Deciphering the toxic effects of antibiotics on denitrification: Process performance, microbial community and antibiotic resistance genes.

The extensive application of antibiotics, and the occurrence and spread of antibiotic resistance genes (ARGs) shade health risks to human and animal. The long-term effects of sulfamethoxazole (SMX) and tetracycline (TC) on denitrification process were evaluated in this study, with the focus on nitrogen removal performance, microbial community and ARGs. Results showed that low-concentration SMX and TC (<0.2 mg L-1) initially caused a deterioration in nitrogen removal performance, while higher concentrations (0.4-20 mg L-1) of both antibiotics had no further inhibitory influences. The abundances of ARGs in both systems generally increased during the whole period, and most of them had significant correlations with intI1, especially efflux-pump genes. Castellaniella, which was the dominant genus under antibiotic pressure, might be potential resistant bacteria. These findings provide an insight into the toxic effects of different antibiotics on denitrification process, and guides future efforts to control antibiotics pollution in ecosystems.

How anammox process resists the multi-antibiotic stress: Resistance gene accumulation and microbial community evolution.

The effects of multiple antibiotics on the anaerobic ammonia oxidation (anammox) process were investigated. The resistance of the anammox system to high-concentration antibiotics was also demonstrated through gradual acclimation experiments. Inhibition of the anammox process (R1) occurred when the concentrations of erythromycin (ERY), sulfamethoxazole (SMX) and tetracycline (TC) were 0.1, 5.0 and 0.1 mg L-1, respectively. The nitrogen removal efficiency (NRE) of R1 was reduced from 97.2% to 60.7% within 12 days and then recovered to 88.9 ± 9.5% when the nitrogen loading declined from 4.52 ± 0.69 to 2.11 ± 0.58 kg N m-3 d-1. Even when the concentrations of ERY, SMX and TC were as high as 1.0, 15.0 and 1.0 mg L-1, respectively, R1 maintained stable operation. The increases in the abundance of antibiotic resistance genes (ARGs) and in extracellular polymeric substances (EPS) content showed that the anammox process alleviated stress from multiple antibiotics mainly by producing ARGs and secreting EPS. The molecular docking simulation results illustrated the potential binding sites between ammonium transporter and different antibiotics. The upregulation of functional gene expression and the stable abundance of Candidatus Kuenenia in R1 compared with that in the control suggested that the R1 reactor generally maintained more stable long-term operation. This work provides a new understanding of the application of the anammox process to treat wastewater containing multiple antibiotics.

Ultrasensitive electrochemical detection of DNA hybridization using Au/Fe3O4 magnetic composites combined with silver enhancement.

A novel method is described for the highly effective amplifying electrochemical response of DNA based on oligonucleotides functionalized with Au/Fe(3)O(4) nanocomposites by the aid of silver (Ag) enhancement. Via electrostatic layer-by-layer (LBL) assembly, the prepared Fe(3)O(4) nanoparticles form nano-clusters coated with a bilayer composed of polystyrene sulfonate sodium salt (PSS) and poly(diallyldimethylammonium chloride) (PDDA), which are in favor of adsorbing lots of gold nanoparticles (AuNPs) on the surface. The application of magnetic Fe(3)O(4) made the procedures much more simple, convenient and feasible. The resulting composites were then used as labels via the Au-S bond for the DNA hybridization, followed by catalytic deposition of silver on the gold tags. Such an assay is then combined with a sensitive anodic stripping voltammetry (ASV) measurement of multiple silver nanoparticle tracers. A 27-mer sequence DNA target is detected at a glassy carbon (GC) electrode with a detection limit down to ca. 100 aM, which is 800 times lower than that obtained using gold nanoparticles only as labels in the control experiments. This Fe(3)O(4)/PSS/PDDA/Au composite offers a great promising future for the ultrasensitive detection of other biorecognition events.

Whether glycine betaine improves the thermotolerance of mesophilic anammox consortia.

While the application of mesophilic anammox process is currently the state of the art, the feasibility of a thermophilic anammox bioprocess is still unclear. In this study, we investigate whether glycine betaine (GB) addition can enhance the thermotolerance of mesophilic anammox biomass in the upflow anaerobic sludge blanket (UASB) reactors fed with synthetic wastewater at a nitrogen loading of approximately 4 kg N m-3 d-1. The results showed that during a long-term operation at 45°C with GB (0, 0.1, 1, 2 mM) addition, anammox performance became worse with the final effluent concentrations of NO2 -N of 145 ± 11.6 mg L-1 and nitrogen removal efficiency decreased from 92.3-6.9%. Specific anammox activity decreased from 392.1 ± 12.1-6.0 ± 0.8 mg N g-1 VSS d-1, which were not significantly higher than those in the control reactor. The content of heme c showed a stronger downward trend in T1 (with GB addition) than in the control reactor T0. The qPCR results showed that the relative abundance of Candidatus Kuenenia decreased in both the experimental (from 53.5-28.8%) and control reactors (from 54.1-35.1%). Overall, continuous addition of exogenous GB did not improve the thermotolerance of mesophilic anammox consortia at 45°C.

Exogenous extracellular polymeric substances as protective agents for the preservation of anammox granules.

The preservation of anammox granules is of great significance for the rapid start-up of the anammox process and improvement of performance stability. Therefore, it is necessary to explore an economical and stable preservation strategy. Exogenous extracellular polymeric substances (EPS) were used as protective agents for the preservation of anammox granules in this study. In brief, EPS from anammox sludge (A-EPS) and denitrifying sludge (D-EPS) were added to preserve anammox sludge at 4 °C and room temperature (15-20 °C). The results showed that A-EPS addition at 4 °C was the optimal condition for the preservation of anammox granules. After 90 days of preservation, the specific anammox activity (SAA) of the anammox granules remained at 92.7 ± 2.2 mg N g-1 VSS day-1 (remaining ratio of 33.4%), while that of the sludge with D-EPS addition at the same temperature was only 77.1 ± 3.2 mg N g-1 VSS day-1 (remaining ratio of 27.8%). The nitrogen removal efficiency of the experimental group with D-EPS at room temperature was 85.9%, and that of the A-EPS group reached 90.6% under the same temperature conditions. The abundance of the functional genes hzsA, hdh and nirS of the sludge (4 °C; A-EPS addition) after recovery were 138.5%, 317.1%, and 375.9%, respectively, of those of sludge from the D-EPS-added group at the same temperature. RDA revealed the contribution of proteins to the preservation process. Overall, this study provides an economical and robust strategy for the preservation of anammox granules.

Deciphering the microbial and genetic responses of anammox biogranules to the single and joint stress of zinc and tetracycline.

The feasibility of using anaerobic ammonium oxidation (anammox) process to treat wastewaters containing antibiotics and heavy metals was evaluated in this study. The nitrogen removal performance and characteristic parameters were monitored during the whole experimental period of 258 d. The single and joint effects of zinc and tetracycline on the microbial community were studied in upflow anaerobic sludge blanket (UASB) reactors. The anammox performance remained at levels comparable with the initial state at the lower inhibitor concentrations (zinc, 0-2.26 mg L-1; tetracycline, 0-0.5 mg L-1). When the concentrations of zinc and tetracycline increased to 3.39 mg L-1 in R1 and 1.0 mg L-1 in R2, an obvious deterioration in performance was observed. Dual inhibitors with a total concentration of ≥3 mg L-1 caused dramatic decreases in the nitrogen removal efficiency of R3. The quantification results showed that the abundances of eight antibiotic resistance genes (ARGs), czcA and intI1 in the experimental reactors generally increased under stress from metals or/and antibiotics, with final values higher than in the control, while the functional gene abundances were lower. Moreover, most genes exhibited significant correlations. Microbial community analysis indicated that Planctomycetes (represented by Candidatus Kuenenia) was inhibited by both zinc and tetracycline, but still held the dominant position. Furthermore, Caldilinea (belonging to Chloroflexi) maintained a higher abundance during the inhibitory period, implying its potential resistance to both inhibitors. These findings suggested that anammox could be inhibited by metals and antibiotics, but it has the potential to remove nitrogen from wastewaters containing both of them within the concentration threshold.

Expression of the nirS, hzsA, and hdh genes and antibiotic resistance genes in response to recovery of anammox process inhibited by oxytetracycline.

The inhibitory effects of oxytetracycline (OTC) on the anaerobic ammonium oxidation (anammox) performance were relieved by employing bio-augmentation (BA) tactics. However, the recovery mechanism was vague. The response of specific anammox activity (SAA), heme c, functional genes, extracellular polymeric substance (EPS) and antibiotics resistance genes (ARGs) to OTC inhibition and BA aid were traced in the present study. The results indicated that response of SAA, heme c content and functional genes, such as nirS, hzsA and hdh to OTC inhibition were not synchronous. The presence of the tetC, tetG, tetX, and intI1 genes enhanced the resistance of anammox sludge to OTC, thus accelerating the performance recovery when aided by BA. A significant correlation existed between number of anammox 16S rRNA gene copies and protein level in the soluble microbial products (SMP), between tetG gene relative abundance and polysaccharose in SMP and between tetG gene relative abundance and protein in bound EPS (EPSs). In nutshell, the current findings provide the first description of a recovery mechanism regarding OTC-inhibited anammox performance aided by BA based on functional genes and highlights the contribution of ARGs and the self-resistance ability of EPS.

Microbial community evolution and fate of antibiotic resistance genes in anammox process under oxytetracycline and sulfamethoxazole stresses.

The microbial community characteristics, functional and antibiotic resistance genes (ARGs), anammox performance under individual and combined oxytetracycline (OTC) and sulfamethoxazole (SMX) were tested under environmentally relevant levels. The results showed that anammox performance was inhibited when the OTC or SMX concentration increased from 0.5 to 1.0 mg L-1. The absolute abundance of tetX in OTC (3.03 × 106 copies mg-1), SMX (2.80 × 106 copies mg-1) and OTC + SMX (2.03 × 106 copies mg-1) was the highest and one more order of magnitude higher than that of tetG, tetM, intI1, or sul2. The anammox performance in the presence of OTC or SMX was lower than that sum of their independent effects. The enrichment of sludge resistomes with prolonged exposure time and increasing OTC and SMX doses might be due to succession of bacterial hosts and potential elevation of ARGs by horizontal transfer.

Anammox granules show strong resistance to engineered silver nanoparticles during long-term exposure.

The increasing use of engineered Ag nanoparticles (NPs) in consumer products raises great concerns about their environmental impacts, but their potential impact on anaerobic ammonium oxidation (anammox) bacteria in wastewater treatment remains unclear. In this study, the response of anammox consortia inhabiting in granules to long-term exposure of AgNPs was investigated. The gradually increased stress of AgNPs (1, 10, 50 mg L-1) showed no adverse effects on the nitrogen removal performance and on the relative abundance of Ca. Kuenenia inhabiting in granules. Notably, in response to the escalating burden of AgNPs, anammox consortia tended to secrete more protein but less polysaccharide, and meanwhile increase the relative abundances of Ag+ efflux protein SilA and regulatory protein SilR. Overall, these results suggested that the strong resistance of anammox granules to AgNPs might be mainly attributed to the defense role of extracellular polymeric substances and gene-based regulation of ion transport.

Effect of chromium on granule-based anammox processes.

In this study, the feasibility of application of anaerobic ammonium oxidation (anammox) to teat wastewater containing chromium was assessed. Anammox granule activity decreased with increasing Cr(VI) concentration and pre-exposure time in batch tests, and the 50% inhibition concentration of Cr(VI) on anammox biomass was 296.5 mg L-1. Approximately 93.9% chromium was absorbed by loosely bound-extracellular polymeric substances and tightly bound-extracellular polymeric substances when less than 60 mg L-1 Cr(VI) was added. During long-term operation in up-flow anaerobic sludge blanket reactor, significant inhibitory effects anammox performance were observed for Cr(VI) concentrations up to 2 mg L-1. The nitrogen removal rate (NRR) rapidly decreased to 1.49 ±â€¯0.89 kg N m-3 d-1, whereas the NRR was 11.37 ±â€¯1.30 kg N m-3 d-1 in a control reactor. Compared with initial levels, specific anammox granule activity was 22%. The tolerance of the anammox process to Cr(VI) can be enhanced after a long-term adaptive phase.