FBREW english version

Wednesday, December 3, 2025

Efficient fluidized bed reactor with co-immobilized enzymes for treatment of real wastewater containing bisphenols and dyes

Project Summary

The project scope is to create and perform experiments on a purification system for contaminated water containing phenols and dyes. The system will consist of a fluidized bed reactor (FBR) and a catalyst made of co-immobilized enzymes on solid supports composed of recycled polyethylene terephthalate (rPET) as core, and chitosan (Cs) and polyacrylic acid (PAA) as shell. Also, the project aims to implement an ecological approach utilizing biocatalysis to effectively eliminate hazardous or persistent substances from water. The project team desires to generate a biocatalyst by co-immobilizing two enzymes - Laccase from Aspergillus (Lc) and Tyrosinase from mushrooms (Tyr) - on solid supports composed of a rPET core and a polymeric shell. One of the innovative solutions is the support activation with carbodiimide alternated with the enzyme immobilization, in repetitive procedures. The project encompasses 4 objectives: obtaining and characterizing co-immobilized tyrosinase and laccase on recycled polyethylene terephthalate coated with polyacrylic acid and chitosan; operational stability of co-immobilized rPET-Cs-PAA-Lc-Tyr products in synthetic and real wastewaters; design of a lab-scale fluidized bed reactor with co-immobilized enzymes to be applied in wastewater treatment; validation of the operational efficiency of the fluidized bed reactor and co-immobilized enzymes system for treatment of bisphenols and dyes from real wastewater.

Stage Summary

Research Stage (as per Funding Application): Stage 1.

Stage Title: Obtaining and Characterizing Co-immobilized Tyrosinase and Laccase on Recycled Polyethylene Terephthalate Coated with Polyacrylic Acid and Chitosan; Operational Stability of the Co-immobilized rPET-Cs-PAA-Lc-Tyr Product in Synthetic and Real Wastewater (PARTIAL); Design of a Laboratory-Scale Fluidized Bed Reactor with Co-immobilized Enzymes for Wastewater Treatment Applications (PARTIAL).

Period of Activities for this Research Report: January 2025 – December 2025 (12 months).

Stage Activities:

O1. Obtaining and Characterizing Co-immobilized Tyrosinase and Laccase on Recycled Polyethylene Terephthalate Coated with Polyacrylic Acid and Chitosan

A1. Production of recycled polyethylene terephthalate (rPET) coated with polyacrylic acid and chitosan.
A2. Immobilization of Lc on rPET-Cs-PAA.
A3. Immobilization of Tyr on rPET-Cs-PAA.
A4. Co-immobilization of Lc and Tyr on rPET-Cs-PAA.

O2. Operational Stability of the Co-immobilized rPET-Cs-PAA-Lc-Tyr Product in Synthetic and Real Wastewater (Stage 1 excludes O2.A2.T3 and O2.A3.T3/T4)

A1. Evaluation of the performance of co-immobilized enzymes in synthetic wastewater containing bisphenols and dyes.
A2. Analysis of water at the outlet of wastewater treatment plants in Ilfov and Galați.
A3. Evaluation of the performance of co-immobilized enzymes in real wastewater from the Ilfov and Galați treatment plants.

O3. Design of a Laboratory-Scale Fluidized Bed Reactor with Co-immobilized Enzymes for Wastewater Treatment Applications (Stage 1 excludes O3.A2.T3)

A1. Conceptual geometry design of the 2 L FBR.
A2. Fabrication of Reactor Version 1.

Verifiable Outcomes of Activities

Project website development.
1 ISI article submitted for publication.
1 patent application submitted for evaluation.
Participation in 2 scientific conferences.
Participation in 1 innovation exhibition.
Functionalized rPET; Thin films of Cs-PAA deposited on functionalized rPET; Well-characterized immobilization support (rPET-Cs-PAA).
rPET-Cs-PAA-Lc biocatalyst obtained; complete study of the influence of pH, temperature, reuse, and storage on the kinetic profiles of the immobilized enzyme using ABTS.
rPET-Cs-PAA-Tyr biocatalyst obtained; complete study of the influence of pH, temperature, reuse, and storage on the kinetic profiles of the immobilized enzyme using ABTS.
rPET-Cs-PAA-Tyr-Lc biocatalyst obtained; complete study of the influence of pH, temperature, reuse, and storage on the kinetic profiles of co-immobilized Lc and Tyr using ABTS and L-tyrosine.
Reactor geometry (Version 1) established.
Components acquired/fabricated and assembly of Reactor Version 1.
Synthetic wastewater formulated and characterized.
Product kinetically characterized in synthetic water; degradation products identified.
Real wastewater from Galați and Ilfov analyzed; quantity of bisphenols and dyes quantified.
Product kinetically characterized in real wastewater.

Summary of Progress

During Stage 1 of the FBREW project, all outcome indicators anticipated in the funding application were fully achieved, namely:

✔ 1 / 1 ISI Paper submitted for publication and published:

Published article: Adsorption of Bisphenol A from Water Using Chitosan-Based Gels, Ludmila Aricov, Anca Ruxandra Leontieș*, Gels, 237, 11, 3, 180 (IF 5.3)

✔ 5 / 2 International conference presentations:

Modulation of Trametes versicolor laccase activity with sodium sulfate and its potential application in the degradation of naphthol green, L. Aricov*, R. M. Toma, A. Raducan, A. Precupaş, A.R. Leontieș, 27th Materials, Methods & Technologies, Burgas, Bulgaria (poster).
Catalytic decomposition of vegetable oil using nanoreactors with entrapped lipase and additives, Anca-Ruxandra Leontieș*, Aurica Precupas, Raluca-Marieta Toma, Adina Raducan, Ludmila Aricov, MEDICTA 2025, Thessaloniki, Greece (poster).
Olive oil degradation using nanoreactors with entrapped lipase and additives, Leontieș Anca-Ruxandra*, Precupas Aurica, Toma Raluca-Marieta, Răducan Adina, Aricov Ludmila, 27th Materials, Methods & Technologies, Burgas, Bulgaria (poster).
Optical Microscopy Coupled with DSC: Insights into Composite and Advanced Materials, Raul Augustin Mitran, 8th Central and Eastern European Conference on Thermal Analysis and Calorimetry, Mostar, Bosnia and Herzegovina (oral presentation).
Nonessential activation of Laccase from Trametes versicolor with sodium sulfate, Ludmila Aricov*, Raluca-Marieta Toma, Adina Raducan, Aurica Precupas, Anca-Ruxandra Leontieș, MEDICTA 2025, Thessaloniki, Greece (poster).

✔ 1 / 1 Patent submitted to OSIM

Mixed polymeric materials for enzymatic immobilization, Ludmila Aricov, Anca Ruxandra Leontieș, and Raul Augustin Mitran, submission code A2025/00564

✔ 1 / 1 Participation in an Innovation Exhibition

Gold Medal at the UGAL INVENT Innovation and Research Exhibition (23–24 October 2025), Galați, awarded to ARCIF AG SRL together with the “Ilie Murgulescu” Institute of Physical Chemistry for the project “Design and construction of a fluidized bed reactor with co-immobilized enzymes for the removal of bisphenols and dyes from wastewater (FBREW)” – Andrei Terente, Puiu Ciobanu, Dragoș Ionuț Sandu, Ludmila Aricov, Anca Ruxandra Leonties.

Additionally, the following materials were obtained:

Functionalized rPET; thin Cs-PAA films deposited on functionalized rPET; well-characterized immobilization support (rPET-Cs-PAA).
Biocatalyst rPET-Cs-PAA-Lc obtained; complete study of pH, temperature, reuse, and storage effects on the kinetic profiles of the immobilized enzyme using ABTS.
Biocatalyst rPET-Cs-PAA-Tyr obtained; complete study of pH, temperature, reuse, and storage effects on the kinetic profiles of the immobilized enzyme using ABTS.
Biocatalyst rPET-Cs-PAA-Tyr-Lc obtained; complete study of pH, temperature, reuse, and storage effects on the kinetic profiles of co-immobilized Lc and Tyr using ABTS and L-tyrosine.

Furthermore, the following were developed and delivered:

Reactor geometry version 1 established.
Components purchased/manufactured and assembly of reactor version 1 completed.

Additionally, the following materials and solutions were characterized:

Formulated and characterized synthetic wastewater.
Kinetic characterization of the product in synthetic wastewater; degradation products identified.
Real wastewater from Galați and Ilfov analyzed; quantities of bisphenols and dyes quantified.
Kinetic characterization of the product in real wastewater; degradation products identified.
Storage and reusability profiles evaluated; operational stability assessed.

Executive Summary

In this stage, we aimed to develop, characterize, and test the operational stability of innovative materials based on immobilized enzymes on a recycled polyethylene terephthalate (rPET) support.

Objective O1: Preparation and Characterization of Enzymatic Materials

The main objective (O1) was to prepare and characterize rPET supports with chemically immobilized enzymes (laccase and tyrosinase).

A1.O1: Involved the functionalization of recycled rPET and subsequent coating with polyacrylic acid (PAA) and chitosan (Cs), yielding the rPET-Cs-PAA support.
A2.O1 and A3.O1: Focused on the separate immobilization of laccase and tyrosinase, respectively, onto this support.
A4.O1: Focused on the co-immobilization of both enzymes onto the same support, resulting in the composite material rPET-Cs-PAA-Lc-Tir.

Objective O2: Operational Stability Evaluation

The goal of Objective O2 was to evaluate the operational stability of the co-immobilized product (rPET-Cs-PAA-Lc-Tir) in the treatment of both synthetic and real wastewater.

A1.O2: Evaluated the efficiency of the co-immobilized enzymes in the treatment of synthetic wastewater containing bisphenols and dyes.
A2.O2: Consisted of obtaining and analyzing the water profile at the outlet of treatment plants in Ilfov and Galați.
A3.O2: Completed this section by evaluating the performance of the co-immobilized enzymes in real wastewater sourced from the mentioned plants.

Note: Objective O2 was partially achieved in this stage, excluding activities O2.A2.T3 and O2.A3.T3/T4.

Objective O3: Reactor Design

Objective O3 aimed to design a laboratory-scale fluidized bed reactor (FBR) using the co-immobilized enzymes.

A1.O3: Involved developing the conceptual geometry design for a 2 L reactor.
A2.O3: Consisted of fabricating the first version of this reactor.

Note: Objective O3 was partially completed in this stage, excluding activity O3.A2.T3.