LANCE: Laccase-nanoparticle conjugates for the elimination of micropollutants (endocrine disrupting chemicals) from wastewater in bioreactors

Elimination of recalcitrant chemicals during wastewater treatment is a difficult problem for both developing and industrialized countries. The biological elimination of very persistent xenobiotics such as endocrine disrupting chemicals from municipal and industrial sewage treatment plants is an ambitious challenge as existing physico-chemical methods, such as advanced oxidation processes, are energy-intensive and consume high amounts of chemicals. Through the entry into force of strict legislative measures, such as the Water Framework Directives (EU WFD in Directive 2000/60/EC of the European Parliament and of the Council establishing a framework for the Community action in the field of water policy, 2000) and REACH (REACH EU in European Community Regulation on chemicals and their safe use (EC 1907/2006), 2007), the market for wastewater treatment is exploding. For instance the European market potential for the membrane bioreactor technology is estimated to 57M€ per year. Based on recent progresses in nanotechnology, new developments in catalysis and environmental applications can be foreseen for the near future. Indeed, because of high surface area-to-volume ratio in nano-systems, heterogeneous enzymatic or catalytic reactions can be greatly enhanced. In the LANCE project a nanoparticle (NP)-based technology is under development. Cheap and resistant oxidative enzymes, i.e. laccases are immobilized onto the surface of the particles in order to produce systems possessing a broad substrate spectrum for the degradation of cocktails of recalcitrant pollutants. One of the objectives is to produce NPs that are compatible with wastewater treatment and can be synthesised in a cost-effective and large-scale fashion, e.g. silica-based NPs using flame spray pyrolysis and emulsion-based techniques. The modified particles are applied in bioreactors where they are retained, i.e. membrane bioreactors or perfusion basket reactors to eliminate pollutants from the wastewater. Such reactors allow multi-cycle use of the NPs coated with active enzymes and thus contribute to decrease the treatment costs. The two-year activities of the LANCE project encompass the synthesis of various NP systems, the immobilization of selected low cost industrial laccases on the latter, and the technical and scientific proof of the "depollution” concep

MINOTAURUS: microorganism and enzyme immobilization: novel techniques and approaches for upgraded remediation of underground-, wastewater and soil

The European project MINOTAURUS aims to deliver innovative bio-processes to eliminate emerging and classic organic pollutants. These bio-processes are all based on the concept of immobilization of biocatalysts (microorganisms and enzymes) and encompass bioaugmentation, enzyme technology, rhizoremediation with halophytes, and a bioelectrochemical remediation process. The immobilization-based technologies are applied to engineered ex situ and natural systems in situ for the bioremediation of groundwater, wastewater and soil. The selection and application of modern physico-chemical, biological and ecotoxicological monitoring tools combined with a rational understanding of engineering, enzymology and microbial physiology is a pertinent approach to open the black-box of the selected technologies. Reliable process-monitoring constitutes the basis for developing and refining biodegradation kinetics models, which in turn will improve the predictability of performances to be achieved with our technologies. A key strength of MINOTAURUS is the possibility of direct implementation of our technologies at five European reference sites that are confronted with pollutants (two technologies will be tested on-site starting from the first year). We will deliver not only a set of tools, techniques, and processes, which will enhance the ability of our communities to respond to the challenges of organic pollutants but also frameworks for structuring and making evidence-based decisions for the most sustainable and appropriate bioremediation measures. The MINOTAURUS consortium includes fifteen partners from eight European countries. Eight research & education institutions, five SMEs covering the whole chain of our bioremediation approaches (production, and monitoring of biocatalysts, bioremediation and engineering), one large end-user operating wastewater treatment plants and one environmental agency work together with the support of an advisory board mainly consisting of environmental decision-maker

Factors affecting the removal of organic micropollutants from wastewater in conventional treatment plants (CTP) and membrane bioreactors (MBR)

As a consequence of insufficient removal during treatment of wastewater released from industry and households, different classes of organic micropollutants are nowadays detected in surface and drinking water. Among these micropollutants, bioactive substances, e.g., endocrine disrupting compounds and pharmaceuticals, have been incriminated in negative effects on living organisms in aquatic biotope. Much research was done in the last years on the fate and removal of those compounds from wastewater. An important point it is to understand the role of applied treatment conditions (sludge retention time (SRT), biomass concentration, temperature, pH value, dominant class of micropollutants, etc.) for the efficiency of conventional treatment plants (CTP) and membrane bioreactors (MBR) concerning the removal of micropollutants such as pharmaceuticals, steroid- and xeno-estrogens. Nevertheless, the removal rates differ even from one compound to the other and are related to the physico-chemical characteristics of the xenobiotic

Multi-catalysis reactions: new prospects and challenges of biotechnology to valorize lignin

Considerable effort has been dedicated to the chemical depolymerization of lignin, a biopolymer constituting a possible renewable source for aromatic value-added chemicals. However, these efforts yielded limited success up until now. Efficient lignin conversion might necessitate novel catalysts enabling new types of reactions. The use of multiple catalysts, including a combination of biocatalysts, might be necessary. New perspectives for the combination of bio- and inorganic catalysts in one-pot reactions are emerging, thanks to green chemistry-driven advances in enzyme engineering and immobilization and new chemical catalyst design. Such combinations could offer several advantages, especially by reducing time and yield losses associated with the isolation and purification of the reaction products, but also represent a big challenge since the optimal reaction conditions of bio- and chemical catalysis reactions are often different. This mini-review gives an overview of bio- and inorganic catalysts having the potential to be used in combination for lignin depolymerization. We also discuss key aspects to consider when combining these catalysts in one-pot reaction

Sorption-assisted surface conjugation: a way to stabilize laccase enzyme

Enyzme immobilization on solid surfaces is one of the most relevant methods to improve enzyme activity and stability under harsh conditions over extended periods. A typically interesting application is the immobilization of laccases, multicopper enzymes oxidizing aromatic compounds, to solid surfaces in order to develop valuable tools for the elimination of micropollutants in wastewater. Laccase of the white-rot fungus Coriolopsis polyzona has been successfully immobilized on fumed silica nanoparticles using a novel method. It consists in the sorption of the enzyme to amino-modified silica nanoparticles and the subsequent covalent cross-linking using glutaraldehyde as a homobifunctional linker. The so-produced nanoparticulate material has been characterized by means of scanning electron microscopy and Brunauer-Emmett-Teller surface area analysis revealing modifications of the surface structure and area during the coupling procedure. Laccase immobilization on spherical nanoparticles produced according to the method of Stöber has been shown to be much less efficient than on fumed silica nanoparticles. Long-term stability assays revealed that the novel developed method allows a drastic stabilization of the enzyme. In real wastewater, 77% of the laccase activity remained on the nanoparticles over 1month, whereas the activity of free laccase dropped to 2.5%. The activity loss on the nanoparticles resulted from partial inactivation of the immobilized enzymes and additional release into the surrounding solution with subsequent fast inactivation of the free enzymes, since almost no activity was found in the supernatant

Radio (14C)- and fluorescent-doubly labeled silica nanoparticles for biological and environmental toxicity assessment

A new and efficient synthetic route to fluorescent and 14C-double-labeled silica-based nanoparticles (NPs) is described. The synthesis has been carried out using the "oil-in-water” micro-emulsion technique. Fluorescent and radioactive labeling have been achieved entrapping labeled poly(ethylene glycol) (PEG) molecules in the NPs. The produced particles have been analyzed by means of scanning electron microscopy, photon correlation spectroscopy, confocal microscopy, scintillation counting and oxidation/combustion experiments. Fluorescence quenching experiments confirm that the label is entrapped in the particles. The results presented suggest that the silica matrix does not block the β-radiations emitted from the labeled PEG molecules entrapped in the NP

Reversibility of enzymatic reactions might limit biotransformation of organic micropollutants

This is the accepted manuscript version of the published article: Gonzalez-Gil, L., Carballa, M., Corvini, P., & Lema, J. (2019). Reversibility of enzymatic reactions might limit biotransformation of organic micropollutants. Science Of The Total Environment, 665, 574-578. doi: 10.1016/j.scitotenv.2019.02.143Biotransformation of many organic micropollutants (OMPs) in sewage treatment plants is incomplete leading to their release into the environment. Recent findings suggest that thermodynamic aspects of the reaction as chemical equilibrium limit biotransformation, while kinetic parameters have a lower influence. Reversibility of enzymatic reactions might result in a chemical equilibrium between the OMP and the transformation product, thus impeding a total removal of the compound. To the best of our knowledge, no study has focused on proving the reversible action of enzymes towards OMPs so far. Therefore, we aimed at demonstrating this hypothesis through in vitro assays with bisphenol A (BPA) in the presence of kinase enzymes, namely acetate kinase and hexokinase, which are key enzymes in anaerobic processes. Results suggest that BPA is phosphorylated by acetate kinase and hexokinase in the presence of ATP (adenosine 5-triphosphate), but when the concentration of this co-substrate decreases and the enzymes loss their activity, the backward reaction occurs, revealing a reversible biotransformation mechanism. This information is particularly relevant to address new removal strategies, which up to now were mainly focused on modifying the kinetic parameters of the reactionThis research was funded by the Spanish Government (AEI) through COMETT project (CTQ2016-80847-R) and by FPU program with a contract (FPU13/01255) and a short research stay grant (EST16/00138). Authors from Universidade de Santiago de Compostela belong to CRETUS Strategic Partnership (AGRUP2015/02) and to Galicia Competitive Research Group (GRC ED431C 2017/29) which are co-funded by FEDER (EU)S

Laccases to take on the challenge of emerging organic contaminants in wastewater

The removal of emerging organic contaminants from municipal wastewater poses a major challenge unsatisfactorily addressed by present wastewater treatment processes. Enzyme-catalyzed transformation of emerging organic contaminants (EOC) has been proposed as a possible solution to this major environmental issue more than a decade ago. Especially, laccases gained interest in this context in recent years due to their broad substrate range and since they only need molecular oxygen as a cosubstrate. In order to ensure the stability of the enzymes and allow their retention and reuse, either immobilization or insolubilization of the biocatalysts seems to be the prerequisite for continuous wastewater treatment applications. The present review summarizes the research conducted on EOC transformation with laccases and presents an overview of the possible immobilization techniques. The goal is to assess the state of the art and identify the next necessary steps that have to be undertaken in order to implement laccases as a tertiary wastewater treatment process in sewage treatment plants

Impact of bio-augmentation with Sphingomonas sp. strain TTNP3 in membrane bioreactors degrading nonylphenol

This study evaluates the potential of bio-augmentation to improve the degradation of recalcitrant nonylphenol during the wastewater treatment in membrane bioreactors (MBR). One MBR containing activated sludge was bio-augmented using multistep inoculation with freeze dried Sphingomonas sp. strain TTNP3, whereas a second control reactor contained activated sludge solely. The 14C-labeled-nonylphenol isomer (4-[1-ethyl-1,3-dimethylpentyl]phenol) was applied as a single pulse. Bio-augmentation resulted in an immediate increase of dissolved radioactivity in the effluent in comparison to the control reactor (13% and 2% of initially applied radioactivity after 1day, respectively). After 5days of operation, the retentate of the bio-augmented reactor contained only 7% of the initial radioactivity in contrast to 50% in the control reactor. The radioactivity associated to the mixed liquor suspended solids, i.e., the suspension of biomass and other solids on the retentate side of the membrane, was mainly found as non-extractable residues that were increasingly formed during prolonged reactor operation, especially for the control MBR. HPLC-LSC and GC-MSn analyses revealed that the bio-augmented reactor produced more polar hydroquinone as main degradation intermediate, whereas the control reactor effluent contained a complex mixture of apolar compounds with shortened oxidized alkyl chains. Thus, the apparent differences in the behavior of nonylphenol between the reactors were due to the catabolism of nonylphenol conferred by bio-augmentation with Sphingomonas sp. strain TTNP

Immobilization of an Artificial Imine Reductase within Silica Nanoparticles Improves its Performance

Silica nanoparticles equipped with an artificial imine reductase display remarkable activity towards cyclic imine- and NAD + reduction. The method, based on immobilization and protection of streptavidin on silica nanoparticles, shields the biotinylated metal cofactor against deactivation yielding over 46 000 turnovers in pure samples and 4000 turnovers in crude cellular extracts