Effects of combined antibiotics on nitrification, bacteria and antibiotic resistance genes in activated sludge: Insights from legacy effect of antibiotics.

The effect of combined antibiotics exposure on nitrogen removal, microbial community assembly and proliferation of antibiotics resistance genes (ARGs) is a hotspot in activated sludge system. However, it is unclear that how the historical antibiotic stress affects the subsequent responses of microbes and ARGs to combined antibiotics. In this study, the effects of combined sulfamethoxazole (SMX) and trimethoprim (TMP) pollution on activated sludge under legacy of SMX or TMP stress with different doses (0.005-30 mg/L) were investigated to clarify antibiotic legacy effects. Nitrification activity was inhibited under higher level of combined exposure but a high total nitrogen removal (∼70%) occurred. Based on the full-scale classification, the legacy effect of past antibiotic stress had a marked effect on community composition of conditionally abundant taxa (CAT) and conditionally rare or abundant taxa (CRAT). Rare taxa (RT) were the keystone taxa in the microbial network, and the responses of hub genera were also affected by the legacy of antibiotic stress. Nitrifying bacteria and genes were inhibited by the antibiotics and aerobic denitrifying bacteria (Pseudomonas, Thaurea and Hydrogenophaga) were enriched under legacy of high dose, as were the key denitrifying genes (napA, nirK and norB). Furthermore, the occurrences and co-selection relationship of 94 ARGs were affected by legacy effect. While, some shared hosts (eg., Citrobacter) and hub ARGs (eg., mdtD, mdtE and acrD) were identified. Overall, antibiotic legacy could affect responses of activated sludge to combined antibiotic and the legacy effect was stronger at higher exposure levels.

Protein-bound calcium phosphate in uremic rat serum: a quantitative study.

Protein-bound calcium (prCa) constitutes about 40% of serum total calcium, in which albumin is the most dominant protein. Given the chemical interaction between calcium and phosphate (Pi), the increased serum Pi in chronic kidney disease may cause changes in the composition and structure of the prCa fraction. Here, we report the phosphate binding on the protein-bound calcium in uremic rat serum. Using adenine-fed rats as a uremic model, we separated the calcium and phosphate fractions in rat serum by ultrafiltration, and found that the level of protein-bound phosphate (prPi) in the uremic serum was markedly higher than in control. The elevated prPi level was comparable to the prCa level, consistent with the presence of protein-bound calcium phosphate pr(Ca)j-m(CaPi)m. We then confirmed its presence by ex vivo X-ray absorption near-edge structure spectroscopy, revealing the discrete state of the calcium phosphate clusters associated with protein. Finally, in a quantitative investigation using Ca- and Pi-boosted serum, we discovered the threshold concentration for the Pi binding on prCa, and determined the binding constant. The threshold, while preventing Pi from binding to prCa in normal condition, allows the reaction to take place in hyperphosphatemia conditions. The protein-bound calcium phosphate could act as a link between the metabolism of serum proteins and the homeostasis of phosphate and calcium, and it deserves further investigation whether the molar ratio of (prPi/prCa)⋅100% may serve as a serum index of the vascular calcification status in chronic kidney disease.

Correction to: Protein-bound calcium phosphate in uremic rat serum: a quantitative study.

In the original article, few equations and units were published incorrectly.

Ex vivo detection of calcium phosphate and calcium carbonate in rat blood serum.

The total calcium (tCa) in blood serum comprises free Ca2+ ions (fCa), protein-bound calcium (prCa), and complexed calcium by small anions (cCa). The cCa fraction, in addition to fCa, has been indicated to have some physiological activity. However, there is little evidence for the structure of its constituents. Here we report an ex vivo detection of the cCa constituents by synchrotron X-ray absorption near-edge structure spectroscopy. We collected the data directly on rat blood serum and, by making use of the reference samples, derived a spectrum that exhibits the features of cCa constituents. Among the features are those of the complexes of calcium phosphate and calcium carbonate. The detected complexes in the cCa fraction are mainly Ca(η2-HPO4)(H2O)4 and Ca(η1-HCO3)(H2O)5+, in which HPO42- and HCO3- serve as bidentate and unidentate ligands, respectively. The remained H2O molecules on the coordination sphere of Ca2+ enable these complexes to behave partially like aquated Ca2+ ions in protein-binding. Besides, as the dominant part of prCa, albumin-bound calcium (albCa) exhibits a spectrum that closely resembles that of fCa, indicating weak interactions between the protein carboxyl groups and calcium. The weak-bound cCa and albCa, along with fCa and the relevant anions, compose a local chemical system that could play a role in maintaining the calcium level in blood.

Pseudo-equilibrium equation of calcium phosphate precipitation from aqueous solution.

An X-ray amorphous phase is frequently present at the early stage of calcium phosphate crystallization, and the relevant solution chemistry is essential for understanding the mechanism of reaction. Here, we report a quantitative study of a series of reaction systems at pseudo-equilibrium states. We determined the composition of solutions and the quantities of the precipitate samples, and characterized the long- and short-range order of the precipitate using X-ray diffraction and synchrotron X-ray absorption near-edge structure spectroscopy, respectively. We found that, in a particle with multiple structural units, only a fraction of the units was able to reach pseudo-equilibrium with the solution composition, which represents the average number of surficial clusters per unit. These findings enabled us to propose a general form of the equilibrium constant equation. The equation fits the pseudo-equilibrium data well, and it converts to the "solubility product (Ksp)" and the conventional "reaction quotient" in two limit cases, respectively. Further, using a cube model, we derived a "particle equation" that reveals the connection between the particle structure and the form of equilibrium constant equation. The dependency of the form of pseudo-equilibrium equation on the structure and size of the precipitate reveals a fundamental relation in chemistry, and its applicability remains to be examined in other reaction systems, such as those involving nanocrystals and porous materials.

Impact of ciprofloxacin and copper combined pollution on activated sludge: Abundant-rare taxa and antibiotic resistance genes.

This study aimed to explore the impacts of ciprofloxacin (CIP, 0.05-40 mg/L) and copper (3 mg/L) combined pollution on nitrification, microbial community and antibiotic resistance genes (ARGs) in activated sludge system during stress- and post-effect periods. Higher CIP concentration inhibited nitrification and an average of 50% total nitrogen removal occurred under 40 mg/L of CIP pressure. The stress- and post-effects on bacterial diversity and structure were obviously distinct. Abundant genera were more sensitive to combined pollution than rare genera based on full-scale classification and conditionally rare or abundant taxa were keystone taxa in their interactions. Ammonia oxidation genes were inhibited under high CIP level, but some aerobic denitrifying bacteria (Thauera, Comamonas and Azoarcus) and key genes increased. 96 ARG subtypes were detected with complex positive relationships and their potential hosts (abundant-rare-functional genera) changed in two periods. This study highlights the different stress- and post-effects of combined pollution on activated sludge.

The dissimilarity of antibiotic and quorum sensing inhibitor on activated sludge nitrification system: Microbial communities and antibiotic resistance genes.

Effects of antibiotics (azithromycin, AZM, 1-40 mg/L) and quorum sensing inhibitor (QSI, 2(5H)-furanone, 1-40 mg/L) combined pollution with environmental concentration of copper on bacterial/archaeal community and antibiotic resistance genes (ARGs) in activated sludge system were explored. QSI inhibited nitrification more obviously than AZM. AZM and QSI were synergistic inhibitions on bacterial diversity, and AZM inhibited bacterial compositions more than QSI. While, QSI had more impacts on archaeal diversity/compositions. Less interactions among bacteria and archaea communities with Aquimonas as keystone genus. Functional differences in bacteria/archaea communities were little, and AZM had more effects on metabolism. AZM mainly affected nitrifying bacteria (Candidatus Nitrospira nitrificans and Nitrosomonas). Specific denitrifying bacteria were enriched by AZM (Brevundimonas, 1.76-31.69%) and QSI (Comamonas, 0.61-9.61%), respectively. AZM enriched ARGs more easily than QSI and they were antagonistic to proliferation of ARGs. Bacteria were main hosts of ARGs (macrolide-lincosamide-streptogramin B, other/efflux, etc.) and archaea (Methanosphaerula, Methanolobus) carried multiple ARGs.

Impacts of combined pollution under gradient increasing and gradient decreasing exposure modes on activated sludge: Microbial communities and antibiotic resistance genes.

The responses of microbial communities and antibiotic resistance genes (ARGs) to azithromycin and copper combined pollution under gradient increasing (from 0.5 to 10 mg/L) and decreasing exposure (from 10 to 0.5 mg/L) modes were investigated. Nitrification was inhibited more obviously under gradient increasing exposure mode. Responses of archaeal community and function structure were more obvious than bacteria under both exposure modes. The dominant bacterial and archaeal compositions (Hyphomicrobium, Euryarchaeota, etc.) were affected by two exposure modes, except some rare archaea (Methanoregula and Methanosarcina). There were more positive correlations between bacteria and archaea, and Nitrospira was keystone genus. Ammonia-oxidizing archaea (0.37-3.06%) and complete ammonia oxidizers (Nitrospira_ENR4) were enriched, and Nitrososphaera_viennensis was closely related to denitrifying genes (napA/B, nosZ, etc.). 50 ARG subtypes were detected and specific ARG subtypes (aac, ImrA, etc.) proliferated in two exposure modes. Bacteria and archaea were common hosts for 24 ARGs and contributed to their shifts.

Fates of antibiotic resistance genes and bacterial/archaeal communities of activated sludge under stress of copper: Gradient increasing/decreasing exposure modes.

Effect of copper (Cu) on antibiotic resistance genes (ARGs) and bacterial/archaeal community of activated sludge under gradient increasing (0.5-10 mg/L) or decreasing exposure (10-0.5 mg/L) modes was explored. Here, 29 genes were detected among 48 selected ARGs and mobile gene elements (MGEs). Two exposure modes showed dissimilar effects on ARGs and distribution was more affected by environmental concentrations of Cu, which promoted transmission of ARGs (multiple drug resistance and sulfonamide). Cellular protection was main resistance mechanism, which was less inhibited than efflux pumps. The tnpA-02, as main MGE, interacted closely with ARGs (sul2, floR, etc.). Gradient increasing exposure mode had more effects on bacterial/archaeal structure and composition. Bacteria were main hosts for specific ARGs and tnpA-02, while archaea carried multiple ARGs (cmx(A), adeA, etc.), and bacteria (24.24 %) contributed more to changes of ARGs than archaea (19.29 %). This study clarified the impacts of Cu on the proliferation and transmission of ARGs.

[Historical Antibiotic Stress Changed the Effects of Sulfamethoxazole and Trimethoprim on Activated Sludge: ARGs and Potential Hosts].

The co-exposure of antibiotics has important effects on antibiotic resistance genes (ARGs) and microbial community aggregation in wastewater treatment plants (WWTPs). However, it is unclear whether differences in historical antibiotic exposure stress can determine responses of microbes and ARGs to combined antibiotics. By selecting a high concentration (30 mg·L-1) of sulfamethoxazole (SMX) and trimethoprim (TMP) as historical exposure stress conditions, the effects of SMX and TMP-combined pollution on ARGs, bacterial communities, and their interactions were explored in short-term experiments. Based on high-throughput quantitative PCR, a total of 13 ARGs were detected, and the absolute abundance was 2.21-5.42 copies·µL-1 (logarithm, DNA, the same below). Among them, sul2, ermB, mefA, and tetM-01 were the main subtypes in the samples, and the absolute abundance was between 2.95 and 5.40 copies·µL-1. The combined contamination of SMX and TMP could cause the enrichment of ARGs and mobile genetic elements (MGEs); however, their effects on each subtype were different, and the historical legacy effect of SMX was higher than that of TMP. Under the different exposure histories, the co-occurrence and co-exclusion patterns existed between ARGs. Moreover, MGEs (especially intI-1) were significantly correlated with sulfonamides (sul1 and sul2), tetracyclines[tet(32)], and macrolide-lincosamide-streptogramin (MLSB) resistance genes (ermB). Based on the full-scale classification of microorganisms, it was found that the microbial community structure of various groups responded differently to combined pollution, and the conditionally abundant taxa (CAT) were obviously enriched. Thauera, Pseudoxanthomonas, and Paracoccus were the dominant resistant bacterial genera. Furthermore, a total of 31 potential hosts of ARGs were identified with network analysis, which were dominated with conditionally rare taxa (CRT). Particularly, Candidatus_Alysiosphaera and Fusibacter were positively correlated with most of the ARGs, being the common protentional hosts. Importantly, some rare genera (RT, Variibacter, Aeromonas, Cloacibacterium, etc.) were potential hosts of transposon IS613, which played an important role in the proliferation and spread of ARGs. In conclusion, this study revealed the legacy effects of historical antibiotic stress on ARGs and their hosts, which could provide new ideas and theoretical basis for reducing ARGs pollution in WWTPs.

Multi-metabolism regulation insights into nutrients removal performance with adding heterotrophic nitrification-aerobic denitrification bacteria in tidal flow constructed wetlands.

Constructed wetlands (CWs) usually exhibit limits in functional redundancy and diversity of microbial community contributing to lower performances of nutrients removal in decentralized domestic sewage treatment. To address this quandary, heterotrophic nitrification-aerobic denitrification (HN-AD) bacteria was added in tidal flow CWs (TFCWs) developing for nitrogen (N) and phosphorus (P) removal. With addition of HN-AD bacteria, TFCWs could be setup more rapidly and obtained better removal efficiencies of 66.9%-70.1% total nitrogen (TN), and 88.2%-92.4% total phosphorus (TP) comparing with control systems (TN: 53.9%; TP: 83.9%) during stable operation. Typical-cycles variations showed that TFCWs with addition of HN-AD bacteria promoted NO3--N and NH4+-N removal respectively under hydraulic retention time (HRT) of 14 h and 8 h with slight NO2--N accumulation. Activated alumina (AA) coupled with HN-AD bacteria decreased P release and relieved its poor removal performance in CWs. Based on metagenomic taxa and functional annotation, Pseudomonas and Thauera played pivotal roles in N removal in TFCWs. Furthermore, gradient oxic environments by 8 h-HRT promoted co-occurrence of heterotrophic nitrifiers (mostly Pseudomonas stutzeri) and autotrophic nitrifiers (mostly Nitrosomonas europaea. and Nitrospira sp.) which potentially accelerated NH4+-N transformation by elevated nitrification and denitrification related genes (e. g. amoABC, hao, napA and nirS genes). Meanwhile, the addition of HN-AD bacteria stimulated nirA and gltD genes of N assimilation processes probably leading to NH4+-N directly removal. The conceptual model of multi-metabolism regulation by HN-AD process highlighted importance of glk, gap2 and PK genes in glycolysis pathway which were vital drivers to nutrients metabolism. Overall, this study provides insights into how ongoing HN-AD bacteria-addition effected microbial consortia and metabolic pathways, serving theoretical basis for its engineered applications of TFCWs in decentralized domestic sewage treatment.

Response of microbial communities based on full-scale classification and antibiotic resistance genes to azithromycin and copper combined pollution in activated sludge nitrification laboratory mesocosms at low temperature.

This study aimed to investigate the short-term response of abundant-rare genera and antibiotic resistance genes (ARGs) to azithromycin (AZM, 0.05-40 mg/L) and copper (1 mg/L) combined pollution in activated sludge nitrification system at low temperature. Nitrification was as expected inhibited in stress- and post-effects periods under AZM concentration higher than 5 mg/L. Abundant and rare taxa presented dissimilar responses based on full-scale classification. Conditionally rare or abundant taxa (CRAT) were keystone taxa. Relative abundance of ammonia-oxidizing archaea increased, and three aerobic denitrifying bacteria (Brevundimonas, Comamonas and Trichococcus) were enriched (from 9.83% to 68.91% in total). Ammonia nitrogen assimilating into Org-N and denitrification may be nitrogen pathways based on predict analysis. 29 ARGs were found with more co-occurrence patterns and high concentration of AZM (greater than 5 mg/L) caused their proliferation. Importantly, expect for some abundant taxa, rare taxa, potential pathogens and nitrogen-removal functional genera were the main potential hosts of ARGs.

An Approach to the Uniform Dispersion of Graphene Nanosheets in Powder Metallurgy Nickel-Based Superalloy.

In this paper, a wet-chemical based method was adopted to acquire the uniform dispersion of graphene nanosheets (GNSs) in a powder metallurgy nickel-based superalloy (FGH96) to fabricate a new GNSs reinforced FGH96 metal matrix composite. The surface of the FGH96 powder was modified using a hydrophilic surfactant named polyvinyl alcohol (PVA), which has good wettability and strong hydrogen bonding between the ⁻OH groups of PVA and oxygen groups of GNSs such as ⁻COOH, ⁻CHO, and ⁻OH. It was shown that the GNSs displayed much better dispersion uniformity on the PVA modified FGH96 powder than the unmodified one. The existence of PVA improved the adsorptive capacity of the GNSs attached on the powder surface and prevented the agglomeration in the following thermal preparation process. Consequently, the micro-hardness of PVA modified composite with 0.1 wt.% GNSs reached 497.9 HV, 3.4% higher than the unmodified FGH96 alloy. Therefore, this preparation process could act as the foundation of a common strategy for the fabrication of GNSs in metal matrix composites with good dispersion uniformity, which may have great potential application in aerospace applications.

[Influence of intranasal instilled titanium dioxide nanoparticles on monoaminergic neurotransmitters of female mice at different exposure time].

OBJECTIVE: To investigate the influence of intranasal instilling titanium dioxide (TiO2) nanoparticles on monoaminergic neurotransmitters at different-time exposure. METHODS: CD female mice were intranasally instilled three different-sized (25 nm, 80 nm and 155 nm) TiO, suspension every other day in a dose of 50 mg/kg body weight. The control group was instilled the same volume of Milli-Q water. Inductively coupled plasma-mass spectrometry (ICP-MS) was used to analyze the titanium contents in murine brain after exposure to TiO2 particles 2 days, 10 days, 20 days and 30 days. The monoaminergic neurotransmitters such as norepinephrine (NE), dopamine (DA), 5-hydroxytryptamine (5-HT), 5-hydroxyindole acetic acid (5-HIAA), 3, 4-dihydroxyphenylacetic acid (DOPAC), and homovanillic (HVA), were determined by reversed-phase high performance liquid chromatography (RP-HPLC) with electrochemical detector. RESULTS: After exposure to TiO, nanoparticles 10 days, the titanium contents in murine brain were increased, the titanium content in the 25 nm group was up to (1059.3 +/- 293.5) ng/g. In 20 days, the titanium content decreased slowly with the metabolism of titanium in vivo, but it kept at a high level, the content decreased to (654.7 +/- 269.2) ng/g in the 25 nm group. After exposure to TiO2 nanoparticles 30 days, the titanium contents had no obviously change. Because of the accumulation of TiO, in the brain, the contents of NE and 5-HT increased significantly after exposure to 80 nm and 155 nm TiO, nanoparticles 20 days, while the decreased contents of DA, DOPAC, HVA and 5-HIAA were observed. CONCLUSION: The inhaled TiO2 nanoparticles could be translocated to and deposited in murine brain after absorbing by nasal mucosa, and further influence the releases and metabolisms of monoaminergic neurotransmitters in brain.

[Rapid analysis of methylmercury and total mercury in biological samples by ultrasonic-assisted solvent extraction coupled with inductively coupled plasma-mass spectrometry].

The analytical method for the determination of total mercury and methylmercury in biological samples was studied using ultrasonic-assisted solvent extraction coupled with inductively coupled plasma-mass spectrometry. Various extraction conditions like solvent types and concentrations, different assisted methods, and ultrasonic radiation time were optimized. Samples were digested with 6 mol x L(-1) HCl, ultrasonicated for 2 hours, extracted by organic solvent, then back-extracted by water and directly measured after dilution. With the optimized method, the total mercury and methylmercury were simultaneously measured. The detection limit was 0. 01 ng x mL(-1) , the relative standard deviation (RSD) was 3. 44%, the linearity of calibration curve of mercury was in the concentrations of 0-50 ng x m(L-1), and the recovery of mercury given by standard addition method was 80%-97%. Mercury in five different biological standard reference materials was determined by the described method. The experimental results were in good agreement with the certified values. The proposed method takes the advantage of ultrasonic-assisted extraction, micro-solvent extraction, and ICP-MS, which is especially suitable for the analysis of mercury in different biological samples.

[Analysis of total mercury and methylmercury concentrations in four commercially important freshwater fish species obtained from Beijing markets].

OBJECTIVE: The present paper makes an attempt to evaluate the risk of mercury exposure to human by analyzing the total mercury and methylmercury concentrations of four commercially important freshwater fish species in Beijing market. METHODS: Fish samples of common carp, grass carp, bighead carp, and snakehead were purchased from Beijing market. Then their muscle, liver and gills were taken out. The total mercury and methylmercury concentrations were determined using ultrasonic-assisted solvent extraction coupled with inductively coupled plasma-mass spectrometry. Samples were digested with 6mol/L HCl and sonicated for 2 hours. Subsequently, some aliquots were directly diluted for determination of total mercury concentrations; the remained extracts were extracted with CH2 C12 , and then back extracted with H20 for determination of methylmercury concentrations. To evaluate the validity of the method, the standard materials of DORM-2 and DOLT-3 were also measured. RESULTS: According to the concentrations oof total mercury and methylmercury in the muscles, an order was obtained as: snakehead > bighead carp > grass carp > common carp, which showed that the accumulation of mercury in fish was closely related to food chain. Methylmercury were about 80% of the total mercury, whereas there was a positive relationship between total mercury and methylmercury. The mercury distributions in the bighead carp and snakehead fish showed that the order of mercury concentrations was: muscle > liver > gill, the ratio of methylmercury to total mercury was: muscle > gill > liver. Methylmercury was mainly accumulated in the muscle of fish. CONCLUSION: mercury concentrations of the four fishes in Beijing markets were all below the standard level according to the National Standard of China, therefore, it is safe to human health.

[Trends in chemical speciation of selenium in living organisms by hyphenated techniques].

In the environment and living organisms, limited concentrations and complexity of selenium compounds constitute challenges to the analytical techniques. Since their biological activities are dependent on their chemical states and structure, the key issue lies in the selective determination of the particular species of selenium, which is beneficial to clearly understanding its biochemical roles in metabolism, physiology, toxicology, nutrition, and clinical chemistry. For these purposes, the advanced analytical techniques are greatly indispensable. In the present paper, the current status and development trends in the speciation analysis of selenium by hyphenated techniques is selectively reviewed with the emphasis on the coupling of electrophoretic or chromatographic separation with atomic spectroscopy and mass spectrometry, which render element-selective detection and identification of selenium compounds.