Metalloenzymes play major roles to achieve high-rate nitrogen removal in N-damo communities: Lessons from metaproteomics.

Nitrite-driven anaerobic methane oxidation (N-damo) is a promising biological process to achieve carbon-neutral wastewater treatment solutions, aligned with the sustainable development goals. Here, the enzymatic activities in a membrane bioreactor highly enriched in N-damo bacteria operated at high nitrogen removal rates were investigated. Metaproteomic analyses, with a special focus on metalloenzymes, revealed the complete enzymatic route of N-damo including their unique nitric oxide dismutases. The relative protein abundance evidenced that "Ca. Methylomirabilis lanthanidiphila" was the predominant N-damo species, attributed to the induction of its lanthanide-binding methanol dehydrogenase in the presence of cerium. Metaproteomics also disclosed the activity of the accompanying taxa in denitrification, methylotrophy and methanotrophy. The most abundant functional metalloenzymes from this community require copper, iron, and cerium as cofactors which was correlated with the metal consumptions in the bioreactor. This study highlights the usefulness of metaproteomics for evaluating the enzymatic activities in engineering systems to optimize microbial management.

How efficiently does a metabolically enhanced system with denitrifying anaerobic methane oxidizing microorganisms remove antibiotics?

In this work, the novel N-damo (Nitrite dependent anaerobic methane oxidation) process was investigated at high biomass activities for its potential to remove simultaneously nitrite and methane, as well as selected antibiotics commonly found in sewage in trace amounts. For this purpose, two MBRs were operated at three high nitrite loading rates (NLRs), namely 76 ± 9.9, 161.5 ± 11.4 and 215.2 ± 24.2 mg N-NO⁻2 L-1 d-1, at long-term operation. The MBRs performance achieved a significantly high nitrite removal activity for an N-damo process (specific denitrifying activity of up to 540 mg N-NO⁻2 g-1 VSS d-1), even comparable to heterotrophic denitrification values. In this study, we have implemented a novel operational strategy that sets our work apart from previous studies with similar bioreactors. Specifically, we have introduced Cerium as a trace element in the feeding medium, which serves as a key differentiating factor. It allowed maintaining a stable reactor operation at high NLRs. Microbial community composition evidenced that both MBRs were dominated with N-damo bacteria (67-87% relative abundance in period III and I, respectively). However, a decrease in functional N-damo bacteria (Candidatus Methylomirabilis) abundance was observed during the increase in biomass activity and concentration, concomitantly with an increase of the other minor families (Hypomicrobiaceae and Xanthobacteraceae). Most of the selected antibiotics showed high biotransformation such as sulfamethoxazole, trimethoprim, cefalexin and azithromycin, whereas others such as roxithromycin and clarithromycin were only partially degraded (20-35%). On the contrary, ciprofloxacin showed almost no removal. Despite the metabolic enhancement, no apparent increase on the antibiotic removal was observed throughout the operation, suggesting that microbiological composition was of greater influence than its primary metabolic activity on the removal of antibiotics.

Influence of metabolism and microbiology on organic micropollutants biotransformation in anoxic heterotrophic reactors.

There is scarce information about the biotransformation of organic micropollutants (OMPs) under anoxic conditions. In this study, a heterotrophic denitrifying bioreactor was set up to study the fate of several OMPs from metabolic and microbiological points of view. Primary metabolic activity was increased by adding progressively higher nitrogen loading rates during the operation (from 0.075 to 0.4 g N-NO3- L-1 d-1), which resulted in an important shift in the microbial population from a specialized biomass to a more diverse community. Such a change provoked a significant increase in the removal efficiency of erythromycin (ERY), roxithromycin (ROX) and bisphenol-A (BPA), and some bacterial taxa, such as Rhodoplanes, were identified as possible indicators related to the biodegradation of these compounds. The increasing primary metabolic activity in the reactor did not enhance the OMP-specific removal rates, suggesting that the bacterial composition is more influential than cometabolism.

Cryopreservation of Holm Oak Embryogenic Cultures for Long-Term Conservation and Assessment of Polyploid Stability.

Holm oak populations are severely affected by oak decline syndrome, and reliable methods of conserving the plant material are required. A vitrification-based cryopreservation method was used for the first time for the long-term conservation of holm oak embryogenic cultures. Successful cryopreservation was achieved after determining the best developmental stage of the somatic embryos used and the optimal incubation period in plant vitrification solution 2 (PVS2). Embryos were recovered from individual nodular embryogenic structures (NES) derived from four embryogenic lines after preculture on a medium containing 0.3 M sucrose, incubation in PVS2 vitrification solution for 15 min at 25 °C and direct immersion in liquid nitrogen (LN). Embryo recovery rates of 16.7-63.3% were obtained after cryostorage for four years in LN. In addition to the embryo developmental stage and the PVS2 treatment time, the genotype can also significantly affect embryo recovery after LN storage. There were no significant differences in plant regeneration or polyploid stability between somatic embryos and plants derived from control embryos (not cryopreserved) and cryopreserved embryos. The findings indicate that embryo proliferation, plant conversion and polyploid stability are maintained in material recovered from the vitrification solution and subsequently cryopreserved.

Cometabolic removal of organic micropollutants by enriched nitrite-dependent anaerobic methane oxidizing cultures.

The innovative and recently discovered n-damo process, based on anaerobic methane oxidation with nitrite, was developed in a membrane-based bioreactor and evaluated in terms of organic micropollutants (OMPs) removal. The main singularity of this study consisted in the evaluation of organic micropollutants (OMPs) removal in the biological reactor. A strategy consisting on progressively increasing the nitrogen loading rate in order to increase the specific denitrification activity was followed to check if the selected OMPs were co-metabolically biotransformed. Significant nitrite removal rate (24.1 mg N L-1 d-1) was achieved after only 30 days of operation. A maximum specific removal of 186.3 mg N gVSS-1 d-1 was obtained at the end of the operation, which is one of the highest previously reported. A successfully n-damo bacteria enrichment was achieved, being Candidatus Methylomirabilis the predominant bacteria during the whole operation attaining a maximum relative abundance of about 40 %. The natural hormones (E1 and E2) were completely removed in the bioreactor. The specific removal rates of erythromycin (ERY), fluoxetine (FLX), roxithromycin (ROX) and sulfamethoxazole (SMX) were successfully correlated with the specific nitrite removal rates, suggesting a co-metabolic biotransformation.

A new decentralized biological treatment process based on activated carbon targeting organic micropollutant removal from hospital wastewaters.

Although hospital wastewaters (HWWs) are usually discharged in urban sewage systems, their separate treatment has several benefits, such as the specific treatment of potential toxics as well as avoidance of further dilutions. In this work, an integrated industrial pilot plant (2200 L) corresponding to the technology SeMPAC® is proposed and validated for such purpose. The process consists of a sequential batch reactor (SBR) connected to an external submerged microfiltration membrane, in which powdered activated carbon (PAC) is directly added into the biological reactor to enhance the removal of the organic micropollutants (OMPs). The combination of different redox conditions in the SBR, as well as the operation at long sludge retention times (SRTs) and high biomass concentrations favored OMP biotransformation in the SBR, being their final removal efficiencies enhanced clearly after PAC addition, especially for the recalcitrant compounds. A periodical renewal of the adsorbent is necessary to overcome its gradual saturation. The main operational conditions were influenced by (i) the recalcitrant OMP carbamazepine, which defines the PAC dosage; (ii) the easily degradable OMP ibuprofen, which can be used to optimize the duration of the aerobic cycle; and (iii) the denitrification efficiency, which defines the correct time length of the anoxic period.

A Comparison of Population-Averaged and Cluster-Specific Approaches in the Context of Unequal Probabilities of Selection.

Sampling designs of large-scale survey studies are typically complex, involving multiple design features such as clustering and unequal probabilities of selection. Single-level (i.e., population-averaged) methods that use adjusted variance estimators and multilevel (i.e., cluster-specific) methods provide two alternatives for modeling clustered data. Although the literature comparing these methods is vast, comparisons have been limited to the context in which all sampling units are selected with equal probabilities (thus circumventing the need for sampling weights). The goal of this study was to determine under what conditions single-level and multilevel estimators outperform one another in the context of a two-stage sampling design with unequal probabilities of selection. Monte Carlo simulation methods were used to evaluate the impact of several factors, including population model, informativeness of the design, distribution of the outcome variable, intraclass correlation coefficient, cluster size, and estimation method. Results indicated that the unweighted estimators performed similarly across conditions, whereas the weighted single-level estimators tended to outperform the weighted multilevel estimators, particularly under nonideal sample conditions. Multilevel weight approximation methods did not perform well when the design was informative. An empirical example is provided to demonstrate how researchers might investigate the implications of the simulation results in practice.

Inhibition of biomass activity in the via nitrite nitrogen removal processes by veterinary pharmaceuticals.

The inhibitory effect of two veterinary pharmaceuticals was studied for different types of biomass involved in via nitrite nitrogen removal processes. Batch tests were conducted to determine the inhibition level of acetaminophen (PAR) and doxycycline (DOX) on the activity of short-cut nitrifying, denitrifying and anoxic ammonium oxidation (anammox) biomass and phosphorus accumulating organisms (PAOs). All biomass types were affected by PAR and DOX, with anammox being the most sensitive bacteria. DOX inhibited more the biomass treating high strength nitrogenous effluents (HSNE) than low strength nitrogenous effluents (LSNE). The phosphorus uptake inhibition under anoxic conditions was lower than 25% in the presence of PAR up to 400 mg L(-1). The same DOX concentration inhibited anoxic phosphorus uptake more than 65% for biomass treating LSNE and HSNE. Heterotrophic denitrifying bacteria seem to be more robust at high DOX and PAR concentrations than anammox. Both veterinary products inactivated ammonium oxidizing, Accumulibacter phosphatis and denitrifying bacteria.

Mass balance of pharmaceutical and personal care products in a pilot-scale single-sludge system: influence of T, SRT and recirculation ratio.

The influence of operation condition on the fate of 16 pharmaceutical and personal care products (PPCPs) in a single-sludge nitrifying/denitrifying pilot plant was assessed. Volatilisation, sorption and degradation were included in the mass balances to determine the most relevant removal mechanisms during PPCP treatment. Sludge retention time (SRT) was an important factor for the removal of compounds that significantly sorb onto sludge, as ethinylestradiol, whose removal increased 11% when working at SRT above 20 d. The internal recirculation ratio was significant for the removal of moderately biodegradable compounds, as citalopram. The positive effect of operating at warmer temperatures was particularly significant for two antibiotics, implying a 30% increase in their transformations. In the case of naproxen, an influence of sludge acclimation and concentration was observed, leading to removal efficiencies from 27% to 99%. Concerning removal mechanisms, most compounds were removed due to biotransformation, although for fragrances sorption and volatilisation played a role.

Removal of pharmaceutical and personal care products (PPCPs) under nitrifying and denitrifying conditions.

The contribution of volatilization, sorption and transformation to the removal of 16 Pharmaceutical and Personal Care Products (PPCPs) in two lab-scale conventional activated sludge reactors, working under nitrifying (aerobic) and denitrifying (anoxic) conditions for more than 1.5 years, have been assessed. Pseudo-first order biological degradation rate constants (k(biol)) were calculated for the selected compounds in both reactors. Faster degradation kinetics were measured in the nitrifying reactor compared to the denitrifying system for the majority of PPCPs. Compounds could be classified according to their k(biol) into very highly (k(biol)>5Lg(SS)(-1)d(-1)), highly (175%) and anoxic (>65%) conditions, whereas naproxen (NPX), ethinylestradiol (EE2), roxithromycin (ROX) and erythromycin (ERY) were only significantly transformed in the aerobic reactor (>80%). The anti-depressant citalopram (CTL) was moderately biotransformed under both, aerobic and anoxic conditions (>60% and >40%, respectively). Some compounds, as carbamazepine (CBZ), diazepam (DZP), sulfamethoxazole (SMX) and trimethoprim (TMP), manifested high resistance to biological transformation. Solids Retention Time (SRT(aerobic) >50d and <50d; SRT(anoxic) >20d and <20d) had a slightly positive effect on the removal of FLX, NPX, CTL, EE2 and natural estrogens (increase in removal efficiencies <10%). Removal of diclofenac (DCF) in the aerobic reactor was positively affected by the development of nitrifying biomass and increased from 0% up to 74%. Similarly, efficient anoxic transformation of ibuprofen (75%) was observed after an adaptation period of 340d. Temperature (16-26 degrees C) only had a slight effect on the removal of CTL which increased in 4%.

Pre-treatment of hospital wastewater by coagulation-flocculation and flotation.

Coagulation-flocculation and flotation processes were evaluated for the pre-treatment of hospital wastewater, including the removal of 13 pharmaceutical and personal care products (PPCPs). Coagulation-flocculation assays were performed in a Jar-Test device and in a continuous pilot-scale plant. Raw hospital wastewater as well as the effluent from the continuous coagulation plant were treated in a flotation cell. Removal of total suspended solids (TSS) during pre-treatment was very effective, reaching an average removal efficiency of 92% in the combined coagulation-flotation process. Musk fragrances were eliminated to a high degree during batch coagulation-flocculation (tonalide: 83.4+/-14.3%; galaxolide: 79.2+/-9.9%; celestolide: 77.7+/-16.8%), presumably due to their strong lipophilic character which promotes the interaction of these compounds with the lipid fraction of solids. For diclofenac (DCF), naproxen (NPX) and ibuprofen (IBP) maximum removals of 46%, 42% and 23%, respectively, were obtained, while the rest of PPCPs were not affected by the physico-chemical treatment. Flotation of raw wastewater led to slightly worse results compared to coagulation-flocculation, although the combined action of both improved the overall efficiency of the process. The proposed pre-treatment strategy for hospital wastewater is useful for assimilating its conventional physico-chemical characteristics to that of municipal wastewater as well as for reducing the load of some PPCPs into the sewer system.

Procedimiento general para purificar a pequeña escala las fimbrias expresadas por cepas porcinas de Escherichia coli enterotoxigenicas / A general procedure for small-scale purification of fimbriae expressed by porcine enterotoxigenic Escherichia coli strains

La fimbriacion en cepas de Escherichia coli enterotoxigénicas es un proceso complejo controlado por varios mecanismos: reguladores globales y locales de la transcripción y el control postranscripcional, e influenciado por factores como velocidad de crecimiento bacteriano, composición del medio de cultivo, temperatura y pH, entre otros. Estas características propician que la expresión fimbrial se pierda con alta frecuencia. De allí que se requieran procedimientos de cultivo que favorezcan el mantenimiento de dicha expresión. Con ese objetivo, en el trabajo la población bacteriana fimbriada se enriquecio mediante cultivo estático en caldo Mueller-Hinton. Luego, la expresión fimbrial se mantuvo mediante el crecimiento en forma consecutiva en agar CFA, y el caldo Minca o Mínimo, según el tipo fimbrial, en los que se alcanzó la máxima expresión a las 4-5h de cultivo. Las fimbrias se extrajeron mediante tratamiento térmico y se precipitaron con sulfato de amonio al 40 por ciento. La purificación se realizó mediante exclusión molecular y el tratamiento con deoxicolato de sodio. La metodología propuesta integra procedimientos conocidos en un proceso simple y reproducible para la obtención de las fimbrias F4, F5, F6 y F41 en cantidades suficientes para su posterior uso en la generación de anticuerpos, el desarrollo de inmunoensayos y otros estudios a escala de laboratorio, que necesiten de preparaciones con una pureza superior al 80 por ciento, que mantengan su estructura nativa

Kinetics of triclosan oxidation by aqueous ozone and consequent loss of antibacterial activity: relevance to municipal wastewater ozonation.

Oxidation of the antimicrobial agent triclosan by aqueous ozone (O(3)) was investigated to determine associated reaction kinetics, reaction site(s), and consequent changes in antibacterial activity of triclosan. Specific second-order rate constants, k(O(3)), were determined for reaction of O(3) with each of triclosan's acid-base species. The value of k(O(3)) determined for neutral triclosan was 1.3(+/-0.1)x10(3)M(-1)s(-1), while that measured for anionic triclosan was 5.1(+/-0.1)x10(8)M(-1)s(-1). Consequently, triclosan reacts very rapidly with O(3) at circumneutral pH (the pH-dependent, apparent second-order rate constant, K(app,O(3)) , is 3.8x10(7)M(-1)s(-1) at pH 7). The pH-dependence of K(app,O(3)) and comparison of triclosan reactivity toward O(3) with that of other phenolic compounds indicates that O(3) reacts initially with triclosan at the latter's phenol moiety. k(O(3)) values for neutral and anionic triclosan were successfully related to phenol ring substituent effects via Brown-Okamoto correlation with other substituted phenols, consistent with electrophilic attack of the triclosan phenol ring. Biological assay of O(3)-treated triclosan solutions indicates that reaction with O(3) yields efficient elimination of triclosan's antibacterial activity. In order to evaluate the applicability of these observations to actual wastewaters, triclosan oxidation was also investigated during ozonation of effluent samples from two conventional wastewater treatment plants. Nearly 100% triclosan depletion was achieved for a 4 mg/L(8.3x10(-5)mol/L)O(3) dose applied to a wastewater containing 7.5 mg/L of DOC, and approximately 58% triclosan depletion for dosage of 6 mg/L(1.3x10(-4)mol/L)O(3) to a wastewater containing 12.4 mg/L of DOC. At O(3) doses greater than 1mg/L(2.1x10(-5)mol/L), hydroxyl radical reactions accounted for <35% of observed triclosan losses in these wastewaters, indicating that triclosan oxidation was due primarily to the direct triclosan-O(3) reaction. Thus, ozonation appears to present an effective means of eliminating triclosan's antibacterial activity during wastewater treatment.