential for its practical application in real processes. The potential application was evaluated for 6 oxidation reaction cycles of BPA and MG. When the nanobiocatalayst was considered for the biotransformation of BPA in repeated batch operation of 6 h, above 95% of BPA biotransformation was attain in each cycle, 10% more than in previous studies with Chapter 4 94 Trametes versicolor laccase (Moldes-Diz et al. 2018) (Figure 4.8). MG biotransformation in 4 h-oxidation cycles (Figure 4.8) showed that the decolorization was maintained throughout the six cycles (>85%). Moreover, the nanobiocatalyst activity in both cases was maintained constant after 6 cycles. The above results imply that the nanobiocatalyst is expected to be meaningful in largescale applications. Figure 4.8. Biotransformation (%, solid bars) and residual activity (open circles) of BPA (blue) and MG (yellow) in subsequent cycles of enzymatic treatment with laccase-sMNP conjugates The determination of the toxicity of nanobiocatalysts appears to be an important parameter, as unlike traditional catalysts, there has been no systematic risk characterization of nanomaterials (Jiang et al. 2014). The results obtained from the E. coli culture in the presence of the different samples and concentrations of nanoparticles showed that the current nanobiocatalyst is not toxic to gramnegative bacteria because no inhibitory effect on growth was detected in all scenarios (data not shown). Therefore, the nanobiocatalyst can be used for environmental applications without risk of damage. Successful detoxification of the treated wastewater 4.3.6. The detoxification of model compounds by the nanobiocatalyst is an important parameter to consider, since, as previously reported, transformation products can lead to more toxic metabolites (Champagne et al. 2010, Donner et al. An eco-friendly nanobiocatalyst: Pycnoporus sanguineus laccase for environmental applications 95 2013). Microtox® was carried out to evaluate the toxicity of the laccase-catalyzed transformation products from BPA and MG. Control (untreated solution) and 24-h treated samples showed EC 50%(15 min) values of 5 and 15% for MG, respectively. In the case of BPA, a EC 50% (15 min) value of 28% was obtained for the untreated solution, but no toxicity was detected after enzymatic treatment. Similar results were observed by Dudziak et al. (2015). Therefore, these results suggest the generation of biotransformation products less toxic than the parent ones. Conclusions 4.4. In the present work, an agricultural residue was used for the production of Pycnoporus sanguineus laccase and successfully immobilized onto silica-coated superparamagnetic nanoparticles. The behavior of the immobilized system was compared with that of free laccase. The results obtained showed that the stability of the laccase increased due to the immobilization in terms of pH, deactivating agents and storage. In addition, the immobilized laccase was successfully reused in 6 consecutive biotransformation cycles of BPA and MG and retained approximately 90% of the initial activity. The easy recovery of the nanobiocatalyst from the reaction media is a remarkable advantage from an operational prospecting. Regarding the toxicity impacts associated with the nanobiocatalyst and the biotransformation products, no-toxicity was observed for the nanobiocatalyst and the biotransformation products were less toxic that the parent ones. Therefore, Pycnoporus sanguineus laccase produced for agro-industrial wastes and immobilized onto sMNP could be a promising alternative for improving its stability, cost and large-scale reuse. References 4.5. Arca-Ramos, A., Kumar, V.V., Eibes, G., Moreira, M.T., Cabana, H., 2016a. Recyclable cross-linked laccase aggregates coupled to magnetic silica microbeads for elimination of pharmaceuticals from municipal wastewater, Environ Sci Pollut Res. 23, 8929-8939 Arca-Ramos, A., Ammann, E.M., Gasser, C.A., Nastold, P., Eibes, G., Feijoo, G., Lema, J.M., Moreira, M.T., Corvini, P.F.-X., 2016b. Assessing the use of Chapter 4 96 nanoimmobilized laccases to remove micropollutants from wastewater, Environ Sci Pollut Res. 23, 3217-3228 Bharathiraja, S., Suriya, J., Krishnan, M., Manivasagan, P., Kim. S-K., 2017. Production of enzymes from agricultural wastes and their potential industrial applications, Adv Food Nutr Res. 80, 125-148 Bocchini, D., Ferreira, H., Ribeiro, R., Ferreira, H., Moretti, M., Gomes, E., 2011. Agroindustrial wastes as substrates for microbial enzymes production and source of sugar for bioethanol production, in: Kumar, S. (Ed.), Integrated waste managementVolume II. Intech, New York, pp. 319-360 Cabana, H., Ahamed, A., Leduc, R., 2011. Conjugation of laccase from the white rot fungus Trametes versicolor to chitosan and its utilization for the elimination of triclosan, Bioresorce Technol. 102, 1656-1662 Champagne, P.P., Ramsay, J.A., 2010. Dye decolorization and detoxification by laccase immobilized on porous glass beads, Bioresource Technol. 101, 2230-2235 Chandra, R., Kumar, V., Yadav, S., 2017. Extremophilic Ligninolytic Enzymes. In: Sani, R., Krishnaraj, R. (eds) Extremophilic Enzymatic Processing of Lignocellulosic Feedstocks to Bioenergy. Springer. Dai, Y., Yao, J., Song, Y., Wang, S., Yuan, Y., 2016. Enhanced adsorption and degradation of phenolic pollutants in water by carbon nanotubes modified laccasecarrying electrospun fibrous membranes, Environ. Sci.: Nano. 3, 857-868 Donner, E., Kosjek, T., Qualmann, S., Kusk K.O., Heath, E., Revitt, D.M., Ledin, A., Andersen, H.R., 2013. Ecotoxicity of carbamazepine and its UV photolysis transformation products, Sci Total Environ. 443, 870-876 Dudziak, M., 2015. Microtox as a tool to evaluate unfavorable phenomenon occurrences during micropollutants decompositions in AOPs, ACEE Archit Civ Eng Environ. 8(2), 85-90 An eco-friendly nanobiocatalyst: Pycnoporus sanguineus laccase for environmental applications 97 Gasser, C.A., Ammann, E.M., Schäffer, A., Shahgaldian, P., Corvini, P.F., 2016. Production of superparamagnetic nanobiocatalysts for green chemistry applications, Appl Microbiol Biotehcnol. 100 (16), 7281-7296 González-Coronel, L.A., Cobas, M., Rostro-Analis, M.J., Parra-Saldivar, R., Hernández-Luna, C., Pazos, M., Sanromán M.A., 2017. Immobilization of laccase of Pycnoporus sanguineus CS43, N Biotechnol. 39 A, 141-149 Jiang, C., Jia, J., Zhai, S., 2014. Mechanistic understanding of toxicity from nanocatalysts, Int J Mol Sci. 15, 13967-13992 Kappor, M., Panwar, D., Kaira, G.S., 2016. Bioprocesses for enzyme production using agro-industrial wastes: technical challenges and commercialization potential, in: Dhillon, G., Kaur, S. (Eds.), Agro-Industrial wastes as feedstock for enzyme production. Academic Press, San Diego, pp. 61-93 Kumar, V., Sivanesan, S., Cabana, H., 2014. Magnetic cross-linked laccase aggregates-Bioremediation tool for decolorization of distinct classes of recalcitrant dyes, Sci Total Environ. 487, 830-839 Kunamneni, A., Ghazi, I., Camarero, S., Ballesteros, A., Plou, F.J., Alcalde, M., 2008. Decolorization of synthetic dyes by laccase immobilized on epoxyactivated carriers. Process Biochem. 43, 169-178. Lloret, L., Hollmann, F., Eibes, G., Moreira, MT., Lema, JM., 2012. Immobilisation of laccase on Eupergit supports and its application for the removal of endocrine disrupting chemicals in a packed-bed reactor. Biodegradation. 23(3), 373-386 Moldes-Diz, Y., Gamallo, M., Eibes, G., Vargas-Osorio, Z., Vázquez-Vázquez, C., Feijoo, G., Lema, J.M., Moreira, M.T., 2018. Development of a superparamagnetic laccase nanobiocatalyst for the enzymatic biotransformation of xenobiotics, J Enviro Eng. In press, DOI: 10.1061/(ASCE)EE.1943-7870.0001333 Chapter 5 104 Introduction 5.1. In Chapter 3 and 4, silica coated superparamagnetic nanoparticles demonstrated to be a good support for immobilization of two different laccases (Trametes versicolor and Pycnoporus sanguineus) and the nanobiocatalysts performed high transformation rates of different compounds present in wastewater treatment plants. However, the versatility and robustness of the nanobiocatalyst for different technologies was no clarified. For many years, fossil fuels have been the main source for plastic building blocks. However, due to the growing environmental concerns, there is an enormous interest to gradually move from traditional fossil fuel derived feedstocks towards a more sustainable and renewable biomass. In this context, 5-hydroxymethylfurfural (HMF) has emerged as one of the 14 top biomass platform molecules for the sustainable future (Wrigsted et al. 2017, Cherubini 2010). In fact, recently new catalytic routes have been developed to transform HMF into building blocks for plastics such as dimethylfuran (DFF) or 5-formyl-2-furancarboxylic acid (FFCA). New unexploited enzymes as Galactose oxidase (GAO) and Aryl alcohol oxidase (AAO) reported to oxidize of primary alcohols to corresponding aldehydes while reducing molecular oxygen to hydrogen peroxide (Ito et al. 1994, Parikka et al. 2010) It presents a broad substrate tolerance for alcohols, but strict stereospecificity (Pickl et al. 2015). Oxidation of alcohols to carbonyl compounds is one of the most important reactions in synthetic chemistry; thus, enzymatic biocatalysts requiring only molecular oxygen as an oxidant is a valuable alternative to chemicals. It has recently been claimed that some variants of galactose oxidase and aryl alcohol oxidase transform 5-HMF in DFF and FFCA (Karich et al. 2018) (Figure 5.1). Figure 5.1. Pathway of 5-HMF oxidation by enzymatic treatment Superparamagnetic nanobiocatalyst to biotransform micropollutants from wastewater 105 Nevertheless, to scale-up the process, the reusability of the biocatalyst is a crucial point and therefore, an immobilization strategy should be selected. This study aims to produce a robust biocatalyst for the transformation of HMF to DFF and FFCA with GAO and AAO, respectively. For this purpose, enzymes were immobilized on silica-coated superparamagnetic nanoparticles that permits an easy recovery of the biocatalyst while maintaining an acceptable degree of enzymatic activity. Materials and methods 5.2. Chemicals, nanoparticles and enzymes 5.2.1. 3-(aminopropyl)triethoxysilane (APTES) (≥98%), glutaraldehyde (25%). Silicacoated magnetic nanoparticles (smNP) were supplied by Nanogap (Ames, Spain). Galactose oxidase (GAO) from Aspergillus oryzae and recombinant Aryl alcohol oxidase from Pleurotus ostreatus were provided by Novozymes and Environmental Biotechnology Laboratory (IHI Zittau, Technical Dresden University, Germany), respectively. Immobilization of GAO and AAO on superparamagnetic silica coated 5.2.2. nanoparticles Prior to immobilization smNP were aminofunctionalized under the reaction of APTES (0.8 mmol APTES g -1 fsNP/smNP) for 24 h at room temperature. When the reaction was completed APTES excess was removed from aminofuctionalized nanoparticles by 4 washed cycles with phosphate buffer (100 mM, ph 7). The support (5 g L -1 ) was incubated with GAO and AAO enzyme (1.88 U mg -1 smNP) at 4°C for 2h and 100 rpm in an orbital shaker (C24 Icubator shaker, New Brunswick Scientific, NJ). Thereafter, 8 mmol glutaradehyde per gram of nanoparticles was added and the reaction was completed at 4°C for at least 12 h. Once formed, the nanoparticles were washed 5 times with phosphate buffer (100 mM, pH 7). Immobilization efficiency was calculated as previously described in Chapter 2. HMF biotransformation with GAO and AAO 5.2.3. A screening of the different enzymes (1000 U L -1 ), at variable pH values (6-8), was investigated for the transformation of 5-HMF (2 mM), in order to evaluate the Chapter 5 106 selection of the enzyme to immobilized and use in different cycle, in 10-mL flasks for 24 h. Thereafter, the oxidation of 5-HMF was conducted by GAO and AAO immobilized on smNP at phosphate buffer (100 mM, pH 6) for 24 h and 1000 U L -1 of initial activity. In parallel, controls lacking enzyme but supports were also carried out to verify that oxidation took place only by enzymatic treatment. Consecutive cycles of batch biotransformation of 5-HMF by AAO 5.2.4. immobilized on smNP The operation of the enzymatic system was conducted in a tank reactor (100 mL) under stirring at room temperature for several consecutive cycles. The reaction medium consisted of 5-HMF (2 mM), phosphate buffer (100 mM, pH 6), and a single initial pulse of AAO (1000 U L -1 ) immobilized onto smNP. The effluent of the reactor was withdrawn at the end of the cycle and the nanobiocatalyst was recovered by an external magnetic field before a new cycle started. Samples were withdrawn at the beginning and at the end of each cycle to measure laccase activity and 5-HMF oxidation. Analysis of 5-HMF transformation products 5.2.5. The oxidation products from HMF were determined by high-performance liquid chromatography (HPLC). The analysis was performed on a Jasco XLC HPLC (Jasco Analitica) equipped with a 3110 MD diode array detector (detection at 280 nm) and an Aminex HPX-87H column maintained at 60°C. Gradient elution (flow rate of 0.6 mL min -1 ), with 5 mM of H 2 SO 4. Genotoxicity of the nanobiocatalyst 5.2.6. The genotoxicity of the nanobiocatalyst was assessed. For this purpose, nine different concentrations of enzyme-sMNPs conjugates (12.5, 25, 50, 100, 200, 400, 600, 800 and 1000 mg L -1 ) were dispersed in a final volume of 1 mL, containing 200 µg mL -1 of DNA in 100 mM of PBS. A control was run in parallel containing only DNA in PBS. The mixtures were incubated for 1 h at 37°C. After incubation, the testing solutions were subjected to electrophoresis in 0.75% (w/v) agarose gels, prepared with 40 mM Tris buffer (pH 7.6) containing 20 mM acetic acid and 1 mM EDTA. Gels were run in the above buffer, at 2.5 A for 1.25 h and the DNA bands were visualized using a molecular imager GELDOC XR+ (BioRad, Hercules, California, USA) and the Superparamagnetic nanobiocatalyst to biotransform micropollutants from wastewater 107 resulting image was processed using Image Lab Software v5.1 (BioRad, Hercules, California, USA). The band area for DNA positive control was manually defined (to measure the band intensity) and then copied into each sample lane, with the decrease in band intensity being considered as a result of a reduction of the amount of DNA present. The results were expressed as the percentage of DNA degradation calculated as the difference of the intensity of each sample and intensity of the background divided by the intensity of the intact DNA solution. All incubations were made in triplicate and loaded twice into the gel. Results and discussion 5.3. HMF biotransformation transformation by GAO and AAO 5.3.1. Due to the remarkable effect of pH on 5-HMF conversion reported in previous works (Karich et al. 2018), the biotransformation was assessed at different pH levels (6, 7 and 8) for free enzymes. The conversion efficiencies are shown in Table 5.1. An improvement of 5-HMF conversion was observed when pH decreased to 7 or 6, which lead to an increase of 10%. Furthermore, the enzymatic activity was maintained constant in all the experiments. Therefore, in subsequent experiments, pH 6 was selected as the optimal for 5-HMF transformation. Table 5.1. 5-HMF transformation rates for GAO and AAO at different pH. pH Biotransformation rate (mmoles L -1 h -1 ) GAO AAO 6 0.044±0.05 2.52±0.36 7 0.035±0.05 2.10±0.22 8 0.042±0.03 1.74±0.08 GAO and AAO was successfully immobilized, as related in Chapter 2, with enzyme loadings of 1.19±0.02 U mg -1 smNP and 1.08±0.03 U mg -1 smNP, respectively. In Figure 5.3 and 5.4 show the time-dependent formation of HMF oxidation products catalyzed by the nanobiocatalysts. The reaction was chosen based on the previous results presented above for free enzymes. Complete biotransformation of HMF on FFCA was observed by AAO in less than 4 hours Chapter 5 108 (Figure 5.2), whereas GAO biotransform up to 48% of HMF into DFF after 18 h (Figure 5.3). These results are in concordance with previous works where it was found that free GAO did not oxidize DFF into FFCA, but produced other unknown products (Karich et al. 2018). However, and enhance on biotransformation rates was observed in comparison with free enzyme. Figure 5.2. HMF concentration (●), HMF control lacking laccase (o), DFF concentration (●) and FFCA concentration (●) by AAO immobilized on smNP(). Figure 5.3. HMF concentration (●), HMF control lacking laccase (o), DFF concentration (●) and FFCA concentration (●) by GAO immobilized on smNP(). Superparamagnetic nanobiocatalyst to biotransform micropollutants from wastewater 109 Biotransformation of 5-HMF by AAO immobilized on silica-coated 5.3.2. superparamagnetic nanoparticles in sequential batch reaction The reusability of the nanobiocatalyst was assessed in consecutive cycles of 4 h. It was observed that HMF transformation was higher than 90% and was maintained constant after 6 cycles (Figure 5.4). Moreover, the immobilized enzyme retained 97% of its initial activity after the consecutive batch treatments of 5-HMF with magnetic separation. Figure 5.4. HMF transformation () in consecutive cycles by AAO immobilized on smNP (●). Evaluation of genotoxicity 5.3.3. This study contributes to an understanding of the potential effects that the catalysis based on iron-based nanoparticles may have on the environment and human health. To guarantee its safety, nanobiocatalyst must not be toxic to the cells at concentrations suitable for the biotransformation of products. In previous studies was reported that silica-coated magnetic nanoparticles exhibit genotoxicity activity when the concentrations of the nanoparticles remaining below 100 µg L -1 (Fernández-Bertólez et al. 2018, Laurent et al. 2014). The genotoxicity results in this case indicate that no DNA damage was found when applying the smNP-based nanobiocatalyst (Figure 5.5) in the range of 12.5-1000 mg L -1 . This result is contradictory to findings stated in the study published by Královec et al. (2019) where silica-coated iron oxide nanoparticles strongly increased the levels of DNA damage. However, the DNA damage is directly related to iron release which is not Chapter 5 110 detected in our nanobiocatalyst, suggesting that surface engineering of the silica coated superparamagnetic nanoparticles created a safe nanosystem for diverse applications. Figure 5.5. Schematic representation of the processing of the electrophoretic results and DNA damage for the nanobiocatalyst for different concentrations (12.5-1000 mg L -1 ) at pH 7 Conclusions 5.4. The study demonstrated that it is possible to use the silica-coated superparamagnetic nanoparticles as support of different enzymes, such as GAO and AAO, and used as precursor for enzyme cascade reactions to produce added value bio-based products (DFF and FFCA) with high efficiency rates. One of the main results of this study is that the nanobiocatalyst is magnetically recoverable and can be reused in repeated HMF biotransformation cycles. The easy recovery of the nanobiocatalyst from the reaction media is a significant advantage from an operational perspective. Moreover, the nanobiocatalyst does not present effect on DNA damage, suggesting that it is a safe nanosystem to be use in different applications. Further research should focus on the development of an enzymatic magnetic reactor to demonstrate the scalability of the process. Superparamagnetic nanobiocatalyst to biotransform micropollutants from wastewater 111 References 5.5. Wrigstedt, P., Keskiväli, J., Perea-Buceta, J. E., & Repo, T. 2017. One-pot transformation of carbohydrates into valuable furan derivatives via 5Hydroxymethylfurfural. Chem Cat Chem, 9(22), 4244-4255. Cherubini, F. 2010. The biorefinery concept: Using biomass instead of oil for producing energy and chemicals. Energy Conversion Management, 51: 1412142 Ito,N., S.E.V. Phillips,, K.D.S. Yadav,, P.F. Knowles (1994) Crystal structure of a free radical enzyme, Galactose Oxidase. J. Mol. Biol. 238(5) 704–814. Parikka, K, A.-S. Leppänen,, L. Pitkänen,, M. Reunanen,, S. Willför, M. Tenkanen, J. .2010. Oxidation of Polysaccharides by Galactose Oxidase Agric. Food Chem. 58(1): 262–71. Pickl, M., Fuchs, M., Glueck, S. M., & Faber, K., 2015. The substrate tolerance of alcohol oxidases. Applied Microbiology and Biotechnology, 99(16): 6617–6642. Karich, A.; Kleeberg, S.B.; Ullrich, R.; Hofrichter, M., 2018. Enzymatic preparation of 2,5-Furandicarboxylic acid (FDCA)—A Substitute of Terephthalic Acid—By the Joined Action of Three Fungal Enzymes. Microorganisms 6, 5. Královec, Havelek,R. Kročová,E., Kučírková, L., Hauschke, M., Bartáček,J., Palarčík, J., Sedlák,M. 2019. Silica-coated iron oxide nanoparticles-induced cytotoxicity, genotoxicity and its underlying mechanism in human HK-2 renal proximal tubule epithelial cells. Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 844: 35-45. Laurent, S., Saei, A.A., Behzadi, S., Panahifar, A., Mahmoudi, M., 2014. Superparamagnetic iron oxide nanoparticles for delivery of therapeutic agents: opportunities and challenges. Expert Opin. Drug Deliv. 11, 1449–70. Chapter 5 112 Fernández-Bertólez, N., Costa,C., Brandão, F., Kiliç, G., Duarte,J., Teixeira, J., Pásaro, E., Valdiglesias, C., Laffon,B. 2018. Toxicological assessment of silicacoated iron oxide nanoparticles in human astrocytes, Food and Chemical Toxicology, 118:13-23. Chapter 6 Development of an enzymatic reactor with internal magnetic separation for biotechnological applications A new sequential batch reactor (SBR) coupled to an internal magnetic separator was developed. The separator consists of a set of axially magnetized permanent toroidal magnets, distributed along a non-magnetic steel rod, uniformly spaced with alternate polarity, which provide an external magnetic field of up to 1.2 T. The feasibility of magnetic separation was assessed for the retention of the nanobiocatalyst based on laccase immobilized on silica-coated mNPs. The proof of concept was evaluated for the enzymatic decolorization of the MG dye and the transformation of HMF, with complete recovery of the nanobiocatalyst (99%) and high biotransformation efficiency of both compounds. The characterization of the reaction products of MG by laccase was conducted HPLC-DAD-ESI-MS. Moreover, the biotransformation products showed less microtoxicity than the parent compound and increased biodegradability. Beyond laboratory scale experiments, the reactor proposed here was scaled-up to a volume of 100 L and the environmental performance and cost analysis were estimated. Chapter 6 120 magnets were simulated using the following physical data: relative magnetic permeability (T) of 1.05 and remnant field (BT) of 1.3 T. 6.2.4. Biotransformation of MG and HMF in the magnetic SBR by laccase immobilized onto silica-coated magnetic nanoparticles The influent containing MG (20 mg L-1 in 100 mM phosphate buffer, pH 6) was fed to the magnetic reactor with immobilized laccase added in a single initial pulse of 200 U L-1. The reactor was operated in cycles of 6 h and at the beginning of each cycle, laccase activity was measured. In parallel, a control experiment with functionalized magnetic nanoparticles lacking laccase was also performed. The effluent was freeze-dried to evaluate the effect of the different biotransformation products (BP) at concentrations between 0.5-5 g L-1 on laccase activity after 24 h. The percentage of dye decolorization was calculated from the decrease in the characteristic absorbance of MG (630 nm). Regarding the biotransformation of HMF, the influent (2 mM in 100 mM phosphate buffer, pH 6) was transformed in consecutive cycles with 1000 U L-1 of AAO. The oxidation products from HMF were determined by High-Performance Liquid Chromatography (HPLC). The analysis was performed on a Jasco XLC HPLC (Jasco Analitica) equipped with a 3110 MD diode array detector (detection at 280 nm) and an Aminex HPX-87H column maintained at 60°C. Gradient elution (flow rate of 0.6 mL min-1), with 5 mM of H2 SO4. 6.2.5. Envisioning the biotransformation of MG present in a textile effluent To evaluate the influence of the composition of a secondary effluent on enzymatic stability and the rate of MG biotransformation, experiments were carried out with the secondary effluent of a WWTP with an initial TOC and COD of 6.97 mg L-1 and 15.36 mg L-1, respectively. The secondary effluent was spiked with the dye (20 mg L-1), simulating the composition expected in a real textile effluent. The reactor operated for 6 cycles with immobilized laccase added in a single initial pulse of 100 U L-1. The percentage of dye decolorization was calculated from the decrease in the characteristic absorbance of MG (630 nm). Moreover, the monitoring of the total organic carbon (TOC) concentration using a Shimadzu TOC-L equipment made it possible to determine the degree of dye removal due to enzymatic treatment. Development and modelling of an enzymatic reactor with internal magnetic separation for biotechnological applications 121 6.2.6. Identification of laccase-catalyzed reaction products from MG decolorization The identification of MG oxidation products by laccase was conducted in a batch experiment. Each reactor (20 mL) contained 20 mg L-1 of MG in an ammonium acetate buffer (pH 6) with an initial laccase activity of 1000 U L-1. Samples were periodically withdrawn and analysed by High Performance Liquid Chromatography (HPLC) with a diode array detector (DAD) coupled to Mass Spectrometry (MS) using the Electrospray Ionization Source (ESI) (HPLC-DAD-ESIMS) to follow the appearance of the different metabolites obtained from the transformation of the parent compound. The analysis of MG intermediates was performed according to the methodology developed and described by Mai et al (2008). Two different kinds of solvents were prepared in this study. Solvent A was 25mM aqueous ammonium acetate buffer (pH 6) while solvent B was methanol instead of ammonium acetate. LC was caried out on an Zorbax® Eclipse XDB-C18 column (250 mm x 4.6 mm i.d., dp=5µm). The flow rate of the mobile phase was set at 1.0 mL min-1. A linear gradient was set as follows: t=0, A=95, B=5; t=20, A=50, B=50; t=60, A=10,B=90; t=65, A=95, B=5. Mass spectrometric detection was performed on a G6410B triple quadrupole mass spectrometer (Agilent, USA) equipped with an ESI. The ESI source was operated with heated nebulizer probe at 350°C. ESI was carried out with nitrogen as sheath (240 kPa, 10 L min-1) with the preliminary nebulization and to initiate the ionization process. Capillary voltage was optimized for the maximum response during perfusion of the MG standard (4500 V). 6.2.7. Toxicity and biodegradability assays In order to investigate the potential toxicity of the transformation products obtained after enzyme treatment, a Microtox® test based on the luminescent marine bacterium Vibrium fischeri was performed using a Microtox® model 500 Analyzer according to the protocol defined in the Standard Methods, 1999. The results were expressed as half of the maximum effective concentration at 15 min (EC50, 15min), which corresponds to the concentration of the pollutant that causes a 50% reduction in the light output of Vibrium fischeri after 15 min incubation. Chapter 6 122 Aerobic biodegradability was determined by monitoring oxygen consumption by aerobic sewage sludge in duplicate assays of treated and untreated effluents for 5 days. The concentration of total and volatile solids in aerobic sludge samples was quantified according to Standard Methods (1999), at values ranging from 3.1 to 3.4 g L-1 and 2.1 to 2.4 g L-1, respectively. After sampling, the sludge was washed with phosphate buffer (200 mM, pH 7) and stored at 4°C. Oxygen consumption was measured with an automated OxiTop device (WTW) according to the Standard Methods (1999). The technique was based on the reduction of pressure inside the closed flasks containing the inoculated sample, being proportional to oxygen consumption. Anaerobic biodegradability was determined by monitoring methane production in triplicate assays of treated and untreated effluent for 17 days in 100 mL bottles. Anaerobic sewage sludge was used as inoculum, and the biomass concentration was fixed at 2 g L-1 of volatile suspended solids (VSS). Biogas production was monitored using a pressure transducer (Centrepoint Electronics), and its composition was analyzed by Gas Chromatography (HP, 5890 Series II), using helium as carrier gas. 6.2.8. Environmental performance and cost analysis The design of the reactor was adapted to a scale of 100 L with a daily flow of 400-600 L d-1 for a hydraulic retention time of 4-6 h, consisting on the elements described in Section 2.4 coupled with four magnetic bars inside the reactor to retain the nanobiocatalyst. Environmental performance The environmental performance of the enzymatic treatment was analyzed according to the Life Cycle Analysis (LCA) methodology and compared with a more widespread advanced oxidation process, such as ozonization. LCA has proven to be a versatile methodology for quantitatively assessing the environmental impacts of products and services (Baumann et al. 2004). To apply the LCA methodology, inventory data from laboratory and pilot plant experiments was collected. Background data (electricity and chemical production) were obtained from the Ecoinvent® database (Doka, 2003, Althaus et al. 2007, Dones et al. 2007). To ensure the validity of the comparison, the functional unit of the study was defined as the Development and modelling of an enzymatic reactor with internal magnetic separation for biotechnological applications 123 color removal (90%) from an effluent containing 20 mg L-1 of the dye MG for a treatment volume of 1 m3. The environmental assessment was conducted using ReCiPe Midpoint characterization factors (Goedkoop et al. 2018) and the following impact categories were considered in the analysis: climate change (CC), ozone layer depletion (OD), terrestrial acidification (OT), freshwater eutrophication (EF), marine eutrophication (MEs), human toxicity (HT), photochemical oxidant formation (POF), freshwater ecotoxicity (FET), marine ecotoxicity (MET) and fossil depletion (FD). SimaPro 8.02 (PréConsutants, 2018) was the software used for the computational implementation of life cycle inventory data and the calculation of environmental profiles. Cost analysis The estimation of capital costs included the reactor vessel, the magnetic separation unit, pumps and PLC control, with a depreciation period of 10 years. The operational costs included the production of the nanoparticles and enzyme, processes that differed significantly in terms of production scheme, chemical and energy requirements, performance and availability. Consequently, different supports (silica-coated magnetic nanoparticles and magnetized silica nanoparticles) and enzymes (Laccase and Manganese Peroxidase) were considered. For the electricity cost, a tariff of 0.114 € kWh-1 was considered (Eurostat, 2018) 6.3. Results and discussion 6.3.1. Characterization of the magnetic reactor and modelling of the magnetic field Figure 6.3 shows a simulation of axial slices of the magnet arrays (limited to 4 for better clarity) of both configurations, alternate and series polarity, showing magnetic fields in the range of 50 mT to 1.2 T. In the case of series arrangement, the magnetic field reaches high values but only at the interspaces between the individual magnets and at the top and bottom of the bar. Chapter 6 124 Figure 6.3. Modelling of magnetic fields (T) produced by both configurations of magnets array: alternate and series polarity. The magnetic field goes from the north pole of one magnet to the south pole of the adjacent magnet and there is no effect on the outer volume bounded by the glass sheath. For the alternate configuration, when placing face-to-face poles of the same polarity, the magnetic field lines must extend to find the opposite pole, and consequently, the high gradient area (that defines the intensity of the magnetic field) is remarkably superior. It was found that for the alternate one, the magnetic field at 8.5 mm from the axis (corresponding to the outer face of the sheath) has a maximum of about 400 mT, whereas at 11 mm it is below 100 mT. The resulting gradient is approximately 120 T mm -1 . According to the magnetic force equation: F = (m ·) B (eq. 1) where the magnetic force depends on the magnetic moment (m) and magnetic field (B), the mobility of individual particles of approximately 100 nm would not exceed a few mm s -1 . However, due to agglomeration, the resulting magnetic moment would increase more rapidly than the decrease in mobility, allowing the separation and accumulation in the outside of the sheath (Mandel et al. 2012). In order to demonstrate the retention capacity of the nanobiocatalyst by the magnetic separation system, the concentration of ferrous ions in the effluent was monitored and it was found that the recovery of the magnetic nanoparticles after 2 min was greater than 99%, which was repeatedly maintained throughout the operation (10 cycles). Development and modelling of an enzymatic reactor with internal magnetic separation for biotechnological applications 125 Recent research has also used magnetic enzyme reactors, such as Wang et al. (2012) who applied a magnetically stabilized fluidized bed reactor for phenol degradation in coking wastewater or Duan et al. (2014) who used a reactor with a system of electromagnets and a microcontroller. These reactors require an energy source for the operation of the electromagnet, with the drawbacks of power consumption and temperature increase. In contrast, the use of an internal magnetic separation unit is an alternative to these configurations, with a higher magnetic field on the outside of the sheath, which leads to an efficient recovery of the nanobiocatalyst without associated energy consumption. 6.3.2. Decolorization of MG by laccase immobilized onto silica-coated magnetic nanoparticles As presented in the description of the operational sequence of the enzymatic magnetic reactor, it is mandatory to establish the reaction time required for the biotransformation of the target compound, in this case, to accomplish extensive decolorization of the dye. Moreover, other basic variables such as pH and enzyme activity must be selected prior to reactor operation and will be application-specific. The influence of pH on MG decolorization (20 mg L-1) and enzyme stability was investigated in batch experiments in a range of 3-7 for free and immobilized enzymes (Table 6.1). An improvement in MG transformation was observed when pH was increased from acidic to neutral conditions. However, MG decolorization was also found in the control with silica-coated mNPs lacking laccase at pH 3 and 7. After 6 h of incubation, 40% and 80% of MG were decolorized at pH 3 and 7, respectively. The coloured cation of triphenylmethyl dyes in basic and acidic medium becomes a carbinol (non-resonant) base (Mohammed et al. 2010). Hassan et al. (2011) found that after 4 h of MG incubation at pH 4.5 and 9.6, the concentration decreased by about 10% and 30% respectively. Moreover, a higher MG decolorization was achieved by free laccase for the entire pH range (Table 6.1). The lower oxidation of the immobilized laccase to phenolic compounds was also observed by other authors. Arca-Ramos et al. (2016) found that this lower oxidation capacity was related to the aggregation of the nanobiocatalyst, which could reduce the accessibility of the substrate. Chapter 6 126 Table 6.1. MG biotransformation and enzyme stability at variable pH by free laccase and laccase immobilized onto mNP for 24 h Biotransformation rate (M h-1) Control decolorization (%)a Residual activity (%)b pH Free Immobilized Free Immobilized Free Immobilized 3 2.27 1.96 42.0 35.0 0.67 66.2 5 5.08 2.83 6.0 4.0 53.9 83.8 6 4.67 4.46 8.0 5.0 78.1 89.5 7 5.89 3.50 93.0 85.0 99.5 99.8 The stability of the nanobiocatalyst and consequently its potential for aggregation is associated with attractive and repulsive forces determined by the zeta potential. Figure 6.4 shows the zeta potential profile of the nanobiocatalyst for a pH range between 2 and 12. Thus, the point of zero charge (pHzc) is around pH 5.3. Above this pH, the nanobiocatalyst is negatively charged, which is consistent with previous results where higher biotransformation rates were observed from pH 5-7 (Table 6.1), since the dye is positively charged and there are no repulsive forces between the dye and the nanobiocatalyst. However, the zeta potential values are less than -30 mV (Figure 6.4), which indicates that the nanobiocatalyst is not completely stable and may present some aggregation and therefore the accessibility of the substrate is reduced in this case. Development and modelling of an enzymatic reactor with internal magnetic separation for biotechnological applications 127 Figure 6.4. Zeta potential curve obtained for laccase immobilized onto Fe 3 O 4 @SiO 2 suspended in distilled water environment. Enzymatic stability, similar to biotransformation, was significantly improved in a pH range of 3 to 7. However, a slight difference was observed between the free and immobilized enzyme in acidic conditions. After 24 h, the free enzyme retained only 0.7% of its initial activity at pH 3 while the immobilized enzyme maintained 66.2% of its activity at the beginning of the experiment (Table 6.1). Therefore, in subsequent experiments, pH 6 was selected as the optimal for MG decolorization. 6.3.3. Sequential batch reactor for the biotransformation of methyl green and HMF by laccase immobilized onto magnetic nanoparticles The designed reactor was used to assess the ability of immobilized laccase to biotransform MG and HMF in repeated 6-h cycles. Immobilized laccase reached a percentage of MG decolorization higher than 95% in the first cycle, decreasing slightly to 87% after the tenth cycle which implies that 90 U L -1 is sufficient to achieve high decolourization values (Figure 6.5). Kunamneni et al. (2008) studied the decolorization of MG by laccase immobilized on epoxy-activated carriers, but decolorization was 67% lower. The slight decay on biotransformation after 10 cycles may be due to the accumulation of biotransformation products that influence the oxidation capacity of the nanobiocatalyst. Chapter 6 128 Figure 6.5. MG decolorization (%, yellow bars) and enzyme activity (○) in subsequent cycles of enzymatic treatment with laccase immobilized onto mNP. Different concentrations of biotransformation products between 0.5-5 g L -1 were tested with 200 U L -1 of immobilized enzyme at acetate buffer pH 5 after incubation for 24 h. The results show that higher values of biotransformation products led to a decline in enzyme activity (Figure 6.6). Figure 6.6. Biotransformation products effect on enzyme activity at different concentrations Development and modelling of an enzymatic reactor with internal magnetic separation for biotechnological applications 129 The reactor was also operated for the biotransformation of HMF. Immobilized laccase achieved the complete transformation of HMF (2 mM) into FFCA the two first cycles and it was almost 60% in the all other cycles (Figure 6.7). On the other hand, after the second cycle, activity dropped 20% but remained constant thereafter. Figure 6.6. Biotransformation rates of HMF to FFCA 6.3.4. Envisioning the biotransformation of MG present in a textile effluent The capacity of the nanobiocatalyst to biotransform MG present in a textile effluent was evaluated. The immobilized laccase reached a percentage of MG decolorization higher than 95% (Figure 6.8), and was maintained after 6 cycles (Figure 6.9). The immobilized laccase retained 95% of its initial activity after consecutive batches of MG decolorization with magnetic separation. Although the decolorization of MG was almost complete after 6 h, the removal of TOC was determined to be 38%. These findings imply that mineralization by enzymatic treatment is not successful and that biotransformation products of MG can be obtained.