Project Description

BYEbuprofen. For cleaner water.

An Overview

The increased consumption of nonsteroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen and diclofenac in recent decades has led to the accumulation of these substances in aquatic ecosystems such as rivers and lakes. This contamination with pharmaceutical products can cause significant health damage to animals.

Our project aspires to find an innovative solution to reduce the amount of pharmaceutical pollutants such as ibuprofen, diclofenac, and estrogen in municipal wastewater using immobilized laccase. Laccase is a naturally occurring enzyme found in many organisms, such as bacteria and fungi, with the capability to degrade these chemicals, among others. Furthermore, we aim to develop a reporter system that allows detection of diclofenac and ibuprofen, thereby enhancing the monitoring of these pollutants.

We conceived this project because we are unanimously passionate about preserving our environment. Vienna is known for its strong engagement in maintaining high water quality standards. In addition, the recently issued EU guideline re-emphasized the need for continued improvement and innovation in maintaining water quality. This has motivated us to turn our passion into purpose and action.

What are NSAIDs?

Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) are one type of Pharmaceutical and Personal Care Product (PPCP) pollutants. Improper discharge and disposal from sources, such as factories, households, and healthcare facilities contribute significantly to the presence of these chemicals in natural aquatic systems. Importantly, Sewage systems and water treatment plants are key pathways for the runoff of these chemicals[1][2].

Current problems and challenges regarding NSAIDs and PPCPs

Well-established and characterised toxicity of NSAIDs in aquatic life includes accumulation in bile and liver of many aquatic vertebrates species, histopathological modifications, and reproduction abnormalities. Importantly, the elevated concentration due to accumulation in various aquatic species poses a threat in humans who consume them[3]. The environmental problem is further exacerbated by poorly assessed fate and chemical understanding of many NSAIDs[4]. As a result, many NSAIDs remain and accumulate in various aquatic systems at a global level.

NSAIDs, especially diclofenac, remain a challenge for water treatment. According to previous reported data, diclofenac concentration range up to 380 ng/L in surveyed countries in Europe and some countries in Asia[3][5][6][7][8]. Furthermore, the removal efficiency of diclofenac remains critically low in conventional activated sludges (ASP, 30%), ASP with sand filter (30%), and biological removal followed by disinfection (20%), compared to other surveyed chemicals whose removal efficiency typically ranges from 60 to 100%[3].

Our approaches: enzymatic degradation and biosensors

We intend to tackle this problem using two distinct strategies. First, we have conducted research to identify novel suitable fungal laccases. We have selected six different laccases as candidates for our project. The Laccase 1 (LCC1) and 2 (LCC2) from Trametes versicolor[9][10], are the best studied fungal laccases for pharmaceutical degradation and have been tested in immobilization experiments. Laccase Lac1 from Coriolopsis trogii has been characterized for its biochemical activity against lignin[11]. A laccase from Trametes hirsuta was shown to degrade chloramphenicol efficiently[12]. Furthermore, the laccase 1a from Trametes pubescens was shown to degrade multiple pharmaceuticals, including estrogen[13].

One of our major goals is to compare degradation and immobilization efficiencies of above-mentioned laccases. In the meantime, we will test various immobilization matrices or substrates such as agar agar and activated carbon (Fig.1a). After screening for the best laccases to degrade our candidate products (such as ibuprofen, diclofenac, and estrogen), we will express, purify, and immobilize our best laccase candidate to test removal efficiency with real or simulated wastewater treatment plant (WWTP) samples (Fig.1b). We envision implementing our prototype product to wastewater treatment systems to degrade NSAID and PPCP pollutants.

Fig 1
Fig 1)
a) A concept of our immobilization process. Laccases are eventually immobilized onto candidate immobilization substrates together with the catalytic co-substrate ABTS [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)].
b) A general workflow of the protein purification step before immobilization.

Second, we aim to develop biosensors that allow easy and cost-effective detection of diclofenac and ibuprofen. Previous studies have reported that diclofenac increases reactive oxygen species (ROS) via interaction with mitochondrial respiratory complex III protein Rip1p and complex IV protein Cox9p in Saccharomyces cerevisiae[14], (Fig.2a). DNA micro-array studies have further shown that diclofenac induces gene expression changes in S. cereviae cells. Amongst many upregulated genes, the Pdr5p gene, an ATP Binding Cassette (ABC) multidrug transporter, is essential for diclofenac tolerance[15]. Schuller and colleagues have exploited, modified, and optimised the Pdr5 promoter controlling a downstream reporter gene to establish a reporter cassette for diclofenac detection[16] (Fig.2b).

Fig 2
Fig 2) Our biosensors.
2a) Diclofenac is thought to interfere with the mitochondria respiratory complexes and thus activates the PKC pathway. This leads to the binding of transcription factors Pdr1p and Pdr3p to bind to the pleiotropic drug-resistant responsive elements (PDREs).
2b) Schuller et al. have thus used the promoter of the PDR5 gene to construct a biosensor for diclofenac (modified from Schuller et al.[16] under Creative Commons license).
2c) Ibuprofen has been shown to destabilise the high-affinity aromatic amino acid permease Tat2p. Thus, tryptophan levels in ibuprofen treated cells are low[17]. Many genes are shown to be enriched during tryptophan depletion, including PYK1 and SHM1(modified from He et al.[17] under Creative Commons license).

Similarly, screening of a genome-wide library of deletion strains has revealed that yeast trains lacking genes that function in aromatic acid transport, biosynthesis, nucleotide synthesis, and metabolism are more sensitive to ibuprofen[18]. Follow-up studies have further mechanistically established that ibuprofen promotes the degradation of the high-affinity aromatic amino acid permease Tat2p[17]. In search of candidate promoters that are potentially upregulated in the presence of ibuprofen, we reasoned that genes which are upregulated upon tryptophan depletion maybe be a plausible downstream response to ibuprofen, as ibuprofen has been suggested to keep cellular tryptophan level low[17]. Thus, we selected the PYK1 and SHM1 promoters, whose genes have been shown to be downregulated upon tryptophan enrichment[19], as potential candidates for our biosensor (Fig.2c).

A solution of innovation, compatibility and sustainability

Physical and chemical methods to deplete PPCP contaminants are available, such as advanced oxidation process[20], Fenton oxidation[21], and soil aquifer treatment[22]. Though effective, these procedures may exert a high carbon footprint burden or involve toxic byproduct formation. Recently, many microorganisms, either in monoculture or co-culture, have been tested and reported in laboratory to be able to effectively degrade various NSAIDs[3]. Despite their promising potential in providing eco-friendly and carbon footprint-neutral remediation solutions, challenges remain. These include incompatibility with chlorine treatment and other disinfection procedures, maintenance of the microbial composition, and requirements for additional carbon sources for survival and high water sterility, which may pose cost challenges in upscaling and implementation[3]. Thus, our proposal showcases an innovative approach and holds the key to a solution to water remediation. Our solution advances practicality and cost- effectiveness while still aligning with global sustainability goals.

Preliminary Experiments on Enzyme Stability and Activity

The primary objective of our preliminary experiments is to determine the optimal working conditions for Trametes versicolor and Agaricus bisporus laccases, specifically regarding their pH and temperature stability. By understanding how these factors influence enzymatic activity, we can optimize the use of laccases in degrading pharmaceutical pollutants. To assess this, we incubate the enzymes in a range of pH buffers and measure their activity using ABTS as a substrate, which, when oxidized, produces a detectable product monitored via spectrophotometry.

Identifying the ideal pH and temperature will help determine the conditions that support maximum free enzyme efficiency. Given that wastewater environments typically exhibit neutral pH, we are also exploring strategies, such as enzyme immobilization, to enhance stability and performance under these conditions.

Agar-Agar Immobilization

We aim to explore the immobilization of Trametes versicolor laccase within agar-agar matrices[23] to increase its stability and reusability in degrading pharmaceutical pollutants like Diclofenac and Ibuprofen. Immobilizing the enzyme provides a stable microenvironment that may protect it from denaturation and enhance its activity across different conditions, particularly the neutral pH commonly found in wastewater treatment systems.

Immobilisation with Agar Agar

This approach is expected to offer increased resistance to environmental fluctuations, extending the operational lifespan of the enzyme. Through this immobilization, we plan to enhance the enzyme’s effectiveness in NSAID degradation, contributing to more sustainable solutions for removing pharmaceuticals from wastewater.

Activated Carbon Immobilization

In addition to agar-agar, we are investigating activated carbon[24],[25] as a substrate for immobilizing Trametes versicolor. Activated carbon, with its large surface area and strong adsorptive properties, is well-suited for enzyme attachment. By treating the carbon with HCl[26], we aim to enhance its surface properties to facilitate stronger enzyme binding and improved catalytic efficiency.

This method seeks to provide a stable platform for laccase, ensuring effective enzyme adsorption and enhancing its performance in breaking down pharmaceutical pollutants. We will evaluate this immobilization strategy to determine its viability for large-scale use in wastewater treatment applications.

References

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  2. J. O. Osuoha, B. O. Anyanwu, and C. Ejileugha, ‘Pharmaceuticals and personal care products as emerging contaminants: Need for combined treatment strategy’, Journal of Hazardous Materials Advances, vol. 9, p. 100206, Feb. 2023, doi: 10.1016/j.hazadv.2022.100206.
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