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Content archived on 2024-05-27

ENzymatic DEcontamination TECHnology

Final Report Summary - ENDETECH (ENzymatic DEcontamination TECHnology)

Executive Summary:
Human activities are associated with increasing amounts of waste that ultimately find their ways into European waters and have negative consequences on the environment. With the background of an aging population and increasing urbanization, wastewater treatment must specifically target pharmaceutical products (PPs) and endocrine disrupting chemicals (EDCs) so that these highly biologically-active compounds are eliminated from the water resource.
The global objective of the ENDETECH program was to develop a technology which aims at eliminating persistent pharmaceutical pollutants and EDCs in wastewaters, thanks to an innovative ENzymatic DEcontamination TECHnology, as a complement to existing wastewater treatment. The pollutants targeted with priority during this project were antibiotics, hormones and other endocrine disruptors. The ENDETECH program was articulated around three main steps: (i) enzyme libraries were screened to identify novel enzymes able to inactivate the targeted pollutants; (ii) the discovered enzymes were immobilized on polymeric beads or ceramic membranes and subsequently (iii) used in pilot bioreactors to decontaminate wastewater.
In the course of the program, the partners discovered a couple of enzymes able to inactivate antibiotic compounds as well as EDCs and these activities were finely characterized. Finally, two enzymes were immobilized on conventional and novel carriers whose toxicity (leaching of toxic compounds) was assessed prior to their utilization. The immobilized enzymes were finally implemented in a specially-designed bioreactor. Artificial water and real hospital wastewater spiked with antibiotics and EDCs were introduced in the bioreactor and the enzymatic degradation of the pollutants was thoroughly studied using chemical and ecotoxicological analysis.
In summary, the ENDETECH results highlighted that an enzymatic degradation of antibiotics and EDCs in wastewater is promising and feasible at an R&D scale. In some cases, the ENDETECH technology performed similarly compared to other advanced wastewater treatment technologies while not generating toxic transformation products. However, in contrast to Advanced Oxidative Processes and adsorptive approaches, the enzyme-based technology is in an earlier stage of development, especially in terms of scale-up and implementation in WWTPs. The main improvements to be considered in order to lift limitations to the development of an enzyme-based wastewater treatment are (i) a neat optimization of enzyme activity towards priority pollutants, (ii) a neat improvement of the enzymatic activity recovered after immobilization and (iii) a reduction of the enzyme costs.

Keywords: pharmaceutical pollutants, antibiotic, endocrine disruptor, enzymatic bioreactor, laccase, wastewater treatment

Project Context and Objectives:
1) Context
Clean water is vital for public health and ecosystems: in July 2010, the UN affirmed that the access to clean and drinkable water was a fundamental right for all human beings. However, this is challenging due to strong economic developments, improvements of global life conditions and an important demographic growth that are increasing chemicals’ discharge and emissions worldwide. Furthermore, the European Water Framework Directive (2008/105/EC) explicitly aims at preventing the deterioration of Europe’s aquatic environment.
The ENDETECH project was focused on pharmaceutical products (PPs) and endocrine disrupting chemicals (EDCs) which are resistant to commonly used wastewater treatment and, thus, are emitted in large quantities to surface waters which are partly also used for drinking water production. In surface water, concentrations of PPs and EDCs are often in the low µg/L range and thus exposure levels in the environment are comparable to those resulting in adverse effects in various aquatic species in laboratory experiments.
The main sources of contamination are well known today (Figure A): after administration to humans or animals, some drugs are metabolized whereas others remain intact and are finally excreted. The mixture of metabolites and unaltered drugs enter the wastewater system and, depending on their intrinsic stability, some molecules are not removed during the treatment. Other sources include runoff from agricultural fields or animal husbandries, urban runoff and discharges from manufacturing sites or from the improper disposal of unused drugs.
Traces of PPs are a pressing ecological issue as they are designed to be biologically active at very low concentrations in humans and animals. Moreover, it is important to notice that they are released continuously in the environment: animals or plants (or even humans when drinking water is contaminated) will possibly be exposed during a whole life-time.
In Europe, most of the wastewater is collected and treated in wastewater treatment plants. Conventional wastewater treatments (based on activated sludge) are not efficient to remove PPs and EDCs completely. The rates of removal depend on the compounds and the treatment techniques. Even ozonation, which is considered as one of the most advanced treatment technology, presents variable decontamination yields and does not remove all pollutants (e.g. x-ray contrast media).

2) Objectives of the ENDETECH project
In order to address the issues described above, the ENDETECH project aimed at promoting a technological solution that is able to effectively remove PPs and EDCs from wastewater. A complete degradation would have been difficult to achieve and the decontamination technology developed was rather focused on achieving a major decrease of the concentrations for the most persistent compounds in order to drastically reduce the environmental and health risks associated with the discharges of these compounds.
For this purpose, the ENDETECH consortium has developed a novel decontamination technology based on enzymes. Biological processes are attractive as a viable alternative to known chemico-physical methods as they are cost-efficient, and environmentally friendly. The enzymatic catalysis has shown its capacity to convert complex chemicals under mild conditions with a very high efficiency. In the context of this project, the ENDETECH partners aimed at developing an enzyme-based decontamination technology to be used in bio-reactors specially designed for wastewater treatment.
The ENDETECH technology does not aim at replacing existing treatment technologies. On the contrary, this innovative approach can complement existing decontamination methods where it is the most needed, e.g. to treat effluents from wastewater treatment plants containing PPs or EDCs which conventional treatments do not sufficiently remove.

In this project, the following pollutants classes were targeted:
• Antibiotics, with a focus on the most recalcitrant classes (macrolides, cyclines, quinolones, sulfonamides and streptogramins);
• Hormones and endocrine disrupting compounds.

The ENDETECH-developed bioreactor relies on enzymes able to inactivate the selected molecules in order to reduce the associated toxicity risks. Inactivation may be achieved by enzymatically modifying the active groups of the compounds to render them unable to interact with their usual target. The effectiveness of the ENDETECH technology was demonstrated through a set of key environmental performance indicators such as analytical as well as ecotoxicological analyses.
The workflow of the ENDETECH project was articulated in three steps (Figure B):
1) The discovery of novel enzymes able to inactivate recalcitrant PPs and EDCs;
2) The immobilization of these enzymes on suitable carriers;
3) The implementation of the immobilized enzyme in a bioreactor designed to treat wastewater.

The global objective of the ENDETECH program was, thus, to improve the treatment of wastewater before it reaches the environment, in order to significantly reduce the environmental and public health burden associated with emerging and persistent contaminants and improve safe water reuse.

Project Results:
Screening for novel enzymes to inactivate antibiotic and endocrine disrupting compounds
The partners developed medium-throughput screening assays to identify enzymes able to inactivate each target compounds previously identified as priority, i.e. tetracycline, ciprofloxacin, erythromycin, sulfamethoxazole (antibiotics) as well as 17β-estradiol and bisphenol A (endocrine disrupting compounds).
Two different strategies were applied for the screening of enzymes: sourcing the enzymes from commercial enzymes libraries and exploring metagenomic libraries. These two strategies were complementary and increased the chances of success of the screening by widening the scope of explored enzymatic activities.
The hits identified were laccase enzymes (in particular the laccase from T. versicolor), which inactivate the biological activities of tetracycline, hormones and endocrine disrupting compounds, and an esterase enzyme (EreB), which inactivates the biological activity of erythromycin. Both enzymes were produced and their degradation of the target pollutants was thoroughly characterized. ENDETECH also included an optimization campaign for laccase to improve its characteristics. However, the improved variants were not implemented in the program.

Screening for the best immobilization conditions
The partners explored several options for the immobilization of laccase and EreB on membranes and beads with different techniques and carriers. Prior to their use, all carrier materials were ecotoxicologically characterized and only those were used that did not leach toxic compounds. Enzymes immobilized on different carriers were thoroughly characterized in terms of immobilization rate, recoverable activity and stability. The best set-ups were then implemented in a pilot bioreactor.

Reactor development and design, pilot experiments
First evaluations of the reactor performance with a high concentration of the target pollutants were carried out and a definitive configuration for the laboratory scale pilot unit was selected. The configuration of the reactor was finely optimized and characterized.
A number of tests with enzymatic membrane reactors (EMR) and packed bed membrane reactors was carried out in the pilot unit at high pharmaceutical concentrations. The operation parameters were studied thoroughly and optimized in order to determine the best degradation conditions and enzyme stability. These experiments were completed for several antibiotics and samples were collected and sent to the partners for the characterization of transformation products and toxicity. Specific methodologies for the analysis of transformation products generated during the enzymatic degradation of tetracycline and erythromycin were developed.
Different experiments were also carried out, collaboratively with all partners of ENDETECH, in the pilot bioreactor with a mixture of pharmaceuticals (38 compounds spiked in water) at low concentration as well as actual wastewater from a Spanish hospital. The resulting samples were chemically and ecotoxicologically analyzed to evaluate the pollutants’ degradation.
A mathematical (and economical) model was developed in order to simulate the tetracycline degradation in wastewaters at industrial scale for three different use cases: a hospital, a middle size city and a pharmaceutical production unit and to optimize the process. This model was used as a base for the theoretical estimation of the bioreactor operating costs in order to compare the ENDETECH technology with other available technologies.

Evaluation of the ENDETECH technology
Last but not least, the partners contributed to a comparison of the different reactors tested with the different enzymes and immobilization technologies, producing benchmarking tables to prepare a synthetic overview of the main factors to take into account for the further exploitation of the technology.
A thorough evaluation of the ENDETECH technology was performed, emphasizing the advantages and limitations of the approach.

Key results
- Immobilized laccases effectively degrade tetracycline: we observed 56-65% degradation when using laccase grafted to ceramic membranes compared to 25-30% when using free enzyme.
- Immobilized esterase EreB effectively degrades erythromycin: we observed 50% degradation when using free enzyme and 75% degradation when using EreB immobilized on membranes.
- Laccases immobilized on polymeric beads, alumina and sand remove 30-95% tetracycline with adsorption to the carrier contributing significantly.
- The tetracycline degrading activity of laccase from T. versicolor immobilized on membranes in a pilot reactor is stable over a week and several cycles of operation.
- The toxicological assessment of the carrier materials used for enzyme immobilization guided the selection of materials not leaching any toxic compounds.
- The degradation of antibiotics mixtures in osmosed water using laccase immobilized on membranes is feasible when a redox mediator is used.
- Laccases immobilized on polymeric beads degrade selected EDCs when applied to EDC mixtures in ultrapure and wastewater.
- Laccases immobilized on polymeric beads effectively degrade the endocrine activity present in raw hospital wastewater.
- The enzymatic degradation of target compounds and wastewater by laccases (without redox mediator) and esterase EreB did not generate toxic transformation products inducing effects in a battery of bioassays.
The ENDETECH results highlight that an enzymatic degradation of antibiotics and EDCs in wastewater is promising and feasible at a R&D scale. In some cases, the ENDETECH technology showed a similar performance compared to existing advanced wastewater treatment technologies while not generating toxic transformation products. But the overall efficiency (noteworthy the cost efficiency) of the technology was judged to be too low for further implementation in spite of encouraging results. The main improvements to be considered in order to lift limitations to the development of an enzyme-based technology for wastewater treatment are (i) a neat optimization of enzyme activity towards priority micropollutants, (ii) a neat improvement of the enzymatic activity recovered after immobilization and (iii) a reduction of the enzyme costs.

Potential Impact:
The results of the project highlight that the ENDETECH technology is not expected to be implemented to treat the Community wastewaters in the short term. The research conducted in the ENDETECH project was of high quality and was the subject of an important number of publications in peer-reviewed journals and oral presentations in specialized meetings and congresses. This strong dissemination effort will undoubtedly help and boost the research in the area of enzymatic remediation in the European Community.
The partial success of the ENDETECH project should not discourage further research in the area, especially given the low ecological footprint promised by enzymatic approaches to tackle the urging issue of persistent pollutants. Waste treatment is among of the greatest challenge of the 21st century worldwide and deserves a continued interest of the scientific community and innovators.

List of Websites:
Pierre-Alain Bandinelli, ENDETECH Project Coordinator
ENDETECH project public website: available at