Decomposition of non-steroidal anti-inflammatory drugs by activated sludge supported by biopreparation in sequencing batch reactor.

The presence of non-steroidal anti-inflammatory drugs in wastewater from sewage treatment plants indicates that they are not completely biodegradable. The designed biopreparation based on immobilized bacteria enables the degradation of paracetamol, ibuprofen, naproxen and diclofenac at a rate of 0.50 mg/L*day, 0.14 mg/L*day, 0.16 mg/L*day and 0.04 mg/L*day, respectively. Lower degradation of drugs in the mixture than in monosubstrate systems indicates their additive, antagonistic effect, limiting the degradative capacity of microorganisms. The biopreparation is stable for at least 6 weeks in bioreactor conditions. Biochemical parameters of activated sludge functioning showed increased oxygen demand, which was related to increased ammonia concentration caused by long-term exposure of activated sludge to drugs. Reduced metabolic activity was also observed. The preparation enables decomposing drugs and their metabolites, restoring the activated sludge's functionality. The tested biopreparation can support activated sludge in sewage treatment plants in degrading non-steroidal anti-inflammatory drugs and phenolic compounds.

Evaluation of the Defined Bacterial Consortium Efficacy in the Biodegradation of NSAIDs.

Due to the increasing pollution of wastewater with non-steroidal anti-inflammatory drugs, preparations need to be developed to decompose these drugs. This work aimed to develop a bacterial consortium with a defined composition and boundary conditions for the degradation of paracetamol and selected non-steroidal anti-inflammatory drugs (NSAIDs), including ibuprofen, naproxen, and diclofenac. The defined bacterial consortium consisted of Bacillus thuringiensis B1(2015b) and Pseudomonas moorei KB4 strains in a ratio of 1:2. During the tests, it was shown that the bacterial consortium worked in the pH range from 5.5 to 9 and temperatures of 15-35 °C, and its great advantage was its resistance to toxic compounds present in sewage, such as organic solvents, phenols, and metal ions. The degradation tests showed that, in the presence of the defined bacterial consortium in the sequencing batch reactor (SBR), drug degradation occurred at rates of 4.88, 10, 0.1, and 0.05 mg/day for ibuprofen, paracetamol, naproxen, and diclofenac, respectively. In addition, the presence of the tested strains was demonstrated during the experiment as well as after its completion. Therefore, the advantage of the described bacterial consortium is its resistance to the antagonistic effects of the activated sludge microbiome, which will enable it to be tested in real activated sludge conditions.

Immobilized Stenotrophomonas maltophilia KB2 in Naproxen Degradation.

Immobilization is a commonly used method in response to the need to increase the resistance of microorganisms to the toxic effects of xenobiotics. In this study, a plant sponge from Luffa cylindrica was used as a carrier for the immobilization of the Stenotrophomonas maltophilia KB2 strain since such a carrier meets the criteria for high-quality carriers, i.e., low price and biodegradability. The optimal immobilization conditions were established as a temperature of 30 °C, pH 7.2, incubation time of 72 h, and an optical density of the culture of 1.4. The strain immobilized in such conditions was used for the biodegradation of naproxen, and an average rate of degradation of 3.8 µg/hour was obtained under cometabolic conditions with glucose. The obtained results indicate that a microbiological preparation based on immobilized cells on a luffa sponge can be used in bioremediation processes where it is necessary to remove the introduced carrier.

Non-steroidal anti-inflammatory drugs in the era of the Covid-19 pandemic in the context of the human and the environment.

From 2019, life in the world has mainly been determined by successive waves of the COVID-19 epidemic. During this time, the virus structure, action, short- and long-term effects of the infection were discovered, and treatments were developed. This epidemic undoubtedly affected people's lives, but increasing attention is also being paid to the effects of the epidemic on the environment. Following PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-analyses) guidelines, a global scoping review of peer-reviewed information has been conducted on the use of over-the-counter non-steroidal anti-inflammatory drugs in the treatment of symptoms of SARS-CoV-2 infections and their positive and negative effects on the human body, the effects of non-steroidal anti-inflammatory drugs (NSAIDs) on aquatic organisms, and their adverse effects on non-target organisms. The literature from 1998 to 2021 was analysed using the Scopus®, Web of Science™ (WoS) and Google Scholar databases. As non-steroidal anti-inflammatory drugs place a heavy burden on the environment, all reports of the presence of these drugs in the environment during the pandemic period have been thoroughly analysed. Of the 70 peer-reviewed records within the scope, only 14% (n = 10) focussed on the analysis of non-steroidal anti-inflammatory drugs concentrations in wastewater and surface waters during the pandemic period. The percentage of these works indicates that it is still an open topic, and this issue should be supplemented with further reports in which the results obtained during the pandemic, which has been going on for several years, will be published. The authors hope this review will inspire scientists to investigate the problem of non-steroidal anti-inflammatory drugs in the environment to protect them for the next generation.

Use of xanthan gum for whole cell immobilization and its impact in bioremediation - a review.

Xanthan gum is one of the exo-polysaccharides produced by bacteria and is characterized by unique non-Newtonian properties. Its structure and conformation strongly depend on the fermentation conditions and such factors as temperature and ions concentration. The properties of the xanthan gum were appreciated in the controlled drug delivery but in the crosslinked form. Due to its ability to enhance the survival rate of immobilized bacteria, the potential of a crosslinked form is promising. Unfortunately, xanthan gum crosslinking procedures often require toxic substances or harsh environmental conditions, which cannot be used in the entrapment of living cells. In this study, we summarised a crosslinking method that could potentially be modified to reduce its toxicity to living cells. Moreover, this review also includes using xanthan gum in bioremediation studies and possible utilization methods to avoid carrier accumulation in the environment.

Effect of Pseudomonas moorei KB4 Cells' Immobilisation on Their Degradation Potential and Tolerance towards Paracetamol.

Pseudomonas moorei KB4 is capable of degrading paracetamol, but high concentrations of this drug may cause an accumulation of toxic metabolites. It is known that immobilisation can have a protective effect on bacterial cells; therefore, the toxicity and degradation rate of paracetamol by the immobilised strain KB4 were assessed. Strain KB4 was immobilised on a plant sponge. A toxicity assessment was performed by measuring the concentration of ATP using the colony-forming unit (CFU) method. The kinetic parameters of paracetamol degradation were estimated using the Hill equation. Toxicity analysis showed a protective effect of the carrier at low concentrations of paracetamol. Moreover, a pronounced phenomenon of hormesis was observed in the immobilised systems. The obtained kinetic parameters and the course of the kinetic curves clearly indicate a decrease in the degradation activity of cells after their immobilisation. There was a delay in degradation in the systems with free cells without glucose and immobilised cells with glucose. However, it was demonstrated that the immobilised systems can degrade at least ten succeeding cycles of 20 mg/L paracetamol degradation. The obtained results indicate that the immobilised strain may become a useful tool in the process of paracetamol degradation.

Xanthan gum as a carrier for bacterial cell entrapment: Developing a novel immobilised biocatalyst.

Xanthan gum (XAN) is a widely used polysaccharide in various industries. Because of its unique properties, in this study, an attempt was made to adopt the procedure of xanthan gum cross-linking for the entrapment of bacterial cells that are able to biodegrade naproxen. The developed procedure proved to be completely neutral for Bacillus thuringiensis B1(2015b) cells, which demonstrated a survival rate of 99%. A negative impact of entrapment was noted for strain Planococcus sp. S5, which showed a survival rate in the 93-51% range. To achieve good mechanical properties of the composites, they were additionally hardened using polydopamine (PDA). XAN/PDA composites revealed a high stability in a wide range of pH, and their sorption capacity included both cationic and anionic molecules. Analysis of the survival rate during storage at 4 °C in 0.9% NaCl showed that, after 35 days, 98-99% of B1(2015b) and 47% of S5 cells entrapped in XAN/PDA remained alive. This study also presents the results of naproxen biodegradation conducted using XAN/PDA/B1(2015b) in a trickling filter with autochthonous microflora. Hence, owing to the significant acceleration of drug biodegradation (1 mg/L in 14 days) and the chemical oxygen demand removal, the entrapped B1(2015b) cells in XAN/PDA composites showed a promising potential in bioremediation studies and industrial applications.

Degradation of diclofenac by new bacterial strains and its influence on the physiological status of cells.

Diclofenac (DCF) is one of the most commonly utilized non-steroidal anti-inflammatory drugs (NSAIDs), which is known to pose an ecotoxicological threat. In this study, from activated sludge and contaminated soil, we isolated four new bacterial strains able to degrade DCF under mono-substrate and co-metabolic conditions with glucose supplementation. We found that the effectiveness of DCF removal is strictly strain-specific and the addition of the primary substrate is not always beneficial. To assess the multidirectional influence of DCF on bacterial cells we evaluated the alterations of increasing concentrations of this drug on membrane structure. A significant increase was observed in the content of 17:0 cyclo fatty acid, which is responsible for reduced fluidity and profound changes in membrane rigidity. The cell injury and oxidative stress were assessed with biomarkers used as endpoints of toxicity, i.e. catalase (CAT), superoxide dismutase (SOD), lipids peroxidation (LPX), and both intra- and extracellular alkaline and acid phosphatase activity. Results indicated that DCF induced oxidative stress, frequently intensified by the addition of glucose. However, the response of the microbial cells to the presence of DCF should not be generalized, since the overall picture of the particular alterations greatly varied for each of the examined strains.

Suitability of Immobilized Systems for Microbiological Degradation of Endocrine Disrupting Compounds.

The rising pollution of the environment with endocrine disrupting compounds has increased interest in searching for new, effective bioremediation methods. Particular attention is paid to the search for microorganisms with high degradation potential and the possibility of their use in the degradation of endocrine disrupting compounds. Increasingly, immobilized microorganisms or enzymes are used in biodegradation systems. This review presents the main sources of endocrine disrupting compounds and identifies the risks associated with their presence in the environment. The main pathways of degradation of these compounds by microorganisms are also presented. The last part is devoted to an overview of the immobilization methods used for the purposes of enabling the use of biocatalysts in environmental bioremediation.

Diclofenac Degradation-Enzymes, Genetic Background and Cellular Alterations Triggered in Diclofenac-Metabolizing Strain Pseudomonas moorei KB4.

Diclofenac (DCF) constitutes one of the most significant ecopollutants detected in various environmental matrices. Biological clean-up technologies that rely on xenobiotics-degrading microorganisms are considered as a valuable alternative for chemical oxidation methods. Up to now, the knowledge about DCF multi-level influence on bacterial cells is fragmentary. In this study, we evaluate the degradation potential and impact of DCF on Pseudomonas moorei KB4 strain. In mono-substrate culture KB4 metabolized 0.5 mg L-1 of DCF, but supplementation with glucose (Glc) and sodium acetate (SA) increased degraded doses up to 1 mg L-1 within 12 days. For all established conditions, 4'-OH-DCF and DCF-lactam were identified. Gene expression analysis revealed the up-regulation of selected genes encoding biotransformation enzymes in the presence of DCF, in both mono-substrate and co-metabolic conditions. The multifactorial analysis of KB4 cell exposure to DCF showed a decrease in the zeta-potential with a simultaneous increase in the cell wall hydrophobicity. Magnified membrane permeability was coupled with the significant increase in the branched (19:0 anteiso) and cyclopropane (17:0 cyclo) fatty acid accompanied with reduced amounts of unsaturated ones. DCF injures the cells which is expressed by raised activities of acid and alkaline phosphatases as well as formation of lipids peroxidation products (LPX). The elevated activity of superoxide dismutase (SOD) and catalase (CAT) testified that DCF induced oxidative stress.

Enhanced Degradation of Naproxen by Immobilization of Bacillus thuringiensis B1(2015b) on Loofah Sponge.

The naproxen-degrading bacterium Bacillus thuringiensis B1(2015b) was immobilised onto loofah sponge and introduced into lab-scale trickling filters. The trickling filters constructed for this study additionally contained stabilised microflora from a functioning wastewater treatment plant to assess the behavior of introduced immobilized biocatalyst in a fully functioning bioremediation system. The immobilised cells degraded naproxen (1 mg/L) faster in the presence of autochthonous microflora than in a monoculture trickling filter. There was also abundant colonization of the loofah sponges by the microorganisms from the system. Analysis of the influence of an acute, short-term naproxen exposure on the indigenous community revealed a significant drop in its diversity and qualitative composition. Bioaugmentation was also not neutral to the microflora. Introducing a new microorganism and increasing the removal of the pollutant caused changes in the microbial community structure and species composition. The incorporation of the immobilised B1(2015b) was successful and the introduced strain colonized the basic carrier in the trickling filter after the complete biodegradation of the naproxen. As a result, the bioremediation system could potentially be used to biodegrade naproxen in the future.

Naproxen in the environment: its occurrence, toxicity to nontarget organisms and biodegradation.

This article summarizes the current knowledge about the presence of naproxen in the environment, its toxicity to nontarget organisms and the microbial degradation of this drug. Currently, naproxen has been detected in all types of water, including drinking water and groundwater. The concentrations that have been observed ranged from ng/L to µg/L. These concentrations, although low, may have a negative effect of long-term exposure on nontarget organisms, especially when naproxen is mixed with other drugs. The biological decomposition of naproxen is performed by fungi, algae and bacteria, but the only well-described pathway for its complete degradation is the degradation of naproxen by Bacillus thuringiensis B1(2015b). The key intermediates that appear during the degradation of naproxen by this strain are O-desmethylnaproxen and salicylate. This latter is then cleaved by 1,2-salicylate dioxygenase or is hydroxylated to gentisate or catechol. These intermediates can be cleaved by the appropriate dioxygenases, and the resulting products are incorporated into the central metabolism. KEY POINTS: •High consumption of naproxen is reflected in its presence in the environment. •Prolonged exposure of nontargeted organisms to naproxen can cause adverse effects. •Naproxen biodegradation occurs mainly through desmethylnaproxen as a key intermediate.

A new pathway for naproxen utilisation by Bacillus thuringiensis B1(2015b) and its decomposition in the presence of organic and inorganic contaminants.

Bacillus thuringiensis B1 (2015b) is a bacterial strain that is able to degrade naproxen. However, the potential effect of water co-contaminations on the degradation process and its pathway have not yet been evaluated. The results of our study show that in the presence of aromatic compounds, the B1 (2015b) strain utilised naproxen with an efficiency that was similar to what it was with no aromatic co-contaminations. In the presence of methanol, biodegradation of naproxen was inhibited, while the addition of ethanol increased the decomposition of naproxen. Among the metal ions that were tested, only cobalt (II) and cadmium (II) negatively affected the degradation of the drug. An analysis of the intermediates and enzymes that are engaged in degrading naproxen revealed that the key metabolites are O-desmethylnaproxen, which is the product of tetrahydrofolate-dependent O-demethylase activity, and salicylic acid. Salicylic acid can then be hydroxylated to catechol or gentisic acid or can be cleaved to 2-oxo-3,5-heptadienedioic acid. The high activity level of catechol 1,2-dioxygenase indicated that the main degradative pathway of naproxen in the B1 (2015b) strain is via catechol cleavage.

Naproxen ecotoxicity and biodegradation by Bacillus thuringiensis B1(2015b) strain.

High level of naproxen consumption leads to the appearance of this drug in the environment but its possible effects on non-target organisms together with its biodegradation are not well studied. The aim of this work was to evaluate naproxen ecotoxicity by using the Microbial Assay for Risk Assessment. Moreover, Bacillus thuringiensis B1(2015b) was tested for both ecotoxicity and the ability of this strain to degrade naproxen in cometabolic conditions. The results indicate that the mean value of microbial toxic concentration estimated by MARA test amounts to 1.66 g/L whereas EC50 of naproxen for B1(2015b) strain was 4.69 g/L. At toxic concentration, Bacillus thuringiensis B1(2015b) showed 16:0 iso 3OH fatty acid presence and an increase in the ratio of total saturated to unsaturated fatty acids. High resistance of the examined strain to naproxen correlated with its ability to degrade this drug in cometabolic conditions. The results of bacterial reverse mutation assay (Ames test) revealed that naproxen at concentrations above 1 g/L showed genotoxic effect but the response was not dose-dependent. Maximal specific naproxen removal rate was observed at pH 6.5 and 30 °C, and in the presence of 0.5 g/L glucose as a growth substrate. Kinetic analysis allowed estimation of the half saturation constant (Ks) and the maximum specific naproxen removal rate (qmax) as 6.86 mg/L and 1.26 mg/L day, respectively. These results indicate that Bacillus thuringiensis B1(2015b) has a high ability to degrade naproxen and is a potential tool for bioremediation.

Organic micropollutants paracetamol and ibuprofen-toxicity, biodegradation, and genetic background of their utilization by bacteria.

Currently, analgesics and nonsteroidal anti-inflammatory drugs (NSAIDs) are classified as one of the most emerging group of xenobiotics and have been detected in various natural matrices. Among them, monocyclic paracetamol and ibuprofen, widely used to treat mild and moderate pain are the most popular. Since long-term adverse effects of these xenobiotics and their biological and pharmacokinetic activity especially at environmentally relevant concentrations are better understood, degradation of such contaminants has become a major concern. Moreover, to date, conventional wastewater treatment plants (WWTPs) are not fully adapted to remove that kind of micropollutants. Bioremediation processes, which utilize bacterial strains with increased degradation abilities, seem to be a promising alternative to the chemical methods used so far. Nevertheless, despite the wide prevalence of paracetamol and ibuprofen in the environment, toxicity and mechanism of their microbial degradation as well as genetic background of these processes remain not fully characterized. In this review, we described the current state of knowledge about toxicity and biodegradation mechanisms of paracetamol and ibuprofen and provided bioinformatics analysis concerning the genetic bases of these xenobiotics decomposition.

Paracetamol - toxicity and microbial utilization. Pseudomonas moorei KB4 as a case study for exploring degradation pathway.

Paracetamol, a widely used analgesic and antipyretic drug, is currently one of the most emerging pollutants worldwide. Besides its wide prevalence in the literature only several bacterial strains able to degrade this compound have been described. In this study, we isolated six new bacterial strains able to remove paracetamol. The isolated strains were identified as the members of Pseudomonas, Bacillus, Acinetobacter and Sphingomonas genera and characterized phenotypically and biochemically using standard methods. From the isolated strains, Pseudomonas moorei KB4 was able to utilize 50 mg L-1 of paracetamol. As the main degradation products, p-aminophenol and hydroquinone were identified. Based on the measurements of specific activity of acyl amidohydrolase, deaminase and hydroquinone 1,2-dioxygenase and the results of liquid chromatography analyses, we proposed a mechanism of paracetamol degradation by KB4 strain under co-metabolic conditions with glucose. Additionally, toxicity bioassays and the influence of various environmental factors, including pH, temperature, heavy metals at no-observed-effective-concentrations, and the presence of aromatic compounds on the efficiency and mechanism of paracetamol degradation by KB4 strain were determined. This comprehensive study about paracetamol biodegradation will be helpful in designing a treatment systems of wastewaters contaminated with paracetamol.

Stenotrophomonas maltophilia: A Gram-Negative Bacterium Useful for Transformations of Flavanone and Chalcone.

A group of flavones, isoflavones, flavanones, and chalcones was subjected to small-scale biotransformation studies with the Gram-negative Stenotrophomonas maltophilia KB2 strain in order to evaluate the capability of this strain to transform flavonoid compounds and to investigate the relationship between compound structure and transformation type. The tested strain transformed flavanones and chalcones. The main type of transformation of compounds with a flavanone moiety was central heterocyclic C ring cleavage, leading to chalcone and dihydrochalcone structures, whereas chalcones underwent reduction to dihydrochalcones and cyclisation to a benzo-γ-pyrone moiety. Substrates with a C-2-C-3 double bond (flavones and isoflavones) were not transformed by Stenotrophomonas maltophilia KB2.

Exploring the Degradation of Ibuprofen by Bacillus thuringiensis B1(2015b): The New Pathway and Factors Affecting Degradation.

Ibuprofen is one of the most often detected pollutants in the environment, particularly at landfill sites and in wastewaters. Contamination with pharmaceuticals is often accompanied by the presence of other compounds which may influence their degradation. This work describes the new degradation pathway of ibuprofen by Bacillus thuringiensis B1(2015b), focusing on enzymes engaged in this process. It is known that the key intermediate which transformation limits the velocity of the degradation process is hydroxyibuprofen. As the degradation rate also depends on various factors, the influence of selected heavy metals and aromatic compounds on ibuprofen degradation by the B1(2015b) strain was examined. Based on the values of non-observed effect concentration (NOEC) it was found that the toxicity of tested metals increases from Hg(II) < Cu(II) < Cd(II) < Co(II) < Cr(VI). Despite the toxic effect of metals, the biodegradation of ibuprofen was observed. The addition of Co2+ ions into the medium significantly extended the time necessary for the complete removal of ibuprofen. It was shown that Bacillus thuringiensis B1(2015b) was able to degrade ibuprofen in the presence of phenol, benzoate, and 2-chlorophenol. Moreover, along with the removal of ibuprofen, degradation of phenol and benzoate was observed. Introduction of 4-chlorophenol into the culture completely inhibits degradation of ibuprofen.

Toxicity and biodegradation of ibuprofen by Bacillus thuringiensis B1(2015b).

In recent years, the increased intake of ibuprofen has resulted in the presence of the drug in the environment. This work presents results of a study on degradation of ibuprofen at 25 mg L-1 in the presence of glucose, as an additional carbon source by Bacillus thuringiensis B1(2015b). In the cometabolic system, the maximum specific growth rate of the bacterial strain was 0.07 ± 0.01 mg mL-1 h-1 and K sµ 0.27 ± 0.15 mg L-1. The maximum specific ibuprofen removal rate and the value of the half-saturation constant were q max = 0.24 ± 0.02 mg mL-1 h-1 and K s = 2.12 ± 0.56 mg L-1, respectively. It has been suggested that monooxygenase and catechol 1,2-dioxygenase are involved in ibuprofen degradation by B. thuringiensis B1(2015b). Toxicity studies showed that B. thuringiensis B1(2015b) is more resistant to ibuprofen than other tested organisms. The EC50 of ibuprofen on the B1 strain is 809.3 mg L-1, and it is 1.5 times higher than the value of the microbial toxic concentration (MTCavg). The obtained results indicate that B. thuringiensis B1(2015b) could be a useful tool in biodegradation/bioremediation processes.

Metabolic Responses of Bacterial Cells to Immobilization.

In recent years immobilized cells have commonly been used for various biotechnological applications, e.g., antibiotic production, soil bioremediation, biodegradation and biotransformation of xenobiotics in wastewater treatment plants. Although the literature data on the physiological changes and behaviour of cells in the immobilized state remain fragmentary, it is well documented that in natural settings microorganisms are mainly found in association with surfaces, which results in biofilm formation. Biofilms are characterized by genetic and physiological heterogeneity and the occurrence of altered microenvironments within the matrix. Microbial cells in communities display a variety of metabolic differences as compared to their free-living counterparts. Immobilization of bacteria can occur either as a natural phenomenon or as an artificial process. The majority of changes observed in immobilized cells result from protection provided by the supports. Knowledge about the main physiological responses occurring in immobilized cells may contribute to improving the efficiency of immobilization techniques. This paper reviews the main metabolic changes exhibited by immobilized bacterial cells, including growth rate, biodegradation capabilities, biocatalytic efficiency and plasmid stability.