Application of Immobilized Biocatalysts in the Biotransformation of Non-Steroidal Anti-Inflammatory Drugs

Featured Application

Given the new EU Water Directive, wastewater treatment plants are interested in increasing the efficiency of wastewater treatment in terms of pharmaceuticals. Diclofenac is one of the most common pollutants indicated in the above directive. In connection with the above, the developed preparation, after passing tests on a semi-technical scale, can be used as a factor supporting the work of the sewage treatment plant to remove non-steroidal anti-inflammatory drugs.

Abstract

Among the micropollutants identified in the environment, non-steroidal anti-inflammatory drugs (NSAIDs) dominate more and more often. This is due to both the high consumption and low efficiency of biological wastewater treatment plants, where the initial transformation of NSAIDs most often takes place. The solution to the problem may be using preparations supporting activated sludge in sewage treatment plants in the biodegradation of NSAIDs. Therefore, the research aimed to develop a biopreparation stimulating the activated sludge of the sewage treatment plant to decompose paracetamol and selected NSAIDs. This biopreparation is based on strains of Stenotrophomonas maltophilia KB2, Planococcus sp. S5, Bacillus thuringiensis B1(2015b), and Pseudomonas moorei KB4 immobilized on a plant sponge. As a result of the tests, it was shown that the optimal species composition of the proposed preparation includes all tested strains immobilized on a carrier with a mass of 1.2 g/L. The system optimization showed that the optimal amount of strains on the carrier was 17 mg/g of the carrier, 15 mg/g of the carrier, 18 mg/g of the carrier, and 20 mg/g of the carrier for KB4, B1(2015b), KB2, and S5, respectively. The presence of phenol stimulated the degradation of the tested drugs, and this effect deepened with increasing phenol concentration. At the same time, the degradation rate of the mixture of NSAIDs in the presence of phenol did not depend on the amount of biomass. The lack of inhibition in the presence of an additional co-contaminant, i.e., phenol, indicates that the preparation constructed in this way has a chance of being used in sewage treatment plant systems, where introduced strains are exposed to various aromatic compounds.

1. Introduction

The presence of drugs in the environment, including non-steroidal anti-inflammatory drugs (NSAIDs), has been increasingly signaled. NSAIDs are analgesics and anti-inflammatory medications, often used for mild to moderate pain. Due to the relatively small number of side effects, they are considered safe, widely used, and available without a prescription. This contributes to their high consumption and, consequently, their presence in sewage and the environment. Among others, naproxen and diclofenac have been found in European waters at concentration ranges of 3–753 ng/L and 1–429 ng/L, respectively [1,2,3,4]. Concentrations observed in the environment are not the root of acute toxicity. However, it has been clearly demonstrated that they lead to chronic toxicity. It manifests itself, among others, in changes in the mating behavior of fish, damage to the gills and liver, cardiac arrhythmias, teratogenic changes, and infertility [3,4,5,6].

The drug’s degradation efficiency in biological wastewater treatment plants ranges from 40 to almost 100%, depending on the type of contamination, its concentration, and activated sludge properties [7,8]. Diclofenac is one of the most difficultly degraded drugs among NSAIDs, also characterized by high toxicity [1]. Due to the incomplete degradation of NSAIDs, microorganisms supporting activated sludge and their effective implementation into the indigenous microbiome of sewage treatment plants are sought. Studies on the effect of bioaugmentation on the indigenous microbiome of sewage treatment plants and its degradation efficiency were carried out on various medicinal substances, including progesterone, where an improvement in the removal of this compound by activated sludge was demonstrated in the presence of an introduced strain of Rhodococcus sp. HYW [8]. Aguilar-Romero et al. [9] successfully introduced the Sphingopyxis granuli RW412 strain into the microbiome of a wastewater treatment plant in Grenada, achieving seven times faster degradation of ibuprofen compared to the indigenous microbiome. At the same time, no accumulation of ibuprofen degradation intermediates was observed. In addition, this strain did not negatively affect the microbial population of the sewage treatment plant while maintaining its own stability [9]. Moreover, after introducing Bacillus thuringiensis B1(2015b), Stenotrophomonas maltophilia KB2, and Pseudomonas moorei KB4 into the activated sludge system, only a minor increase in the metabolic activity and diversity of the initial microbiome were observed. However, even though these bacteria could degrade selected NSAIDs, no significant differences were observed between the degradation rate by indigenous microbiomes and those introduced by these strains. The exception was the degradation of diclofenac, which was more effective in bioaugmented activated sludge [10]. Marchlewicz et al. [11] showed that the Stenotrophomonas maltophilia KB2 strain might negatively affect the remaining strains. Therefore, effective augmentation required only the B1(2015b) and KB4 strains. Such augmented active sludge degraded diclofenac, naproxen, ibuprofen, and paracetamol more efficiently than the native microbiome. In addition, it was observed that these strains survived in bioreactor conditions in the presence of activated sludge for five weeks of the experiment [11].

Despite reports of effective augmentation of the indigenous microbiome by bacterial strains with increased degradation potential, most often, introducing such microorganisms into the microbial cultures of sewage treatment plants leads to their displacement. The reason is the changing environmental conditions in the sewage treatment plant and the competition between microorganisms. The solution to this problem is the construction of preparations based on immobilized bacteria or enzymes [12].

Laccase is often used in bioremediation processes as an enzyme with a broad substrate specificity and high oxidative potential. Among other things, the high oxidation efficiency of such drugs as paracetamol, mefenamic acid, and diclofenac by immobilized laccases was demonstrated, which, after immobilization on polyvinylidene fluoride membrane, were highly stable in sewage conditions at variable temperatures and pH [13,14]. However, the use of enzymes has its drawbacks, one of the most important of which is the need to isolate the enzyme, which increases the cost of the bioremediation process. Hence, it is more economical to use immobilized whole bacterial cells [12]. Wang et al. [15] successfully used an immobilized bacterial consortium on activated carbon to remove pyrene-Cr(VI) from contaminated soil. In turn, Fu et al. [16] demonstrated the effectiveness of immobilized bacteria on ground cinnamon shells in decomposing seawater-polluting diesel. It was indicated that introducing an immobilized bacteria to the system increased the phosphorus and nitrogen content and promoted the growth of oil-degrading bacteria [16].

Significant for technological processes, apart from the ability of microorganisms to decompose a larger group of drugs, is their resistance to the presence of co-contaminants. It was shown that a mixture of drugs could significantly affect the degradation rates of each of them. In addition, compounds such as alcohols, phenols, organic solvents, or heavy metals can considerably reduce the degradation capacity of introduced strains [11,17,18]. Among others, Marchlewicz et al. [11] showed inhibition of the degradation of ibuprofen, diclofenac, and naproxen in the presence of 2-nitrophenol. In turn, copper inhibited the decomposition of paracetamol and diclofenac, and in the presence of acetone, the decomposition of paracetamol, naproxen, and diclofenac was inhibited. In addition, a synergistic effect of the drugs themselves on the microorganisms that decompose them is often observed. For example, Bacillus thuringiensis B1(2015b) showed the ability to degrade 10 mg/L of ibuprofen within 24 h, while the same concentration of the drug administered in a bioreactor with a mixture of naproxen, diclofenac, and paracetamol was degraded within 3 days [11,19].

In previous studies, the authors successfully immobilized strains capable of degrading selected NSAIDs on the plant sponge Luffa cylindrica and characterized their properties [20,21,22,23]. The choice of plant sponge from Loofah cylindrica was dictated by the fact that it is a cheap, biodegradable, and biocompatible material with a hydrophilic surface on which there are many functional groups. The main advantages of this material are high porosity, non-toxicity, simple application and operation technique, and mechanical resistance [20]. The current work continues research aimed at developing an optimized preparation for the degradation of a mixture of NSAIDs and paracetamol. The specific objectives were the selection of appropriate bacterial ratios and the amount of carrier. To determine the effectiveness of the preparation in the presence of co-contaminants, the degradation of the mixture of NSAIDs and paracetamol in the presence of various phenol concentrations was tested.

2. Materials and Methods

2.1. Immobilization of Bacterial Strains

Bacillus thuringiensis B1(2015b) (GenBank Accession Number KP895873.1), Pseudomonas moorei KB4 (GenBank Accession Number GCA_004212425.1), Planococcus sp. S5 (GenBank Accession Number AY028621.1) and Stenotrophomonas maltophilia KB2 (VTT E-113197) were cultivated separately in the nutrient broth (BBL^® Nutrient broth, Becton Dickinson, Franklin Lakes, New Jersey, USA) at 30 °C and 130 rpm for 24 h [19,24,25,26]. The cells were then centrifuged (5000× g at 4 °C for 15 min), washed with a fresh sterile mineral salts medium (MSM) [26], and used in further experiments. Planococcus sp. S5, Bacillus thuringiensis B1(2015b), Pseudomonas moorei KB4, and Stenotrophomonas maltophilia KB2 were immobilized separately through the adsorption on the surface of the loofah sponge, according to Dzionek et al. [20], Dzionek et al. [21], Surma et al. [22], Wojcieszyńska et al. [23], respectively. The initial optical density of the cultures used during the immobilization of Planococcus sp. S5, Pseudomonas moorei KB4, and Stenotrophomonas maltophilia KB2 were 1.2, and Bacillus thuringiensis B1(2015b)—0.2. The amount of immobilized biomass on the carrier is, respectively, for the strain Pseudomonas moorei KB4—17 mg of the dry mass of bacteria per 1 g of carrier, for the strain Bacillus thuringiensis B1(2015b)—15 mg of the dry mass of bacteria per 1 g of carrier, for strain Stenotrophomonas maltophilia KB2—18 mg of the dry mass of bacteria per 1 g of carrier, and for the Planococcus sp. S5 strain—20 mg of the dry mass of bacteria per 1 g of carrier. The individual parts of the carriers were mixed in appropriate ratios.

2.2. NSAIDs Degradation Experiments

Periodic cultures were conducted in an MSM [26]. Drugs at a concentration of 250 µg/L (naproxen, paracetamol, diclofenac, ibuprofen), 150 mg/L phenol, and preparations with the strains arrangement (V1)—B1(2015b), KB4, KB2 (1:1:1); (V2)—B1(2015b), KB4, S5 (1:1:1); (V3)—B1(2015b), KB4, KB2, S5 (1:1:1:1) for selecting the appropriate composition of the preparation were tested. The ratios are expressed in the amounts of carriers with immobilized strains. The cultures were cultivated at 30 °C. Glucose (0.5 g/L) was used as a carbon source. To determine the optimal amount of the tested preparation, MSM with phenol in concentration 0.5 mg/L or 300 mg/L and NSAIDs (in concentration 0.125 mg/L of each drug; summary 0.5 mg/L NSAIDs) was supplemented with preparation with an optimal species composition in the amount of 0.6, 1.2, and 1.8 g carrier/L. The cultures were cultivated at 30 °C, 130 rpm. During the experiment, the concentration of NSAIDs, phenol, and the metabolic activity of the preparations were monitored at specific time intervals. All layouts were performed in 3 replicates.

2.3. Biochemical Analysis

The metabolic activity of immobilized microorganisms was measured using the fluorescein diacetate method (FDA), according to Dzionek et al. [27,28].

2.4. Determination of NSAIDs Concentration

2.4.1. HPLC Analysis

The decomposition of NSAIDs was monitored by HPLC (Merck HITACHI, Darmstadt, Germany) equipped with a LiChromospher^® RP-18 column (4 mm × 250 mm), liChroCART^® 250-4 Nucleosil 5 C18, a DAD detector (concentration of paracetamol, ibuprofen, and naproxen) and a fluorescent detector (ibuprofen concentration). Separation was carried out in a gradient flow of 1 mL/min. The mobile phase with the gradient flow consisted of changes in acetonitrile and 1% acetic acid proportion during the analysis time (first 6 min proportion was 10:90 v/v; next 3.5 min 80:20 v/v; last 3.5 min 10:90 v/v). Samples were taken at the proper period and centrifuged (14,000 rpm, 20 min). The detection wavelength was set: absorption—224 nm; emission—295 nm for ibuprofen; 276 nm for diclofenac and phenol; 260 nm for naproxen; 240 nm for paracetamol.

2.4.2. Gas Chromatography–Mass Spectrometry (GC/MS) Analysis

The study was conducted according to the methodology described [28] with some modifications. Briefly, samples of the culture solution were concentrated in a concentrator (24 h, 30 °C). Then, 0.1 mL of N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) + trimethylchlorosilane (TMCS), 99:1 (Supelco/Merck, Darmstadt, Germany), were introduced to each sample and incubated for 30 min at 60 °C. After that, the samples were dissolved in 1 mL of hexane. For qualitative analysis, a PEGASUS 4D GCxGC-TOFMS gas chromatograph (LECO Corp., St. Joseph, MI, USA) connected to a BPX5 capillary column (5% phenyl equivalent, 59.35 m × 0.25 mm; 0.25 μm) (SGE Int., Melbourne, Australia) was used.

The temperature of the ion source and transfer line was 250 °C. Helium was used as the carrier gas, and the flow rate was set to 1.0 mL/min. The oven temperature was set and maintained at 40 °C for 2 min after injecting 1 μL of the sample (no flux splitting mode). The temperature was then raised to 62 °C (0.5 °C/min rate), then to 90 °C (10 °C/min rate), and, finally, to 250 °C (40 °C/min rate). The ionization source was operated in the positive ion mode (electron energy: 70 V), and the acquisition rate was 10 spectra/s. Quantification was based on previously prepared calibration curves for each compound.

2.5. Carrier Analysis by Scanning Electron Microscopy (SEM)

The immobilized strains on the surface of the loofah sponge were observed after sample preparation, according to Dzionek et al. [20]. Improvement of the electrical conductivity of the sample surface was obtained by sputtering in a Safematic CCU-010 HV SEM Coating System (Safematic GmbH, Zizers, Switzerland) with a thin layer of gold. The tests were observed with a Hitachi SU8010 field emission scanner electron microscope (Hitachi High-Technologies Corporation, Tokyo, Japan).

2.6. Statistical Analysis

STATISTICA 13 PL software was used for statistical analysis (TIBCO Software Inc., Palo Alto, CA, USA).

3. Results and Discussion

3.1. Development of the Qualitative Composition of the Biopreparation

Microorganisms most often transform NSAIDs into their hydroxyl derivatives, and in this form, they get from biological sewage treatment plants to the environment. Few bacteria and fungi break down these drugs into intermediates of basal metabolism [1,2,17,29,30].

One of the best-known mechanisms of drug degradation is the degradation of paracetamol. It is most often transformed into such critical metabolites as 4-aminophenol and hydroquinone. The degradation of these intermediates limits this drug’s degradation rate [31,32]. Moreover, many papers concern the decomposition of ibuprofen, the first step of which is usually hydroxylation of the aromatic ring or the side chain carbon. Complete degradation proceeds with the aromatic ring cleavage, most often carried out by enzymes belonging to the cleaving dioxygenases [33,34,35]. Diclofenac and naproxen are much more difficult to decompose. There are few reports on the degradation of naproxen, and the critical step in its decomposition is demethylation to desmethylnaproxen. Naproxen is often degraded by pathways typical for naphthalene derivatives, where salicylate 1,2-dioxygenase, responsible for the cleavage of the aromatic ring, plays a unique role [3,36]. Hydroxylation is also the first step in the transformation of diclofenac. In the following steps, the amino bond between the rings is hydrolyzed. Monocyclic intermediates can be degraded via various pathways to the final products, which are central metabolism intermediates [1,2].

The bacteria in which the complete decomposition of paracetamol has been described include the Pseudomonas moorei KB4 strain isolated from the activated sludge from the wastewater treatment plant Klimzowiec (Chorzów, Poland), which is also characterized by the ability to degrade diclofenac [24,37]. In turn, the Bacillus thuringiensis B1(2015b) strain, isolated from the soil of the chemical factory Organika-Azot in Jaworzno (Poland), belongs to the bacteria that break down ibuprofen and naproxen [38,39]. These strains were used to construct a preparation composed of immobilized bacteria on a plant sponge from Loofha cylindrica. In addition, the preparation was enriched with the Stenotrophomonas maltophilia KB2 strain, which can degrade naproxen and a wide range of aromatic compounds due to the rich enzyme system [25,40]. The second tested strain is Planococcus sp. S5, showing the ability to decompose phenolic compounds and naproxen [21,41]. Both strains were isolated from the activated sludge of a sewage treatment plant in Bytom Miechowice (Poland) [25,41].

In the conducted research, it was essential to determine the optimal species composition of the preparation. Three systems were selected for the study: V1—containing strains B1(2015b), KB4 and KB2, V2—including strains B1(2015b), KB4 and S5 and V3—containing all four tested strains. In earlier studies [11], the KB2 strain was shown to be antagonistic to the KB4 strain. However, the obtained results indicate that the immobilization of the KB2 strain abolishes this effect, as no significant differences were observed in any of the tested systems (Figure 1, Figure 2 and Figure 3).

Partovinia et al. [42] showed that immobilizing a consortium of microorganisms on support resulted in antagonistic interactions between the microorganisms. This effect was not observed when the microorganisms of the consortium were immobilized separately on the carrier. The antagonistic effect of the KB2 strain on KB4 observed in the consortium of free bacteria was due to the production by the KB2 strain of an alkaline serine protease that degrades Braun’s lipoprotein present in the outer layer of lipopolysaccharide cells of the KB4 strain [11]. Separate immobilization of the strains included in the preparation prevented direct contact of this enzyme with the lipoprotein of the KB4 strain, which limited the antagonistic effects.

In the first stage of the research, the distribution of 250 ug/L of paracetamol and ibuprofen by each tested system was checked. It was shown that the V1 and V3 systems degraded paracetamol within 18 h, while the degradation of this drug by the V2 system took up to 24 h (Figure 1A). However, no significant differences were observed in the degradation of ibuprofen by all the tested systems, which occurred within 28 h (Figure 1B). In the next step, the decomposition rate of individual drugs in the presence of a mixture of paracetamol, ibuprofen, naproxen, and diclofenac was determined. Paracetamol was degraded the fastest in systems designed in this way (28 h) (Figure 2A). The ibuprofen degradation occurred within 31–32 h, regardless of the system used (Figure 2B) (Figure 2C,D). Naproxen was best degraded in the V3 system, while diclofenac was in the V1 system. However, the decomposition of the tested drugs with two aromatic rings took significantly longer than the decomposition of single-ring drugs [2,3]. No complete degradation of any of them was observed after 32 h. This is due to the high durability of such structures. Naproxen is a derivative of naphthalene. Hence, its degradation involves enzymes engaged in the degradation of two fused aromatic rings. Only after the degradation of one of them is the activity of typical dioxygenases cleaving a single aromatic ring possible, which significantly extends the degradation process of naproxen [39,43]. On the other hand, diclofenac has two aromatic rings linked together by an amine bridge. Although the literature indicates a relatively easy cleavage of this compound into two rings, the deactivating effect of chlorine substituents in diclofenac significantly slows down the degradation process [1,2,4].

An interesting phenomenon is the long period of adaptation of the tested systems to the degradation of naproxen. Noticeable decomposition of naproxen occurred only after the 21st day of culture. Alvarado-Gómez et al. [44] indicate the hydrophilic nature of the sponge, which excludes the sorption of naproxen on its surface and diffusion inside [20]. The poor distribution of naproxen is probably related to the hydrophilic nature of the sponge and the large condensed aromatic system of naproxen. As a highly hydrophobic compound, naproxen probably diffuses slowly through the sponge structure, significantly reducing its availability to immobilized cells. Only the complete formation of a biofilm penetrating the structure of the sponge allows better contact of the drug with the bacteria. As a consequence, this leads to faster degradation of naproxen. Distribution analysis of the 1000 ug/L mixture of NSAIDs tested showed only minor differences between the systems (Figure 3A). The highest decomposition rate in 24 h was demonstrated for the V3 system, which was 485.5 µg/L. The analysis of phenol decomposition, as an indicator of the efficiency of the preparation in the treatment plant conditions, also showed no significant differences between the systems (Figure 3B). Due to the lack of apparent differences in the degradation of the analyzed substances, the V3 system containing all four strains was selected for further research. The choice was dictated by the fact that the preparation is ultimately tested as a preparation supporting the activated sludge of sewage treatment plants, which receive various pollutants. The rich species composition ensures greater versatility of the proposed preparation.

3.2. Choosing the Optimal Amount of Carrier with Immobilized Biomass and Effect of Phenol on the Degradation of a Mixture of NSAIDs and Paracetamol

The following research stage included selecting the optimal amount of carrier with immobilized biomass for biodegradation processes. The preparations contained the KB4 strain at 17 mg/g of the carrier, the B1(2015b) strain at 15 mg/g of the carrier, the KB2 strain at 18 mg/g of the carrier, and the S5 strain at 20 mg/g of the carrier. Figure 4 shows the carrier with immobilized strains constituting the basis for the preparation construction.

Three systems were proposed in the study. The first system contained a carrier at 0.6 g/L, the second at 1.2 g/L, and the third at 1.8 g/L. The corresponding biomass of bacteria for these systems is shown in

Table 1. The amount of immobilized biomass on the carrier.

	Carrier Mass
Strain	0.6 g	1.2 g	1.8 g
KB4	10.2 mg	20.4 mg	30.6 mg
B1(2015b)	9.0 mg	18.0 mg	27.0 mg
KB2	10.8 mg	21.6 mg	32.4 mg
S5	12.0 mg	24.0 mg	36.0 mg

.

The presence of co-contaminants in wastewater can significantly impact the decomposition of drugs. Therefore, it was essential to check whether introducing one of the most frequent aromatic pollutants—phenol—into the system would affect the course of degradation of the mixture of NSAIDs and paracetamol by the biopreparation.

The analysis of the degradation of individual drugs in the presence of NSAIDs mixture and 0.1 mg/L of phenol showed only differences in the rate of degradation of ibuprofen and diclofenac (Figure 5, Figures S1 and S2). The degradation of ibuprofen in the systems with 1.2 g and 1.8 g carriers was similar. Still, with the smallest amount of carrier, it was significantly slower. In turn, diclofenac was decomposed the fastest in the system with the largest amount of carrier. In the other systems, the degradation was similar. After using a higher concentration of phenol—300 mg/L—the differences in the system with ibuprofen and diclofenac deepened. No significant differences were observed in the system with paracetamol and naproxen (Figure 6).

Degradation studies indicate a positive effect of biomass growth on the degradation of xenobiotics, especially those as difficult to degrade as diclofenac. This is associated with a greater possibility of xenobiotic adsorption on biomass and, consequently, faster degradation [45,46]. The lack of differences in paracetamol degradation depending on the biomass used is surprising and challenging to explain.

In the system with increased concentration of phenol, stimulation of NSAID degradation was observed, except for ibuprofen, where no difference in degradation time was observed (Figure 5B and Figure 6B).

Changes in the degradation rate in the presence of a simple aromatic compound can be caused by the induction of appropriate degradation enzymes or an increase in biomass in its presence. Phenol is a good carbon source for the B1 strain [17]. However, immobilization significantly affects the growth kinetics of the strains due to the limited availability of the substrate for cells [4,15]. The lack of changes in the rate of ibuprofen degradation in systems with different phenol concentrations may be related to the fact that its degradation proceeds through hydroxylation with the participation of aliphatic monooxygenase and transformation to 1,4-hydroquinone, for which acyl-CoA synthase is responsible, the enzymes of which are not induced by phenol [34,38].

After analyzing the distribution of the mixture of 0.5 mg/L of the tested drugs and 0.1 or 300 mg/L of phenol by the tested test systems, no significant differences in the degradation rate were observed depending on the biomass used (Figure 7). At the same time all systems behaved stably with high degradation efficiency.

4. Conclusions

Summarizing the obtained results, it can be stated that:

• Due to slight differences in the degradation of diclofenac, which is challenging to decompose and is currently recommended for monitoring in wastewater treatment plants, the authors commend a system with 1.2 g carrier/L as the optimal system;
• This system is more economical and less burdensome for the functioning of the sewage treatment plant infrastructure due to the limited amount of the carrier itself;
• The system is based on four strains: Stenotrophomonas maltophilia KB2, Planococcus sp. S5, Bacillus thuringiensis B1(2015b), and Pseudomonas moorei KB4 immobilized on a plant sponge;
• The co-pollution, which is phenol, not only does not inhibit the functioning of the system but is a good stimulator of the system;
• This system ensures stable operation and effective decomposition with a variable load of pollutants.

5. Patents

The patent application at the Patent Office of the Republic of Poland entitled “Microbial preparation supporting the work of activated sludge in sewage treatment plants in the removal of paracetamol or non-steroidal anti-inflammatory drugs or phenolic compounds” under the number P.444493.