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.

Co-interaction of nitrofuran antibiotics and the saponin-rich extract on gram-negative bacteria and colon epithelial cells.

Large-scale use of nitrofurans is associated with a number of risks related to a growing resistance to these compounds and the toxic effects following from their increasing presence in wastewater and the environment. The aim of the study was to investigate an impact of natural surfactant, saponins from Sapindus mukorossi, on antimicrobial properties of nitrofuran antibiotics. Measurements of bacterial metabolic activity indicated a synergistic bactericidal effect in samples with nitrofurantoin or furazolidone, to which saponins were added. Their addition led to more than 50% greater reduction in viable cells than in the samples without saponins. On the other hand, no toxic effect against human colon epithelial cell was observed. It was found that exposure to antibiotics and surfactants caused the cell membranes to be dominated by branched fatty acids. Moreover, the presence of saponins reduced the hydrophobicity of the cell surface making them almost completely hydrophilic. The results have confirmed a high affinity of saponins to the cells of Pseudomonas strains. Their beneficial synergistic effect on the action of antibiotics from the nitrofuran group was also demonstrated. This result opens promising prospects for the use of saponins from S. mukorossi as an adjuvant to reduce the emission of antibiotics into the environment.

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.

A comprehensive review on the influence of light on signaling cross-talk and molecular communication against phyto-microbiome interactions.

Generally, plant growth, development, and their productivity are mainly affected by their growth rate and also depend on environmental factors such as temperature, pH, humidity, and light. The interaction between plants and pathogens are highly specific. Such specificity is well characterized by plants and pathogenic microbes in the form of a molecular signature such as pattern-recognition receptors (PRRs) and microbes-associated molecular patterns (MAMPs), which in turn trigger systemic acquired immunity in plants. A number of Arabidopsis mutant collections are available to investigate molecular and physiological changes in plants under the presence of different light conditions. Over the past decade(s), several studies have been performed by selecting Arabidopsis thaliana under the influence of red, green, blue, far/far-red, and white light. However, only few phenotypic and molecular based studies represent the modulatory effects in plants under the influence of green and blue lights. Apart from this, red light (RL) actively participates in defense mechanisms against several pathogenic infections. This evolutionary pattern of light sensitizes the pathologist to analyze a series of events in plants during various stress conditions of the natural and/or the artificial environment. This review scrutinizes the literature where red, blue, white, and green light (GL) act as sensory systems that affects physiological parameters in plants. Generally, white and RL are responsible for regulating various defense mechanisms, but, GL also participates in this process with a robust impact! In addition to this, we also focus on the activation of signaling pathways (salicylic acid and jasmonic acid) and their influence on plant immune systems against phytopathogen(s).

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.

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.

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.

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.

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 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.

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.

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.

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.

Enzymes Involved in Naproxen Degradation by Planococcus sp. S5.

Naproxen is a one of the most popular non-steroidal anti-inflammatory drugs (NSAIDs) entering the environment as a result of high consumption. For this reason, there is an emerging need to recognize mechanisms of its degradation and enzymes engaged in this process. Planococcus sp. S5 is a gram positive strain able to degrade naproxen in monosubstrate culture (27%). However, naproxen is not a suf-ficient growth substrate for this strain. In the presence of benzoate, 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid or vanillic acid as growth substrates, the degradation of 21.5%, 71.71%, 14.75% and 8.16% of naproxen was observed respectively. It was shown that the activity of monooxygenase, hydroxyquinol 1,2-dioxygenase, protocatechuate 3,4-dioxygenase and protocatechuate 4,5-dioxyegnase in strain S5 was induced after growth of the strain with naproxen and 4-hydroxybenzoate. Moreover, in the presence of naproxen activity of gentisate 1,2-dioxygenase, enzyme engaged in 4-hydroxybenzoate metabolism, was completely inhibited. The obtained results suggest that monooxygenase and hydroxyquinol 1,2-dioxygenase are the main enzymes in naproxen degradation by Planococcus sp. S5.

Activity of a carboxyl-terminal truncated form of catechol 2,3-dioxygenase from Planococcus sp. S5.

Catechol 2,3-dioxygenases (C23Os, E.C.1.13.12.2) are two domain enzymes that catalyze degradation of monoaromatic hydrocarbons. The catalytically active C-domain of all known C23Os comprises ferrous ion ligands as well as residues forming active site pocket. The aim of this work was to examine and discuss the effect of nonsense mutation at position 289 on the activity of catechol 2,3-dioxygenase from Planococcus strain. Although the mutant C23O showed the same optimal temperature for activity as the wild-type protein (35 °C), it exhibited activity slightly more tolerant to alkaline pH. Mutant enzyme exhibited also higher affinity to catechol as a substrate. Its K(m) (66.17 µM) was approximately 30% lower than that of wild-type enzyme. Interestingly, removal of the C-terminal residues resulted in 1.5- to 1.8-fold (P < 0.05) increase in the activity of C23OB61 against 4-methylcatechol and 4-chlorocatechol, respectively, while towards catechol the activity of the protein dropped to about 80% of that of the wild-type enzyme. The results obtained may facilitate the engineering of the C23O for application in the bioremediation of polluted areas.

Bacterial degradation of naproxen--undisclosed pollutant in the environment.

The presence of non-steroidal anti-inflammatory drugs (NSAIDs) in the environment is an emerging problem due to their potential influence on human health and biocenosis. This is the first report on the biotransformation of naproxen, a polycyclic NSAID, by a bacterial strain. Stenotrophomonas maltophilia KB2 transformed naproxen within 35 days with about 28% degradation efficiency. Under cometabolic conditions with glucose or phenol as a carbon source degradation efficiency was 78% and 40%, respectively. Moreover, in the presence of naproxen phenol monooxygenase, naphthalene dioxygenase, hydroxyquinol 1,2-dioxygenase and gentisate 1,2-dioxygenase were induced. This suggests that degradation of naproxen occurs by its hydroxylation to 5,7,8-trihydroxynaproxen, an intermediate that can be cleaved by hydroxyquinol 1,2-dioxygenase. The cleavage product is probably further oxidatively cleaved by gentisate 1,2-dioxygenase. The obtained results provide the basis for the use of cometabolic systems in the bioremediation of polycyclic NSAID-contaminated environments.

Immobilization as a strategy for improving enzyme properties-application to oxidoreductases.

The main objective of the immobilization of enzymes is to enhance the economics of biocatalytic processes. Immobilization allows one to re-use the enzyme for an extended period of time and enables easier separation of the catalyst from the product. Additionally, immobilization improves many properties of enzymes such as performance in organic solvents, pH tolerance, heat stability or the functional stability. Increasing the structural rigidity of the protein and stabilization of multimeric enzymes which prevents dissociation-related inactivation. In the last decade, several papers about immobilization methods have been published. In our work, we present a relation between the influence of immobilization on the improvement of the properties of selected oxidoreductases and their commercial value. We also present our view on the role that different immobilization methods play in the reduction of enzyme inhibition during biotechnological processes.

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.

High activity catechol 1,2-dioxygenase from Stenotrophomonas maltophilia strain KB2 as a useful tool in cis,cis-muconic acid production.

This is the first report of a catechol 1,2-dioxygenase from Stenotrophomonas maltophilia strain KB2 with high activity against catechol and its methyl derivatives. This enzyme was maximally active at pH 8.0 and 40 °C and the half-life of the enzyme at this temperature was 3 h. Kinetic studies showed that the value of K m and V max was 12.8 µM and 1,218.8 U/mg of protein, respectively. During our studies on kinetic properties of the catechol 1,2-dioxygenase we observed substrate inhibition at >80 µM. The nucleotide sequence of the gene encoding the S. maltophilia strain KB2 catechol 1,2-dioxygenase has high identity with other catA genes from members of the genus Pseudomonas. The deduced 314-residue sequence of the enzyme corresponds to a protein of molecular mass 34.5 kDa. This enzyme was inhibited by competitive inhibitors (phenol derivatives) only by ca. 30 %. High tolerance against condition changes is desirable in industrial processes. Our data suggest that this enzyme could be of use as a tool in production of cis,cis-muconic acid and its derivatives.