Physiological responses and molecular mechanisms of biofilm formation induced by extracellular metabolites of euglena in Pseudomonas aeruginosa LNR1 for diesel biodegradation based on transcriptomic and proteomic.

Diesel, as a toxic and complex pollutant, is one of the main components in oily wastewater, and poses serious threats to the aquatic environment and the health of organisms. Employing environmentally friendly biostimulants to enhance the metabolic functions of microorganisms is currently the optimal choice to improve the biodegradation of oil-containing wastewater efficiency. This study takes Pseudomonas aeruginosa LNR1 as the target, analyzing the physiological responses and molecular mechanisms of biofilm formation when enhanced by the extracellular metabolites of euglena (EME) for diesel degradation. The results show that EME not only induces auto-aggregation behavior of strain LNR1, forming aerobic suspended granule biofilm, but also promotes the secretion of signaling molecules in the quorum sensing (QS) system. Transcriptomic and proteomic analyses indicate that the stimulatory effect of EME on strain LNR1 mainly manifests in biofilm formation, substance transmembrane transport, signal transduction, and other biological processes, especially the QS system in signal transduction, which plays a significant regulatory role in biofilm formation, chemotaxis, and two-component system (TCS). This study collectively unveils the molecular mechanisms of biostimulant EME inducing strain LNR1 to enhance diesel degradation from different aspects, providing theoretical guidance for the practical application of EME in oily wastewater pollution control.

Comprehensive assessment of toxicity and environmental risk associated with sulfamethoxazole biodegradation in sulfur-mediated biological wastewater treatment.

Incomplete mineralization of sulfamethoxazole (SMX) in wastewater treatment systems poses a threat to ecological health. The toxicity and environmental risk associated with SMX biodegradation in the sulfur-mediated biological process were examined for the first time through a long-term (180 days) bioreactor study and a series of bioassays. The results indicated that the sulfur-mediated biological system was highly resistant and tolerant to SMX toxicity, as evidenced by the enrichment of sulfate-reducing bacteria (SRB), the improved microbial metabolic activity, and the excellent performance on pollutants removal under long-term SMX exposure. SMX can be effectively biodegraded by the cleavage and rearrangement of the isoxazole ring, hydrogenation and hydroxylation reactions in sulfur-mediated biological wastewater system. These biodegradation pathways effectively reduced the acute toxicity, antibacterial activity, and ecotoxicities of SMX and its biotransformation products (TPs) in the effluent of the sulfur-mediated biological system. The TPs produced via hydrogenation (TP1), hydroxylation, and isoxazole ring cleavage (TP3, TP4, TP5, TP8, and TP9) exhibited lower toxicity than SMX. Under SMX stress, although the abundance of sulfonamide resistance genes increased, the total abundance of ARGs decreased due to the extrusion of some intracellular SMX by the efflux pump genes and the inactivation of some SMX through the biodegradation process. Efflux pump and inactivation, as the main resistance mechanisms of antibiotics in the sulfur-mediated biological system, play a crucial role in microbial self-defense. The findings of this study demonstrate the great potential of the sulfur-mediated biological system in SMX removal, detoxication, and ARGs environmental risk reduction.

Unraveling the Toxicity Associated with Ciprofloxacin Biodegradation in Biological Wastewater Treatment.

Incomplete mineralization of antibiotics in biological sludge systems poses a risk to the environment. In this study, the toxicity associated with ciprofloxacin (CIP) biodegradation in activated sludge (AS), anaerobic methanogenic sludge (AnMS), and sulfur-mediated sludge (SmS) systems was examined via long-term bioreactor tests and a series of bioassays. The AS and AnMS systems were susceptible to CIP and its biotransformation products (TPs) and exhibited performance deterioration, while the SmS system exhibited high tolerance against the toxicity of CIP and its TPs along with excellent pollutant removal. Up to 14 TPs were formed via piperazinyl substituent cleavage, defluorination, decarboxylation, acetylation, and hydroxylation reactions in AS, AnMS, and SmS systems. Biodegradation of CIP in the AS, AnMS, and SmS systems, however, could not completely eliminate its toxicity as evident from the inhibition of Vibrio fischeri luminescence along with Escherichia coli K12 and Bacillus subtilis growth. The anaerobic systems (AnMS and SmS) were more effective than the aerobic AS system at CIP biodegradation, significantly reducing the antibacterial activity of CIP and its TPs in the aqueous phase. In addition, the quantitative structure-activity relationship analysis indicated that the TPs produced via decarboxylation and hydroxylation (TP2 and TP4) as well as by cleavage of piperazine (TP12, TP13, and TP14) exhibited higher toxicity than CIP. The findings of this study provide insights into the toxicity and possible risks associated with CIP biodegradation in biological wastewater treatment.

Enhanced performance and electron transfer of sulfur-mediated biological process under polyethylene terephthalate microplastics exposure.

Microplastics are ubiquitous in estuaries, coasts, sewage and wastewater treatment plants (WWTPs), which could arouse unexpected effects on critical microbial processes in wastewater treatment. In this study, polyethylene terephthalate microplastics (PET-MPs) were selected to investigate the mechanism of its influence on the performance of sulfur-mediated biological process from the perspective of microbial metabolic activity, electron transfer capacity and microbial community. The results indicated that the exposure of 50 particles/L PET-MPs improved the chemical oxygen demand (COD) and sulfate removal efficiencies by 6.6 ± 0.5% and 4.5 ± 0.3%, respectively, due to the stimulation of microbial metabolic activity and the enrichment of sulfate-reducing bacteria (SRB) species, such as Desulfobacter. In addition, we found that the PET-MPs promoted Cytochrome C (Cyt C) production and improved the direct electron transfer (DET) capacity mediated by Cyt C. The long-term presence of PET-MPs stimulated the secretion of extracellular polymeric substance (EPS), especially the proteins and humic substances, which have been verified to be electroactive polymers to act as electron shuttles to promote the interspecies electron transfer pathway in sulfur-mediated biological process. Meanwhile, the transformation products (bis-(2-hydroxyethyl) terephthalate (BHET) and Mono (2-hydroxyethyl) terephthalic acid (MHET) of PET-MPs were detected in sulfur-mediated biological process. These findings indicate that the sulfur-mediated biological process has good adaptability to the toxicity of PET-MPs, which strengthens a deeper understanding of the dual function of microplastics in WWTPs.

Biotransformation of ibuprofen in biological sludge systems: Investigation of performance and mechanisms.

Ibuprofen (IBU), a common non-steroidal anti-inflammatory drug (NSAID), is widely used by humans for controlling fever and pain, and is frequently detected in the influent of wastewater treatment plants and different aquatic environments. In this study, the biotransformation of IBU in activated sludge (AS), anaerobic methanogenic sludge (AnMS) and sulfate-reducing bacteria (SRB)-enriched sludge systems was investigated at three different concentrations of 100, 500 and 1000 µg/L via a series of batch and continuous studies. IBU at concentration of 100 µg/L was effectively biodegraded by AS whereas AnMS and SRB-enriched sludge were less effective in IBU biodegradation at all concentrations tested. However, at higher IBU concentrations of 500 and 1000 µg/L, AS showed poor IBU biodegradation and chemical oxygen demand (COD) removal due to inhibition of aerobic heterotrophic bacteria (i.e., Candidatus Competibacter) by IBU and/or IBU biotransformation products. The microbial analyses showed that IBU addition shifted the microbial community structure in AS, AnMS and SRB-enriched sludge systems, however, the removals of COD, nitrogen and sulfur in both anaerobic sludge systems were not affected significantly (p > 0.05). The findings of this study provided a new insight into biotransformation of IBU in three important biological sludge systems.

Stress-responses of activated sludge and anaerobic sulfate-reducing bacteria sludge under long-term ciprofloxacin exposure.

The activated sludge (AS) and sulfate-reducing bacteria (SRB) sludge systems were continuously operated for 200 days in laboratory to investigate the stress-responses of these two sludge systems under ciprofloxacin (CIP) exposure. It was found that CIP was effectively removed by SRB sludge via adsorption and biodegradation, but little biodegradation in AS system. The CIP biodegradation by SRB sludge made the SRB sludge system more sustainable and tolerant to long-term CIP exposure than AS system with significant (p < 0.05) CIP desorption and decrease of CIP removal. CIP shaped the microbial communities in AS and SRB sludge, and significantly (p < 0.05) inhibited the family Nitrosomonadaceae (ammonia-oxidizing bacteria (AOB)) and genus Nitrospira (nitrite-oxidizing bacteria (NOB)/complete ammonia oxidizer(comammox)) and the nitrogen removal in AS system. Moreover, CIP posed the increase of genus Zoogloea-like organisms and the non-filamentous bulking of AS, e.g. 313 ±â€¯12 mL/g of sludge volume index (SVI) at phase V (influent CIP = 5000 µg/L). The genus Desulfobacter was enriched in SRB sludge system under long-term CIP exposure, and stimulated chemical oxygen demand (COD) removal and sulfate reduction. The increase of genera Zoogloea, Acinetobacter and Flavobacterium in AS, and Zoogloea and Acinetobacter in SRB sludge systems under CIP exposure promoted extracellular polymeric substances (EPS) production and CIP adsorption for self-protection of microbes against CIP toxicity. The functional groups of N-H, O-H, C-O-C and C=O in EPS of AS and SRB sludge provided adsorption sites for CIP and impeded CIP impact on microbial cells. The findings of this study provide an insight into the stress-responses of AS and SRB sludge under long-term CIP exposure, and exhibit the great potential of treating CIP-laden wastewater by SRB sludge system.

Insights into the Fate and Removal of Antibiotics in Engineered Biological Treatment Systems: A Critical Review.

Antibiotics, the most frequently prescribed drugs of modern medicine, are extensively used for both human and veterinary applications. Antibiotics from different wastewater sources (e.g., municipal, hospitals, animal production, and pharmaceutical industries) ultimately are discharged into wastewater treatment plants. Sorption and biodegradation are the two major removal pathways of antibiotics during biological wastewater treatment processes. This review provides the fundamental insights into sorption mechanisms and biodegradation pathways of different classes of antibiotics with diverse physical-chemical attributes. Important factors affecting sorption and biodegradation behavior of antibiotics are also highlighted. Furthermore, this review also sheds light on the critical role of extracellular polymeric substances on antibiotics adsorption and their removal in engineered biological wastewater treatment systems. Despite major advancements, engineered biological wastewater treatment systems are only moderately effective (48-77%) in the removal of antibiotics. In this review, we systematically summarize the behavior and removal of different antibiotics in various biological treatment systems with discussion on their removal efficiency, removal mechanisms, critical bioreactor operating conditions affecting antibiotics removal, and recent innovative advancements. Besides, relevant background information including antibiotics classification, physical-chemical properties, and their occurrence in the environment from different sources is also briefly covered. This review aims to advance our understanding of the fate of various classes of antibiotics in engineered biological wastewater treatment systems and outlines future research directions.

Insights into pharmaceuticals removal in an anaerobic sulfate-reducing bacteria sludge system.

In this study, we examined eight typical and widely detected pharmaceuticals (PhAs) removal in an anaerobic sulfate-reducing bacteria (SRB) sludge system (five antibiotics: sulfadiazine (SD), sulfamethoxazole (SMX), trimethoprim (TMP), ciprofloxacin (CIP) and enoxacin (ENO), and three nonsteroidal anti-inflammatory drugs (NSAIDs): ibuprofen (IBU), ketoprofen (KET) and diclofenac (DIC)). The results showed that the SRB sludge had the higher removal efficacy (20 to 90%) for antibiotics (SD, SMX, TMP, CIP and ENO) than NSAIDs (<20%) via adsorption and biodegradation under different operating conditions. Based on a series of batch studies, fluoroquinolone antibiotics (CIP and ENO) were instantly (<15 min) removed (∼98%) via adsorption on SRB sludge with adsorption coefficient (Kd) as high as 25.3 ±â€¯1.8 L/g-suspended solids (SS). And thermodynamics results indicated that the adsorption of CIP and ENO on SRB sludge was spontaneous (Gibbs free energy change (ΔG°) <0 kJ/mol), exothermic (enthalpy change (ΔH°) <0 kJ/mol), and the adsorption process involved both physisorption and chemisorption (absolute value of ΔH°â€¯= 20 to 80 kJ/mol). Three widely prescribed antibiotics (SMX, TMP and CIP) were further investigated for their possible biodegradation pathways along with functional enzymes involved through a series of batch experiments. The biotransformation intermediates indicated that biotransformations of SMX and CIP in SRB sludge system could be initiated from the cleavage of isoxazole and piperazinyl rings catalyzed by sulfite reductase (SR) and cytochrome P450 (CYP450) enzymes, respectively. TMP was likely biotransformed via O-demethylation and N-acetylation coupled with hydroxylation reactions with CYP450 enzymes as the main functional enzymes. This study provided new insight into PhAs removal in SRB sludge system, and has significant potential of implementing sulfur-mediated biological process for the treatment of PhAs containing wastewater.

A Unique ATPase, ArtR (PA4595), Represses the Type III Secretion System in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an important human pathogen which uses the type III secretion system (T3SS) as a primary virulence factor to establish infections in humans. The results presented in this report revealed that the ATP-binding protein PA4595 (named ArtR, a Regulator that is an ATP-activated Repressor of T3SS) represses T3SS expression in P. aeruginosa. The expression of T3SS genes, including exoS, exoY, exoT, exsCEBA, and exsD-pscB-L, increased significantly when artR was knockout. The effect of ArtR on ExsA is at the transcriptional level, not at the translational level. The regulatory role and cytoplasm localization of ArtR suggest it belongs to the REG sub-family of ATP-binding cassette (ABC) family. Purified GST-tagged ArtR showed ATPase activity in vitro. The conserved aspartate residues in the dual Walker B motifs prove to be essential for the regulatory function of ArtR. The regulation of T3SS by ArtR is unique, which does not involve the known GacS/A-RsmY/Z-RsmA-ExsA pathway or Vfr. This is the first REG subfamily of ATP-binding cassette that is reported to regulate T3SS genes in bacteria. The results specify a novel player in the regulatory networks of T3SS in P. aeruginosa.

Regulatory Effect of DNA Topoisomerase I on T3SS Activity, Antibiotic Susceptibility and Quorum- Sensing-Independent Pyocyanin Synthesis in Pseudomonas aeruginosa.

Topoisomerases are required for alleviating supercoiling of DNA during transcription and replication. Recent evidence suggests that supercoiling of bacterial DNA can affect bacterial pathogenicity. To understand the potential regulatory role of a topoisomerase I (TopA) in Pseudomonas aeruginosa, we investigated a previously isolated topA mutation using genetic approaches. We here report the effects of the altered topoisomerase in P. aeruginosa on type III secretion system, antibiotic susceptibility, biofilm initiation, and pyocyanin production. We found that topA was essential in P. aeruginosa, but a transposon mutant lacking the 13 amino acid residues at the C-terminal of the TopA and a mutant, named topA-RM, in which topA was split into three fragments were viable. The reduced T3SS expression in topA-RM seemed to be directly related to TopA functionality, but not to DNA supercoiling. The drastically increased pyocyanin production in the mutant was a result of up-regulation of the pyocyanin related genes, and the regulation was mediated through the transcriptional regulator PrtN, which is known to regulate bacteriocin. The well-established regulatory pathway, quorum sensing, was unexpectedly not involved in the increased pyocyanin synthesis. Our results demonstrated the unique roles of TopA in T3SS activity, antibiotic susceptibility, initial biofilm formation, and secondary metabolite production, and revealed previously unknown regulatory pathways.

[Adsorption Mechanisms of Ciprofloxacin by Extracellular Polymeric Substances of Sulfate-reducing Bacteria Sludge].

Extracellular polymeric substances (EPS) in microbial sludge, fulfils a key role in removal of micro-organic pollutants during biological wastewater treatment. In this study, the authors evaluated the removal of ciprofloxacin (CIP) by sulfate-reducing bacteria (SRB) sludge in a sulfate-reducing up-flow sludge bed (SRUSB) reactor, and examined the role of EPS on CIP removal in an SRB sludge system. The results indicated that CIP was removed efficiently through adsorption and biodegradation by SRB sludge, with adsorption the major removal pathway. EPS also played an important role in CIP adsorption by SRB sludge, and the adsorption mechanisms of CIP by EPS were investigated using the three-dimensional excitation-emission matrix fluorescence spectroscopy technologies combined with parallel factor analysis. The functional groups binding CIP onto EPS were identified through Fourier transform infrared (FTIR) spectra analysis. The results suggested that the static quenching of EPS following CIP adsorption led to formation of an EPS-CIP complex, and that the CIP was mainly bound with tryptophan and tyrosine-like protein substances in EPS with the binding constants of 1.43×104 L·mol-1 and 1.02×104 L·mol-1, respectively. The FTIR results suggested that hydroxyl, amino and carboxyl functional groups were mainly responsible for binding of CIP onto EPS. The results revealed the adsorption mechanisms of CIP by EPS in SRB sludge, and enhanced understanding of the role of EPS in sulfur-mediated biological processes for the removal of CIP and other organic micro-pollutants.

Understanding the Role of Extracellular Polymeric Substances on Ciprofloxacin Adsorption in Aerobic Sludge, Anaerobic Sludge, and Sulfate-Reducing Bacteria Sludge Systems.

Extracellular polymeric substances (EPS) of microbial sludge play a crucial role in removal of organic micropollutants during biological wastewater treatment. In this study, we examined ciprofloxacin (CIP) removal in three parallel bench-scale reactors using aerobic sludge (AS), anaerobic sludge (AnS), and sulfate-reducing bacteria (SRB) sludge. The results showed that the SRB sludge had the highest specific CIP removal rate via adsorption and biodegradation. CIP removal by EPS accounted up to 35. 6 ± 1.4%, 23.7 ± 0.6%, and 25.5 ± 0.4% of total removal in AS, AnS, and SRB sludge systems, respectively, at influent CIP concentration of 1000 µg/L, which implied that EPS played a critical role in CIP removal. The binding mechanism of EPS on CIP adsorption in three sludge systems were further investigated using a series of batch tests. The results suggested that EPS of SRB sludge possessed stronger hydrophobicity (proteins/polysaccharides (PN/PS) ratio), higher availability of adsorption sites (binding sites ( n)), and higher binding strength (binding constant ( Kb)) between EPS and CIP compared to those of AS and AnS. The findings of this study provide an insight into the role of EPS in biological process for treating CIP-laden wastewaters.

Ciprofloxacin degradation in anaerobic sulfate-reducing bacteria (SRB) sludge system: Mechanism and pathways.

Ciprofloxacin (CIP), a fluoroquinolone antibiotic, removal was examined for the first time, in an anaerobic sulfate-reducing bacteria (SRB) sludge system. About 28.0% of CIP was biodegraded by SRB sludge when the influent CIP concentration was 5000 µg/L. Some SRB genera with high tolerance to CIP (i.e. Desulfobacter), were enriched at CIP concentration of 5000 µg/L. The changes in antibiotic resistance genes (ARGs) of SRB sludge coupled with CIP biodegradation intermediates were used to understand the mechanism of CIP biodegradation for the first time. The percentage of efflux pump genes associated with ARGs increased, while the percentage of fluoroquinolone resistance genes that inhibit the DNA copy of bacteria decreased during prolonged exposure to CIP. It implies that some intracellular CIP was extruded into extracellular environment of microbial cells via efflux pump genes to reduce fluoroquinolone resistance genes accumulation caused by exposure to CIP. Additionally, the degradation products and the possible pathways of CIP biodegradation were also examined using the new method developed in this study. The results suggest that CIP was biodegraded intracellularly via desethylation reaction in piperazinyl ring and hydroxylation reaction catalyzed by cytochrome P450 enzymes. This study provides an insight into the mechanism and pathways of CIP biodegradation by SRB sludge, and opens-up a new opportunity for the treatment of CIP-containing wastewater using sulfur-mediated biological process.

Sulfamethoxazole degradation in anaerobic sulfate-reducing bacteria sludge system.

Sulfamethoxazole (SMX) is one of the most commonly used antibiotics. SMX degradation in sulfate-reducing bacteria (SRB) sludge systems has not been reported so far. This research investigated the SMX degradation using SRB sludge in a sulfate-reducing up-flow sludge bed reactor. Moreover, the mechanisms and kinetics of SMX removal were also investigated using SRB sludge via a series of batch experiments. The results showed that SMX removal was characterized by a rapid sorption onto SRB sludge, and desorption from SRB sludge to aqueous phase until achieving equilibrium, and then followed by slow biodegradation. Biodegradation was the dominant route for SMX removal. The sorption process conformed well to a pseudo-second-order kinetic model, meaning that the sorption occurred primarily via a chemical sorption process. The removal of SMX followed the pseudo-zero-order kinetic model with a specific removal rate of 13.2 ± 0.1 µg/L/d at initial SMX concentration 100 µg/L in batch tests. Based on the analysis of metabolites, most of the SMX biotransformation products' structures altered in the isoxazole ring, which were significantly different from that produced by aerobic and anaerobic sludge systems. Thus, SRB sludge system could play an important role in SMX biodegradation, especially in Sulfate-reduction Autotrophic denitrification and Nitrification Integrated (SANI) process for sewage treatment.

Rapid screening of toxic salbutamol, ractopamine, and clenbuterol in pork sample by high-performance liquid chromatography-UV method.

A rapid and simple high-performance liquid chromatography-UV method was developed for the separation and quantification of salbutamol, ractopamine, and clenbuterol in pork. A mixture of acetonitrile-formic acid-ammonium acetate was used as the mobile phase to separate three ß-agonists on a C18 column with gradient. The effects of the addition of formic acid and ammonium acetate to mobile phases on the separation of ß-agonists were investigated. These additives can greatly improve the resolution and sensitivity. Under the optimized chromatographic condition, this separation does not need extra sample preparation. Complete baseline separation of three ß-agonists was achieved in < 20 minutes; the linear range is 0.2-50 µg/L with a correlation coefficient R2 value of > 0.99. Excellent method reproducibility was found by intra- and interday precisions with a relative standard deviation of < 3%. The detection limit (S/N = 3) was found to be <0.05 µg/L; this method can be used for routine screening of the ß-agonist residues in foods of animal origin before being identified by confirmatory methods.