La presencia de compuestos recalcitrantes en efluentes, suelos y sedimentos es un problema medioa... more La presencia de compuestos recalcitrantes en efluentes, suelos y sedimentos es un problema medioambiental de especial relevancia. Los compuestos de baja solubilidad tales como hidrocarburos aromáticos policíclicos (HAPs) tienen una alta persistencia ya que, debido a sus propiedades físico-químicas, su degradación por las bacterias autóctonas se ve limitada. Con el objetivo de desarrollar un sistema capaz de llevar a cabo la degradación de estos compuestos, se ha planteado la utilización de los hongos ligninolíticos o bien sus enzimas oxidativas. La enzima manganeso peroxidasa (MnP) promueve la oxidación de Mn +2 a Mn +3 , el cual actúa como un poderoso agente oxidante que oxida compuestos orgánicos recalcitrantes de forma inespecífica. La degradación de compuestos de baja solubilidad mediante la enzima MnP requiere la adición de un agente que incremente la solubilidad del compuesto a fin de facilitar la acción de la enzima. En este trabajo se plantea la adición de disolventes miscibles e inmiscibles como alternativas para incrementar la solubilidad de un HAP modelo tal como el antraceno de cara a su degradación enzimática.
The oligomerization of esculin by laccase in aqueous medium resulted in a precipitate fraction wi... more The oligomerization of esculin by laccase in aqueous medium resulted in a precipitate fraction with excellent antioxidant properties.
The oligomerization of esculin by laccase in aqueous medium resulted in a precipitate fraction wi... more The oligomerization of esculin by laccase in aqueous medium resulted in a precipitate fraction with excellent antioxidant properties.
Bioreactor systems have been developed as alternatives to standard culture flasks due to their ho... more Bioreactor systems have been developed as alternatives to standard culture flasks due to their homogeneous nature, easiness of monitoring and increased cell production. Here we investigated the in vitro expansion of bone marrow (BM) mesenchymal stem cells (MSC) in spinner flasks, using gelatin microcarriers (Cultispher S) to support cell adhesion and proliferation. MSC expansion was performed using a low-serum containing medium (2% of fetal bovine serum, FBS).A strategy was defined for the maximization of cell expansion: microcarriers were pre-coated with FBS in order to increase cell seeding efficiency and an adequate feeding regime was established (25% medium exchange everyday). The maximum cell density, 4.2 × 105 cells/mL, was obtained at day 8, corresponding to a fold increase in total cell number of 8.4 ± 0.8. Expanded MSC retained their differentiation potential into adipogenic and osteogenic lineages, as well as their clonogenic ability. Harvested cells expressed >90% of CD73, CD90 and CD105 markers.These results demonstrated that a microcarrier-based stirred culture system is adequate for human MSC expansion, using a low-serum containing medium, allowing the generation of significant cell numbers for potential applications in regenerative medicine.
The presence of recalcitrant compounds in both wastewaters and soils is an important environmenta... more The presence of recalcitrant compounds in both wastewaters and soils is an important environmental problem. Oxidative enzymes from white-rot fungi have been successfully utilised for the in vitro degradation of xenobiotics, such as the azo dye Orange II and the polycyclic aromatic hydrocarbon anthracene (compounds with high and low solubilities, respectively). Two different reactor configurations are proposed: (i) an enzymatic membrane reactor for the treatment of soluble compounds, consisting of a continuous stirred tank reactor coupled to an ultrafiltration membrane to facilitate the retention and recycling of enzyme; and (ii) a two-phase enzymatic reactor for the degradation of poorly soluble compounds, consisting of an immiscible solvent, which contains the contaminant at high concentrations, and the aqueous phase containing the enzyme and cofactors involved in the catalytic cycle. In this paper, factors affecting the design and operation of both systems are discussed, and experimental results concerning the efficiency and stability of the processes are presented.
ABSTRACT Although the removal of steroid estrogens by enzymatic treatment with free fungal enzyme... more ABSTRACT Although the removal of steroid estrogens by enzymatic treatment with free fungal enzymes has been already demonstrated in previous batch experiments, the main limitations associated to this system are the low productivity and degradation yield, typical of batch operations, as well as the unfeasibility of reusing the enzyme. With the aim of overcoming these drawbacks, an enzymatic membrane reactor (EMR) was designed for the continuous removal of estrone (E1) and estradiol (E2) by a commercial laccase from Myceliophthora thermophila. The use of an ultrafiltration membrane allowed the separation of the enzyme according to its high molecular weight while the degradation products could pass through it. Not only the continuous operation was proved, but also different operational parameters were assessed for their relevance on the efficiency of the process. Accordingly, the effects of laccase activity (500-1,000 U/L), hydraulic residence time (1-4 h) and oxygen frequency supply (2-8 pulses of oxygen each HRT) were investigated. Removal efficiencies between 64-100 % and degradation rates of 0.96-2.92 mg/(L.h) were attained. Furthermore, the residual estrogenic activity of the effluent was largely reduced up to 97 %. The present work proves the feasibility of using a laccase-mediated reactor for the continuous removal of estrogenic compounds.
ABSTRACT A Two Phase Partitioning Bioreactor (TPPB) was proposed to carry out the degradation of ... more ABSTRACT A Two Phase Partitioning Bioreactor (TPPB) was proposed to carry out the degradation of the poorly soluble compound anthracene by laccase from Trametes versicolor. The organic phase consisted of silicone oil saturated with anthracene. The surfactant (Triton X-100) was added to the aqueous phase at concentration above the critical micelle concentration (CMC) to enhance anthracene solubility. The feasibility of reusing the aqueous phase, containing the laccase-mediator system was tested in three consecutive cycles of 24 h with an initial laccase activity of 1200 U/L and 1 mM of the mediator 1-hydroxybenzotriazole (HBT). In the first cycle, total degradation of anthracene took place at a high conversion rate: 16 mu mol/L(R)h. The degree of removal achieved in two subsequent cycles with no extra addition of laccase or HBT were 68 % and 40 % for a laccase activity of 150 and 90 U/L in each cycle respectively. These results proved the potential of a TPPB including a surfactant for the removal of poorly soluble compounds by the enzyme laccase and the possibility of exhausting the oxidative potential of the aqueous phase in consecutive cycles to minimize consumption of both laccase and the mediator.
The degradation of anthracene by laccase from Trametes versicolor in enzymatic reactors was evalu... more The degradation of anthracene by laccase from Trametes versicolor in enzymatic reactors was evaluated. The use of a surfactant (Triton X-100) at concentration above critical micelle concentration (CMC) enhanced anthracene solubility and facilitated its degradation. Moreover, Triton exerted a beneficial effect on the laccase stability and protected it from the oxidative action of the mediator 1-hydroxybenzotriazole (HBT). In a further stage, the combined configuration of a two phase partitioning bioreactor (TPPB) operating with silicone oil as an immiscible solvent and the surfactant achieved the degradation of anthracene at higher conversion rate: 16 mol/L R h. Furthermore, a model for anthracene degradation by laccase-mediator system was developed. The first order kinetic constant (k) and the overall mass transfer coefficient (K L a) were estimated by using the method of least squares. The increased K L a value obtained, 788.1 h −1 , proved that Triton also improved mass transfer. Anthracene concentration in aqueous phase was close to that corresponding to equilibrium state suggesting that mass transfer mechanism did not limit the global process. The kinetic constant, which is expected to depend on the initial concentration of enzyme, resulted in 52.2 h −1 . Enzyme inactivation occurred in two stages and could be modeled by using a three parameter biexponential model. The possibility of reusing silicone oil to dissolve more anthracene was proven in three consequent cycles with high percentages of anthracene removal.
Roasting is an essential technological process used to produce high-quality cocoa-based products.... more Roasting is an essential technological process used to produce high-quality cocoa-based products. Response surface methodology (RSM) was employed to optimize the roasting conditions in cocoa beans based on antioxidant compounds (total phenolic, total flavonoids) and their activity (percentage inhibition of 1,1-diphenyl-2picrylhydrazyl [DPPH] radical and ferric-reducing antioxidant power assay) using two variables: temperature and time. Cocoa beans were roasted at temperature ranging between 150 and 250 C for 10-50 min using superheated steam. The effects of the roasting conditions on the antioxidant properties of cocoa beans were investigated using a second-order central composite design. Results showed that roasting temperature and time significantly affect antioxidants in cocoa beans. Numerical optimization and superimposed contour plots suggested the optimal roasting conditions to be 192 C for temperature with 10 min of roasting time (R 2 ¼ 0.99). These conditions can be used for roasting of cocoa beans to produce high-quality cocoa products in terms of antioxidant properties.
The release of pharmaceutical compounds to the environment, such as non-steroidal anti-inflammato... more The release of pharmaceutical compounds to the environment, such as non-steroidal anti-inflammatory drugs (NSAIDs), implies a great concern because of its negative effects, e.g. potential cytotoxicity to liver and kidney of animal species. In the present study, a high-redox potential laccase is proposed as an alternative system for the biotransformation of selected NSAIDs: naproxen (NPX) and diclofenac (DCF). The effects of pH and the use of synthetic and natural mediators (1-hydroxybenzotriazole (HBT) and syringaldehyde (SA), respectively) were evaluated with the aim of optimizing the removal of NSAIDs by laccase. Results proved high removal yields for NPX (70-94%) after 24 h in the presence of HBT at acidic and neutral pH, while DCF was completely transformed at pH 4 within shorter periods, from 30 min to 4 h, both in the absence or presence of mediators. The operation at a higher pH implied the addition of mediators to remove DCF, with values of 50 and 98% for SA and HBT, respectively. Kinetic parameters of the transformation reactions and laccase inactivation were estimated and compared for the different experimental conditions. Besides, the identification of the major biotransformation products of DCF was attempted: decarboxylated compounds were detected by gas chromatography-mass spectrometry and corresponding reaction pathways were proposed. Moreover, laccase-catalyzed treatment was demonstrated to significantly improve the aerobic biodegradability of the medium containing DCF in comparison with untreated solution, suggesting that DCF transformation products are less toxic than the parent compound.
Transformation Products of Emerging Contaminants in the Environment, 2014
ABSTRACT Natural and synthetic endocrine disrupting chemicals (EDCs) are released into the enviro... more ABSTRACT Natural and synthetic endocrine disrupting chemicals (EDCs) are released into the environment, mainly through sewage treatment systems, before reaching the receiving bodies. It is crucial to maintain ongoing efforts to develop, validate and/or update the current approaches for the effective removal of EDCs. White-rot fungi (WRF) secrete mediators of high molecular weight, increasing the range of compounds potentially biodegradable. The enzymatic membrane reactor (EMR) configuration detailed in this chapter consists of a continuous stirred tank reactor coupled to an ultrafiltration polyethersulfone membrane with a nominal molecular weight cut-off of 10kDa, which allows the recovery and recycling of the enzyme to the reaction vessel. The most common transformation products (TPs) from advanced oxidation processes (AOPs) are obtained by the breakdown of the EDC structure, yielding molecules with lower molecular weight than that of the parent compound and reduced estrogenic activity.
The goal of this study is the development of a system based on the use of the ligninolytic enzyme... more The goal of this study is the development of a system based on the use of the ligninolytic enzyme Manganese Peroxidase (MnP) for the degradation of polycyclic aromatic hydrocarbons (PAHs), of which anthracene was selected as an example. A main problem of these compounds is their poor solubility in aqueous media. Therefore, the addition of different water miscible organic solvents
In this chapter, the engineering aspects of processes catalyzed by peroxidases will be presented.... more In this chapter, the engineering aspects of processes catalyzed by peroxidases will be presented. In particular, a discussion of the existing technologies that utilize peroxidases for different purposes, such as the removal of recalcitrant compounds or the synthesis of polymers, is analyzed. In the first section, the essential variables controlling the process will be investigated, not only those that are
Several studies reported that natural and synthetic estrogens are the major contributors to the e... more Several studies reported that natural and synthetic estrogens are the major contributors to the estrogenic activity associated with the effluents of wastewater treatment plants. The ability of the enzyme laccase to degrade these compounds in batch experiments has been demonstrated in previous studies. Nevertheless, information is scarce regarding in vitro degradation of estrogens in continuous enzymatic bioreactors. The present work constitutes an important step forward for the implementation of an enzymatic reactor for the continuous removal of estrone (E1) and estradiol (E2) by free laccase from Myceliophthora thermophila. In a first step, the effect of the main process parameters (pH, enzyme level, gas composition (air or oxygen) and estrogen feeding rate) were evaluated in fed-batch bioreactors. E1 and E2 were oxidized by 94.1 and 95.5%, respectively, under the best conditions evaluated. Thereafter, an enzymatic membrane reactor (EMR) was developed to perform the continuous degradation of the estrogens. The configuration consisted of a stirred tank reactor coupled with an ultrafiltration membrane, which allowed the recovery of enzyme while both estrogens and degradation products could pass through it. The highest removal rates at steady state conditions were up to 95% for E1 and nearly complete degradation for E2. Furthermore, the residual estrogenic activity of the effluent was largely reduced up to 97%.
The feasibility of the operation of a fluidized bed reactor for the removal of estrogens by immob... more The feasibility of the operation of a fluidized bed reactor for the removal of estrogens by immobilized laccase was investigated in order to improve the degradation yields and enzyme stability previously obtained with packed bed reactors. High removal levels (between 76 and 90%) and significantly prolonged stability of the biocatalyst over 16 days were attained. In parallel, a decrease up to 90% in the estrogenic activity of the effluent was measured. Thus, the technology presented seems a promising tool to increase the applicability of laccases in bioremediation processes.
Bioreactor systems have been developed as alternatives to standard culture flasks due to their ho... more Bioreactor systems have been developed as alternatives to standard culture flasks due to their homogeneous nature, easiness of monitoring and increased cell production. Here we investigated the in vitro expansion of bone marrow (BM) mesenchymal stem cells (MSC) in spinner flasks, using gelatin microcarriers (Cultispher S) to support cell adhesion and proliferation. MSC expansion was performed using a low-serum containing medium (2% of fetal bovine serum, FBS).
In the present study, a novel and efficient technology based on the use of an oxidative enzyme wa... more In the present study, a novel and efficient technology based on the use of an oxidative enzyme was developed to perform the continuous removal of estrogenic compounds from polluted wastewaters. A 2 L enzymatic membrane reactor (EMR) was successfully operated for 100 h with minimal requirements of laccase for the transformation of estrone (E1), 17β-estradiol (E2), and 17α-ethinylestradiol (EE2)from both buffer solution and real wastewater (filtered secondary effluent). When the experiments were performed at high and low concentrations of the target compounds, 4 mg/L and 100 μg/L, not only high removal yields (80−100%) but also outstanding reduction of estrogenicity (about 84−95%) were attained. When the EMR was applied for the treatment of municipal wastewaters with real environmental concentrations of the different compounds (0.29−1.52 ng/ L), excellent results were also achieved indicating the high efficiency and potential of the enzymatic reactor system. A second goal of this study relied on the identification of the transformation products to elucidate the catalytic mechanism of estrogens' transformation by laccase. The formation of dimers and trimers of E1, E2, and EE2, as well as the decomposition of E2 into E1 by laccase-catalyzed treatment, has been demonstrated by liquid chromatography atmospheric pressure chemical ionization (LC-APCI) analysis and confirmed by determination of accurate masses through liquid chromatography electrospray time-of-flight mass spectrometry (LC-ESI-TOF). Dimeric products of E2 and EE2 were found even when operating at environmental concentrations. Moreover, the reaction pathways of laccase-catalyzed transformation of E2 were proposed.
The high hydrophobicity of polycyclic aromatic hydrocarbons (PAHs) greatly hamper their degradati... more The high hydrophobicity of polycyclic aromatic hydrocarbons (PAHs) greatly hamper their degradation in liquid media. The use of an organic solvent can assist the degradative action of ligninolytic enzymes from white rot fungi. The enzymatic action of the enzyme manganese peroxidase (MnP) in media containing a miscible organic solvent, acetone (36% v/v), was evaluated as a feasible system for the in vitro degradation of three PAHs: anthracene, dibenzothiophene and pyrene. These compounds were degraded to a large extent after a short period of time (7, 24 and 24 h, respectively), at conditions maximizing the MnP-oxidative system. The initial amount of enzyme present in the reaction medium was determinant for the kinetics of the process. The order of degradability, in terms of degradation rates was as follows: anthracene > dibenzothiophene > pyrene. The intermediate compounds were determined using gas chromatography-mass spectrometry and the degradation mechanisms were proposed. Anthracene was degraded to phthalic acid. A ring cleavage product of the oxidation of dibenzothiophene, 4-methoxybenzoic acid, was also observed.
La presencia de compuestos recalcitrantes en efluentes, suelos y sedimentos es un problema medioa... more La presencia de compuestos recalcitrantes en efluentes, suelos y sedimentos es un problema medioambiental de especial relevancia. Los compuestos de baja solubilidad tales como hidrocarburos aromáticos policíclicos (HAPs) tienen una alta persistencia ya que, debido a sus propiedades físico-químicas, su degradación por las bacterias autóctonas se ve limitada. Con el objetivo de desarrollar un sistema capaz de llevar a cabo la degradación de estos compuestos, se ha planteado la utilización de los hongos ligninolíticos o bien sus enzimas oxidativas. La enzima manganeso peroxidasa (MnP) promueve la oxidación de Mn +2 a Mn +3 , el cual actúa como un poderoso agente oxidante que oxida compuestos orgánicos recalcitrantes de forma inespecífica. La degradación de compuestos de baja solubilidad mediante la enzima MnP requiere la adición de un agente que incremente la solubilidad del compuesto a fin de facilitar la acción de la enzima. En este trabajo se plantea la adición de disolventes miscibles e inmiscibles como alternativas para incrementar la solubilidad de un HAP modelo tal como el antraceno de cara a su degradación enzimática.
The oligomerization of esculin by laccase in aqueous medium resulted in a precipitate fraction wi... more The oligomerization of esculin by laccase in aqueous medium resulted in a precipitate fraction with excellent antioxidant properties.
The oligomerization of esculin by laccase in aqueous medium resulted in a precipitate fraction wi... more The oligomerization of esculin by laccase in aqueous medium resulted in a precipitate fraction with excellent antioxidant properties.
Bioreactor systems have been developed as alternatives to standard culture flasks due to their ho... more Bioreactor systems have been developed as alternatives to standard culture flasks due to their homogeneous nature, easiness of monitoring and increased cell production. Here we investigated the in vitro expansion of bone marrow (BM) mesenchymal stem cells (MSC) in spinner flasks, using gelatin microcarriers (Cultispher S) to support cell adhesion and proliferation. MSC expansion was performed using a low-serum containing medium (2% of fetal bovine serum, FBS).A strategy was defined for the maximization of cell expansion: microcarriers were pre-coated with FBS in order to increase cell seeding efficiency and an adequate feeding regime was established (25% medium exchange everyday). The maximum cell density, 4.2 × 105 cells/mL, was obtained at day 8, corresponding to a fold increase in total cell number of 8.4 ± 0.8. Expanded MSC retained their differentiation potential into adipogenic and osteogenic lineages, as well as their clonogenic ability. Harvested cells expressed >90% of CD73, CD90 and CD105 markers.These results demonstrated that a microcarrier-based stirred culture system is adequate for human MSC expansion, using a low-serum containing medium, allowing the generation of significant cell numbers for potential applications in regenerative medicine.
The presence of recalcitrant compounds in both wastewaters and soils is an important environmenta... more The presence of recalcitrant compounds in both wastewaters and soils is an important environmental problem. Oxidative enzymes from white-rot fungi have been successfully utilised for the in vitro degradation of xenobiotics, such as the azo dye Orange II and the polycyclic aromatic hydrocarbon anthracene (compounds with high and low solubilities, respectively). Two different reactor configurations are proposed: (i) an enzymatic membrane reactor for the treatment of soluble compounds, consisting of a continuous stirred tank reactor coupled to an ultrafiltration membrane to facilitate the retention and recycling of enzyme; and (ii) a two-phase enzymatic reactor for the degradation of poorly soluble compounds, consisting of an immiscible solvent, which contains the contaminant at high concentrations, and the aqueous phase containing the enzyme and cofactors involved in the catalytic cycle. In this paper, factors affecting the design and operation of both systems are discussed, and experimental results concerning the efficiency and stability of the processes are presented.
ABSTRACT Although the removal of steroid estrogens by enzymatic treatment with free fungal enzyme... more ABSTRACT Although the removal of steroid estrogens by enzymatic treatment with free fungal enzymes has been already demonstrated in previous batch experiments, the main limitations associated to this system are the low productivity and degradation yield, typical of batch operations, as well as the unfeasibility of reusing the enzyme. With the aim of overcoming these drawbacks, an enzymatic membrane reactor (EMR) was designed for the continuous removal of estrone (E1) and estradiol (E2) by a commercial laccase from Myceliophthora thermophila. The use of an ultrafiltration membrane allowed the separation of the enzyme according to its high molecular weight while the degradation products could pass through it. Not only the continuous operation was proved, but also different operational parameters were assessed for their relevance on the efficiency of the process. Accordingly, the effects of laccase activity (500-1,000 U/L), hydraulic residence time (1-4 h) and oxygen frequency supply (2-8 pulses of oxygen each HRT) were investigated. Removal efficiencies between 64-100 % and degradation rates of 0.96-2.92 mg/(L.h) were attained. Furthermore, the residual estrogenic activity of the effluent was largely reduced up to 97 %. The present work proves the feasibility of using a laccase-mediated reactor for the continuous removal of estrogenic compounds.
ABSTRACT A Two Phase Partitioning Bioreactor (TPPB) was proposed to carry out the degradation of ... more ABSTRACT A Two Phase Partitioning Bioreactor (TPPB) was proposed to carry out the degradation of the poorly soluble compound anthracene by laccase from Trametes versicolor. The organic phase consisted of silicone oil saturated with anthracene. The surfactant (Triton X-100) was added to the aqueous phase at concentration above the critical micelle concentration (CMC) to enhance anthracene solubility. The feasibility of reusing the aqueous phase, containing the laccase-mediator system was tested in three consecutive cycles of 24 h with an initial laccase activity of 1200 U/L and 1 mM of the mediator 1-hydroxybenzotriazole (HBT). In the first cycle, total degradation of anthracene took place at a high conversion rate: 16 mu mol/L(R)h. The degree of removal achieved in two subsequent cycles with no extra addition of laccase or HBT were 68 % and 40 % for a laccase activity of 150 and 90 U/L in each cycle respectively. These results proved the potential of a TPPB including a surfactant for the removal of poorly soluble compounds by the enzyme laccase and the possibility of exhausting the oxidative potential of the aqueous phase in consecutive cycles to minimize consumption of both laccase and the mediator.
The degradation of anthracene by laccase from Trametes versicolor in enzymatic reactors was evalu... more The degradation of anthracene by laccase from Trametes versicolor in enzymatic reactors was evaluated. The use of a surfactant (Triton X-100) at concentration above critical micelle concentration (CMC) enhanced anthracene solubility and facilitated its degradation. Moreover, Triton exerted a beneficial effect on the laccase stability and protected it from the oxidative action of the mediator 1-hydroxybenzotriazole (HBT). In a further stage, the combined configuration of a two phase partitioning bioreactor (TPPB) operating with silicone oil as an immiscible solvent and the surfactant achieved the degradation of anthracene at higher conversion rate: 16 mol/L R h. Furthermore, a model for anthracene degradation by laccase-mediator system was developed. The first order kinetic constant (k) and the overall mass transfer coefficient (K L a) were estimated by using the method of least squares. The increased K L a value obtained, 788.1 h −1 , proved that Triton also improved mass transfer. Anthracene concentration in aqueous phase was close to that corresponding to equilibrium state suggesting that mass transfer mechanism did not limit the global process. The kinetic constant, which is expected to depend on the initial concentration of enzyme, resulted in 52.2 h −1 . Enzyme inactivation occurred in two stages and could be modeled by using a three parameter biexponential model. The possibility of reusing silicone oil to dissolve more anthracene was proven in three consequent cycles with high percentages of anthracene removal.
Roasting is an essential technological process used to produce high-quality cocoa-based products.... more Roasting is an essential technological process used to produce high-quality cocoa-based products. Response surface methodology (RSM) was employed to optimize the roasting conditions in cocoa beans based on antioxidant compounds (total phenolic, total flavonoids) and their activity (percentage inhibition of 1,1-diphenyl-2picrylhydrazyl [DPPH] radical and ferric-reducing antioxidant power assay) using two variables: temperature and time. Cocoa beans were roasted at temperature ranging between 150 and 250 C for 10-50 min using superheated steam. The effects of the roasting conditions on the antioxidant properties of cocoa beans were investigated using a second-order central composite design. Results showed that roasting temperature and time significantly affect antioxidants in cocoa beans. Numerical optimization and superimposed contour plots suggested the optimal roasting conditions to be 192 C for temperature with 10 min of roasting time (R 2 ¼ 0.99). These conditions can be used for roasting of cocoa beans to produce high-quality cocoa products in terms of antioxidant properties.
The release of pharmaceutical compounds to the environment, such as non-steroidal anti-inflammato... more The release of pharmaceutical compounds to the environment, such as non-steroidal anti-inflammatory drugs (NSAIDs), implies a great concern because of its negative effects, e.g. potential cytotoxicity to liver and kidney of animal species. In the present study, a high-redox potential laccase is proposed as an alternative system for the biotransformation of selected NSAIDs: naproxen (NPX) and diclofenac (DCF). The effects of pH and the use of synthetic and natural mediators (1-hydroxybenzotriazole (HBT) and syringaldehyde (SA), respectively) were evaluated with the aim of optimizing the removal of NSAIDs by laccase. Results proved high removal yields for NPX (70-94%) after 24 h in the presence of HBT at acidic and neutral pH, while DCF was completely transformed at pH 4 within shorter periods, from 30 min to 4 h, both in the absence or presence of mediators. The operation at a higher pH implied the addition of mediators to remove DCF, with values of 50 and 98% for SA and HBT, respectively. Kinetic parameters of the transformation reactions and laccase inactivation were estimated and compared for the different experimental conditions. Besides, the identification of the major biotransformation products of DCF was attempted: decarboxylated compounds were detected by gas chromatography-mass spectrometry and corresponding reaction pathways were proposed. Moreover, laccase-catalyzed treatment was demonstrated to significantly improve the aerobic biodegradability of the medium containing DCF in comparison with untreated solution, suggesting that DCF transformation products are less toxic than the parent compound.
Transformation Products of Emerging Contaminants in the Environment, 2014
ABSTRACT Natural and synthetic endocrine disrupting chemicals (EDCs) are released into the enviro... more ABSTRACT Natural and synthetic endocrine disrupting chemicals (EDCs) are released into the environment, mainly through sewage treatment systems, before reaching the receiving bodies. It is crucial to maintain ongoing efforts to develop, validate and/or update the current approaches for the effective removal of EDCs. White-rot fungi (WRF) secrete mediators of high molecular weight, increasing the range of compounds potentially biodegradable. The enzymatic membrane reactor (EMR) configuration detailed in this chapter consists of a continuous stirred tank reactor coupled to an ultrafiltration polyethersulfone membrane with a nominal molecular weight cut-off of 10kDa, which allows the recovery and recycling of the enzyme to the reaction vessel. The most common transformation products (TPs) from advanced oxidation processes (AOPs) are obtained by the breakdown of the EDC structure, yielding molecules with lower molecular weight than that of the parent compound and reduced estrogenic activity.
The goal of this study is the development of a system based on the use of the ligninolytic enzyme... more The goal of this study is the development of a system based on the use of the ligninolytic enzyme Manganese Peroxidase (MnP) for the degradation of polycyclic aromatic hydrocarbons (PAHs), of which anthracene was selected as an example. A main problem of these compounds is their poor solubility in aqueous media. Therefore, the addition of different water miscible organic solvents
In this chapter, the engineering aspects of processes catalyzed by peroxidases will be presented.... more In this chapter, the engineering aspects of processes catalyzed by peroxidases will be presented. In particular, a discussion of the existing technologies that utilize peroxidases for different purposes, such as the removal of recalcitrant compounds or the synthesis of polymers, is analyzed. In the first section, the essential variables controlling the process will be investigated, not only those that are
Several studies reported that natural and synthetic estrogens are the major contributors to the e... more Several studies reported that natural and synthetic estrogens are the major contributors to the estrogenic activity associated with the effluents of wastewater treatment plants. The ability of the enzyme laccase to degrade these compounds in batch experiments has been demonstrated in previous studies. Nevertheless, information is scarce regarding in vitro degradation of estrogens in continuous enzymatic bioreactors. The present work constitutes an important step forward for the implementation of an enzymatic reactor for the continuous removal of estrone (E1) and estradiol (E2) by free laccase from Myceliophthora thermophila. In a first step, the effect of the main process parameters (pH, enzyme level, gas composition (air or oxygen) and estrogen feeding rate) were evaluated in fed-batch bioreactors. E1 and E2 were oxidized by 94.1 and 95.5%, respectively, under the best conditions evaluated. Thereafter, an enzymatic membrane reactor (EMR) was developed to perform the continuous degradation of the estrogens. The configuration consisted of a stirred tank reactor coupled with an ultrafiltration membrane, which allowed the recovery of enzyme while both estrogens and degradation products could pass through it. The highest removal rates at steady state conditions were up to 95% for E1 and nearly complete degradation for E2. Furthermore, the residual estrogenic activity of the effluent was largely reduced up to 97%.
The feasibility of the operation of a fluidized bed reactor for the removal of estrogens by immob... more The feasibility of the operation of a fluidized bed reactor for the removal of estrogens by immobilized laccase was investigated in order to improve the degradation yields and enzyme stability previously obtained with packed bed reactors. High removal levels (between 76 and 90%) and significantly prolonged stability of the biocatalyst over 16 days were attained. In parallel, a decrease up to 90% in the estrogenic activity of the effluent was measured. Thus, the technology presented seems a promising tool to increase the applicability of laccases in bioremediation processes.
Bioreactor systems have been developed as alternatives to standard culture flasks due to their ho... more Bioreactor systems have been developed as alternatives to standard culture flasks due to their homogeneous nature, easiness of monitoring and increased cell production. Here we investigated the in vitro expansion of bone marrow (BM) mesenchymal stem cells (MSC) in spinner flasks, using gelatin microcarriers (Cultispher S) to support cell adhesion and proliferation. MSC expansion was performed using a low-serum containing medium (2% of fetal bovine serum, FBS).
In the present study, a novel and efficient technology based on the use of an oxidative enzyme wa... more In the present study, a novel and efficient technology based on the use of an oxidative enzyme was developed to perform the continuous removal of estrogenic compounds from polluted wastewaters. A 2 L enzymatic membrane reactor (EMR) was successfully operated for 100 h with minimal requirements of laccase for the transformation of estrone (E1), 17β-estradiol (E2), and 17α-ethinylestradiol (EE2)from both buffer solution and real wastewater (filtered secondary effluent). When the experiments were performed at high and low concentrations of the target compounds, 4 mg/L and 100 μg/L, not only high removal yields (80−100%) but also outstanding reduction of estrogenicity (about 84−95%) were attained. When the EMR was applied for the treatment of municipal wastewaters with real environmental concentrations of the different compounds (0.29−1.52 ng/ L), excellent results were also achieved indicating the high efficiency and potential of the enzymatic reactor system. A second goal of this study relied on the identification of the transformation products to elucidate the catalytic mechanism of estrogens' transformation by laccase. The formation of dimers and trimers of E1, E2, and EE2, as well as the decomposition of E2 into E1 by laccase-catalyzed treatment, has been demonstrated by liquid chromatography atmospheric pressure chemical ionization (LC-APCI) analysis and confirmed by determination of accurate masses through liquid chromatography electrospray time-of-flight mass spectrometry (LC-ESI-TOF). Dimeric products of E2 and EE2 were found even when operating at environmental concentrations. Moreover, the reaction pathways of laccase-catalyzed transformation of E2 were proposed.
The high hydrophobicity of polycyclic aromatic hydrocarbons (PAHs) greatly hamper their degradati... more The high hydrophobicity of polycyclic aromatic hydrocarbons (PAHs) greatly hamper their degradation in liquid media. The use of an organic solvent can assist the degradative action of ligninolytic enzymes from white rot fungi. The enzymatic action of the enzyme manganese peroxidase (MnP) in media containing a miscible organic solvent, acetone (36% v/v), was evaluated as a feasible system for the in vitro degradation of three PAHs: anthracene, dibenzothiophene and pyrene. These compounds were degraded to a large extent after a short period of time (7, 24 and 24 h, respectively), at conditions maximizing the MnP-oxidative system. The initial amount of enzyme present in the reaction medium was determinant for the kinetics of the process. The order of degradability, in terms of degradation rates was as follows: anthracene > dibenzothiophene > pyrene. The intermediate compounds were determined using gas chromatography-mass spectrometry and the degradation mechanisms were proposed. Anthracene was degraded to phthalic acid. A ring cleavage product of the oxidation of dibenzothiophene, 4-methoxybenzoic acid, was also observed.
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Papers by Gemma Eibes