Metagenomic insights into the wastewater resistome before and after purification at large­scale wastewater treatment plants in the Moscow city.

Wastewater treatment plants (WWTPs) are considered to be hotspots for the spread of antibiotic resistance genes (ARGs). We performed a metagenomic analysis of the raw wastewater, activated sludge and treated wastewater from two large WWTPs responsible for the treatment of urban wastewater in Moscow, Russia. In untreated wastewater, several hundred ARGs that could confer resistance to most commonly used classes of antibiotics were found. WWTPs employed a nitrification/denitrification or an anaerobic/anoxic/oxic process and enabled efficient removal of organic matter, nitrogen and phosphorus, as well as fecal microbiota. The resistome constituted about 0.05% of the whole metagenome, and after water treatment its share decreased by 3-4 times. The resistomes were dominated by ARGs encoding resistance to beta-lactams, macrolides, aminoglycosides, tetracyclines, quaternary ammonium compounds, and sulfonamides. ARGs for macrolides and tetracyclines were removed more efficiently than beta-lactamases, especially ampC, the most abundant ARG in the treated effluent. The removal efficiency of particular ARGs was impacted by the treatment technology. Metagenome-assembled genomes of multidrug-resistant strains were assembled both for the influent and the treated effluent. Ccomparison of resistomes from WWTPs in Moscow and around the world suggested that the abundance and content of ARGs depend on social, economic, medical, and environmental factors.

New insight into the granule formation in the reactor for enhanced biological phosphorus removal.

While granulated activated sludge exhibits high productivity, the processes of granule formation are incompletely studied. The processes of granule formation and succession of communities were investigated in a laboratory sequencing batch reactor (SBR) under conditions for enhanced biological phosphorus removal (EBPR) using microbiological and molecular techniques. Active consumption of acetate, primarily by the phosphate-accumulating organisms (PAO), commenced at day 150 of cultivation. This was indicated by the high ratio of molar P-released/acetate uptake (0.73-0.77 P-mol/C-mol), characteristic of PAO. During this period, two types of granule-like aggregates formed spontaneously out of the activated sludge flocs. The aggregates differed in morphology and microbial taxonomic composition. While both aggregate types contained phosphorus-enriched bacterial cells, PAO prevailed in those of morphotype I, and glycogen-accumulating organisms (GAOs) were predominant in the aggregates of morphotype II. After 250 days, the elimination of the morphotype II aggregates from the reactor was observed. The subsequent selection of the community was associated with the development of the morphotype I aggregates, in which the relative abundance of PAO increased significantly, resulting in higher efficiency of phosphorus removal. Metagenomic analysis revealed a predominance of the organisms closely related to Candidatus Accumulibacter IС and IIС and of Ca. Accumulibacter IIB among the PAO. Based on the content of the genes of the key metabolic pathways, the genomes of potential PAO belonging to the genera Amaricoccus, Azonexus, Thauera, Zoogloea, Pinisolibacter, and Siculibacillus were selected. The patterns of physicochemical processes and the microbiome structure associated with granule formation and succession of the microbial communities were revealed.

The structure of microbial communities of activated sludge of large-scale wastewater treatment plants in the city of Moscow.

Microbial communities in wastewater treatment plants (WWTPs) play a key role in water purification. Microbial communities of activated sludge (AS) vary extensively based on plant operating technology, influent characteristics and WWTP capacity. In this study we performed 16S rRNA gene profiling of AS at nine large-scale WWTPs responsible for the treatment of municipal sewage from the city of Moscow, Russia. Two plants employed conventional aerobic process, one plant-nitrification/denitrification technology, and six plants were operated with the University of Cape Town (UCT) anaerobic/anoxic/oxic process. Microbial communities were impacted by the technology and dominated by the Proteobacteria, Bacteroidota and Actinobacteriota. WWTPs employing the UCT process enabled efficient removal of not only organic matter, but also nitrogen and phosphorus, consistently with the high content of ammonia-oxidizing Nitrosomonas sp. and phosphate-accumulating bacteria. The latter group was represented by Candidatus Accumulibacter, Tetrasphaera sp. and denitrifiers. Co-occurrence network analysis provided information on key hub microorganisms in AS, which may be targeted for manipulating the AS stability and performance. Comparison of AS communities from WWTPs in Moscow and worldwide revealed that Moscow samples clustered together indicating that influent characteristics, related to social, cultural and environmental factors, could be more important than a plant operating technology.

New Insight Into the Interspecies Shift of Anammox Bacteria Ca. "Brocadia" and Ca. "Jettenia" in Reactors Fed With Formate and Folate.

The sensitivity of anaerobic ammonium-oxidizing (anammox) bacteria to environmental fluctuations is a frequent cause of reactor malfunctions. It was hypothesized that the addition of formate and folate would have a stimulating effect on anammox bacteria, which in turn would lead to the stability of the anammox process under conditions of a sharp increase in ammonium load, i.e., it helps overcome a stress factor. The effect of formate and folate was investigated using a setup consisting of three parallel sequencing batch reactors equipped with a carrier. Two runs of the reactors were performed. The composition of the microbial community was studied by the 16S rRNA gene profiling and metagenomic analysis. Among anammox bacteria, Ca. "Brocadia" spp. dominated during the first run. A stimulatory effect of folate on the daily nitrogen removal rate (dN) was identified. The addition of formate led to progress in dissimilatory nitrate reduction and stimulated the growth of Ca. "Jettenia" spp. The spatial separation of two anammox species was observed in the formate reactor: Ca. "Brocadia" occupied the carrier and Ca. "Jettenia"-the walls of the reactors. Biomass storage at low temperature without feeding led to an interspecies shift in anammox bacteria in favor of Ca. "Jettenia." During the second run, a domination of Ca. "Jettenia" spp. was recorded along with a stimulating effect of formate, and there was no effect of folate on dN. A comparative genome analysis revealed the patterns suggesting different strategies used by Ca. "Brocadia" and Ca. "Jettenia" spp. to cope with environmental changes.

Novel design and optimisation of a nitritation/anammox set-up for ammonium removal from filtrate of digested sludge.

Although the anammox process is extensively applied for the treatment of NH4-rich wastewater, new technical solutions overcoming the operational difficulties remain an important task. An innovative design of anammox-based set-up was employed to improve sludge settling under high ammonium load. The set-up included a completely mixed bioreactor with suspended and immobilised activated sludge. To prevent sludge flotation, recycled suspended sludge was additionally treated in an aerated tank at dissolved oxygen (DO) concentration of 1.5 ± 0.2 mg/l followed by processing in a flow-homogeniser. Introduction of these elements resulted in a 3.5-fold increase in total nitrogen removal efficiency (TNRE). The bioreactor achieved maximal TNRE of 86% corresponding to total nitrogen removal rate of 0.77 kg N/m3/d under defined optimal conditions: temperature of 35 ± 2°C, DO of 0.6 ± 0.2 mg/l, hydraulic retention time of 12 h, and dose of suspended sludge of 1.5 ± 0.1 g total suspended solids (TSS)/l. A weakly attached sludge was first described as a technologically important factor. Suspended, weakly and firmly attached sludge exhibited the highest heterotrophic, nitrifying, and anammox activities, respectively. New probes were constructed to detect anammox bacteria by fluorescence in situ hybridisation. Probe for Candidatus 'Jettenia' could be recommended for widespread use.

In Vitro Evaluation of the Biocompatibility of Newly Synthesized Bis-Quaternary Ammonium Compounds with Spacer Structures Derived from Pentaerythritol or Hydroquinone.

　With the object of developing new biocides milder for human use than the current antiseptics, we synthesized a series of bis-quaternary ammonium compounds (bis-QACs). The antimicrobial activity of the newly synthesized bis-QACs and common biocides used as antiseptics was compared by examining minimum inhibitory concentrations and minimum bactericidal concentrations (MBCs). Moreover, the cytotoxicity of these compounds against human cells was determined to calculate the biocompatibility index (BI) of these compounds. BI was the ratio of the concentration of a biocide giving a 50% lethal effect on normal human epidermal keratinocytes to its MBC against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. The commonly used antiseptics tested were benzalkonium chloride (BAC), octenidine dihydrochloride (OCT), chlorhexidine digluconate (CHG) and polyhexamethylene biguanide (PHMB). In comparison with these antiseptics, it was shown that some of new bis-QACs exhibited a wider and more potent antimicrobial spectrum than OCT. The cytotoxicity of these bis-QACs was equal or lower compared to that of the quaternary ammonium compounds (BAC and OCT), although these bis-QACs showed higher toxicity than the biguanide-based compounds (CHG and PHMB). Finally, the comparison of BIs revealed that new bis-QACs such as N-dodecyl ｛4,4'-(2,4,8,10-tetraoxaspiro［5.5］undecan-3,9-diyl) ｝dipyridinium dibromide (4TOSU-12), 3,3'-［1,4-Phenylenebis (oxy)］bis (1-dodecylpyridinium) dibromide (3PHBO-12) and 3-(3-Hydroxy-2-(hydroxymethyl)-2-｛［(1-dodecylpyridinium-3-yl) oxy］methyl｝propoxy)-1-dodecylpyridinium dibromide (3HHDMP-12) had equal or greater biocompatibility than the commonly used biocides tested. Thus, these results strongly suggested that 4TOSU-12, 3PHBO-12 and 3HHDMP-12 could be useful as antiseptics for topical application to the skin.

Electrocatalytic multicomponent assembling of isatins, 3-methyl-2-pyrazolin-5-ones and malononitrile: facile and convenient way to functionalized spirocyclic [indole-3,4'-pyrano[2,3-c]pyrazole] system.

Electrochemically induced catalytic multicomponent transformation of isatins, 3-methyl-2-pyrazolin-5-ones and malononitrile in ethanol in an undivided cell in the presence of sodium bromide as an electrolyte results in the formation of spirooxindoles with fused functionalized pyrano[2,3-c]pyrazole system in 78-99} yields. The developed efficient electrocatalytic approach to medicinally relevant spirocyclic [indole-3,4'-pyrano[2,3-c]pyrazoles] is beneficial from the viewpoint of diversity-oriented large-scale processes and represents a novel example of facile environmentally benign synthetic concept for electrocatalytic multicomponent reaction strategy.

Enantiomerically pure bicyclo[3.3.1]nona-2,6-diene as the sole source of enantioselectivity in BIPHEP-Rh asymmetric hydrogenation.

In situ resolution of the rapidly racemising diphosphine BIPHEP and its relatives with the cationic Rh complex of (S,S)-bicyclonona-2,6-diene permits the asymmetric hydrogenation of dehydroamino esters.