Enzymatic bioreactors for the oxidation of estrogenic and anti-inflammatory compounds by laccases

  1. Lucía Lloret Caulonga
Dirixida por:
  1. Gemma M. Eibes Director
  2. Gumersindo Feijoo Costa Director

Universidade de defensa: Universidade de Santiago de Compostela

Ano de defensa: 2013

  1. Juan Manuel Lema Rodicio Presidente
  2. Diego Moldes Moreira Secretario/a
  3. Ana Paula Mora Tavares Vogal
  4. María Teresa Moreira Vilar Vogal
  5. Lara Valentín Carrera Vogal
  1. Departamento de Enxeñaría Química

Tipo: Tese


Over the past decade, public concern about the environmental impact of steroid estrogens present in municipal wastewaters has grown due to their potential for disturbing endocrine systems of animals and humans. Besides, the release of anti-inflammatory drugs to the environment also implies a great concern because of their negative effects, e.g. potential cytotoxicity to liver and kidney of some species. The degradation of these types of compounds implies an important ecological challenge as they have complex structures and low bioavailability. In fact, they have been detected in wastewater treatment plant effluents at different concentrations ranging from nanograms to micrograms per liter, once certain conventional processes can only render partial transformation yields. Hence, alternative treatment methods have been investigate in order to overcome that environmental issue, such as physical methods (e.g. sorption and membrane separation), microbial removal by bacteria and algae and advanced oxidation processes (AOPs) (e.g. ozonation, UV/H2O2, Fenton, etc.). In general, AOPs provide high removal rates but low selectivity; however, these methods imply in some cases considerable high costs associated to reagents and/or investment, the formation of harmful byproducts and even the generation of compounds with estrogenicity higher than that of the parent substrate and/or non-biodegradable. An advanced treatment alternative may be based on the use of white rot fungi cultures. These microorganisms were reported to remove a wide range of xenobiotics by the action of fungal oxidative enzymes such as manganese peroxidase, lignin peroxidise and laccase; thereby, the use of enzyme-catalyzed transformation of estrogens and anti-inflammatories was proposed in the current work. The use of enzymes in industrial processes is commonly linked to a reduce consumption of energy and chemicals and moreover, they can usually react under moderate conditions. In the case of laccases (copper-containing oxidases, EC, the use of oxygen as electron acceptor would represent an additional advantage for the application of these enzymes in comparison with peroxidases. Specifically, the use of laccases from Myceliophthora thermophila and Trametes versicolor for the oxidation of the estrogens estrone (E1), 17ß-estradiol (E2) and 17¿-ethinylestradiol (EE2) and the anti-inflammatories naproxen (NPX) and diclofenac (DCF) was investigated in this research. The main goal of this work is the development of technology to perform the successful laccase-catalyzed oxidation of the compounds mentioned above. In the first stage of the research the use of laccases as well as laccase-mediator systems was investigated in batch reactors (Chapter 2). Two different laccases were evaluated and effects of pH (affecting not only the activity and stability of the enzyme but also the target substrates and the mediator action) and mediator (type and concentration) on both removal efficiency and enzyme stability were investigated aiming to establish the groundwork for the optimum laccase-catalyzed transformation of this type of compounds. In the case of estrogens, these compounds were successfully removed by Myceliophthora thermophila laccase even in the absence of mediator and at neutral pH, which was expected to facilitate the application of the enzymatic treatment on the detoxification of wastewaters. The target anti-inflammatories were transformed by Myceliophthora thermophila and Trametes versicolor laccase with variable yields. Furthermore, the toxicity of the biotransformation products generated from the enzymatic reactions was evaluated: laccase-catalyzed transformation was proved to be effective in reducing the estrogencity of the medium containing the estrogenic compounds by the application of LYES analysis; also, DCF byproducts presented considerable higher aerobic biodegradability and detoxification was also proved by Microtox® assays. In view of the promising obtained results, continuous bioreactors were operated for the successful transformation of the target compounds: for this purpose, enzymes need to be immobilized to enable the recovery of the biocatalyst, or separated from the effluent by membrane modules. Here, both routes were applied and potential technologies were found to be feasible. Laccase from Myceliophthora thermophila was immobilized by different procedures for the application of the biocatalysts in packed bed and fluidized bed reactors (PBRs and FBRs) for the removal of E1, E2 and E2 (Chapter 3). The enzyme was immobilized by its encapsulation in a sol-gel matrix based on silane compounds which hydrolyze and polymerize in the presence of the enzyme, resulting in a hydrogel with the laccase encapsulated inside; also, laccase immobilization was conducted by covalent bonding to commercial solid epoxy-activated acrylic supports, Eupergit C and Eupergit C 250L. Laccase was successfully immobilized by both procedures yielding bound protein percentages of up to 44-99 and 59-83% and activities 1-80 and 5-17 U/g for covalently immobilized and encapsulated laccase, respectively. The somewhat lower catalytic efficiency of laccase immobilized by both studied methods in comparison to that of free form was balanced by its increased stability and broader operational window related to temperature, pH and chemical inhibitors. The PBRs were evaluated by their application on the removal of a synthetic dye used as model compound; moreover, the bioreactors were operated in consecutive continuous cycles aiming to verify the reusability of the biocatalysts. The proposed PBRs were applied in a next step of the research for the continuous removal of E1, E2 and EE2, providing removal yields of 55-75 and 65-80% for the encapsulated and the covalently immobilized laccase, respectively; 6-14 and 11-22% of elimination was attributed to adsorption of the substrates. Nonetheless, a FBR was developed due to the drawbacks related to the use of PBRs (poor or passive aeration, slow mass transfer and formation of preferential paths, etc.). This system was proved to be feasible on the elimination of estrogenic substrates at concentrations in the range of 10-100 µg/L, and the bioreactor and biocatalyst were stable for more than 10 days of operation; moreover, an estrogenicity reduction of 90% was achieved. Afterwards, an enzymatic membrane reactor (EMR) was evaluated for the continuous removal of estrogens by free laccase from Myceliophthora thermophila (Chapter 4). In a first step, fed-batch experiments were carried out with the goal of investigating the effect of aeration/oxygenation, addition rate of the substrates and laccase activity, for conducting preliminary continuous assays in a 370-mL EMR. Nonetheless, the conventional method based on ¿one factor at a time¿ approach for the optimization of the process is not the most adequate procedure and moreover, it is time-consuming, laborious and incomplete. Hence, response surface methodology (RSM) is used as proper alternative. The application of RSM allowed the evaluation of individual and interrelated effects of oxygenation rate, hydraulic residence time (HRT) and laccase activity on the continuous transformation of the substrates; moreover, different response variables were investigated and optimized: i) removal rate, ii) removal rate per units of enzyme used (aiming to minimize the costs of the technology), and iii) the percentage of estrogenic activity reduction (in order to optimize not only the elimination of the parent compounds but also de detoxification of the effluent). Only 100 U/L were found as optimal to maximize the efficacy of the enzyme: E1 was oxidized by 0.06 mg/(L¿h¿U), although the removal of estrogenicity was 60%. On the other hand, the highest values assayed (1,000 U/L, HRT 4 h and 60 mg O2/(L¿h)) provided nearly complete detoxification. Finally, a 2-L EMR was operated, for 100 h and with minimal enzyme requirements (100 U/L), to corroborate the real applicability of the developed technology: the bioreactor was proved for the enzymatic treatment of secondary wastewater effluents, collected in a municipal wastewater treatment plant, containing E1, E2 and EE2 at only100 µg/L as well as at real environmental levels (0.29-1.52 ng/L). High removal yields (80-100%) were attained despite partial inactivation of the enzyme (about 20% within the first hours). On the other hand, there is a significant lack of knowledge in literature regarding the identification of reaction products of the target compounds despite the increasing research on the application of fungal enzymes for the oxidation of pharmaceuticals and endocrine disrupting compounds. Effort was paid in this work to perform the identification of the reaction products of E1, E2 and EE2 as well as those of DCF resulted from laccase-catalyzed transformation, as a first step to elucidate the main reaction pathways; for this purpose, different analytical techniques such as GC-MS, LC-APCI and LC-ESI-TOF were applied (Chapter 5). The formation of dimers and trimers of estrogens from the reaction catalyzed by Myceliophthora thermophila laccase, as well as the transformation of E2 into E1, was proved by analyzing the obtained spectra and the possible fragmentation patters, and corroborated by the determination of accurate masses. Furthermore, DCF transformation products resulted from decarboxylation reactions catalyzed by Trametes versicolor laccase were assessed. In the last stage of the research an emerging technology was explored: laccase-immobilized microreactors were developed, characterized and applied with the objective of improving the efficiency of laccase-catalyzed processes and to broaden the range of application of these enzymes (Chapter 6). Microreactor systems have been demonstrated to present numerous advantages: the use of drastically reduced volumes of reactant solutions, high efficiency and repeatability, increased heat exchange and mass transfer, strict control of the reaction conditions by means of the characteristic laminar flow, etc. Nonetheless, only few previous studies considered these systems for the application of laccases. In the current research, a simple, versatile and inexpensive method was developed for the fabrication of enzymatic microreactors, based on the formation of a Trametes versicolor laccase-immobilized membrane on the inner wall of microtubes as a result of cross-linking polymerization reactions. These microreactors were used for the oxidation of the target compounds, obtaining great transformation yields with reduced residence times. Furthermore, it was designed a two-stage microreactor system for the prevention of the biocatalyst inactivation when operating under adverse conditions for the enzyme.