Despite the higher investment costs of two-stage partial nitrification/anammox (PN/AMX) systems compared to single-reactor configurations, creating more favorable conditions for specific N-cycling microbial groups in different reactors can help more easily maintain the stability of the overall system. In this work, an unusual two-reactor PN/AMX system with brush-type carriers, reverse sequence configuration and internal recirculation was operated for 500 d using both high strength synthetic wastewater and real reject water. Feasibility of the proposed system was confirmed by relatively high (>70%) total N removal without strict control over the nitrification process due to synergistic activity of nitrifying, denitrifying and anammox bacteria. The limiting level of COD in the influent was similar to 600 mg O-2/L, exceeding which sharply deteriorated the process of nitrogen removal. The microbial community of the PN reactor naturally evolved into clearly distinguishable free-floating sludge and 2 types of biofilms (red and brown), which had different relative abundances of key N cycling groups in each phase of PN/AMX system operation. While the AMX community was dominated by the genus Candidatus Jettenia ecosi. Considering the ease of operation and relatively high N removal efficiency, the proposed PN/AMX system may merit further study for process optimization and practical use.

The process of anaerobic ammonium oxidation by nitrite (anammox) is a globally essential part of N cycle. To date, 8 Candidatus genera and more than 22 species of anammox bacteria have been discovered in various anthropogenic and natural habitats, including nitrogen-polluted aquifers. In this work, anammox bacteria were detected for the first time in the groundwater ecosystem with high anthropogenic nitrogen pollution (up to 1760 mg NO3--N/L and 280 mg NH4+-N/L) and low year-round temperature (7-8 degrees C) in the zone of a uranium sludge repository. Further metagenomic analysis resulted in retrieval of metagenome-assembled genomes of 4 distinct anammox bacteria: a new genus named Ca. Frigussubterria, new species in Ca. Kuenenia, and two strains of a new species in Ca. Scalindua. Analysis of the genomes revealed essential genes involved in anammox metabolism. Both strains of Ca. Scalindua chemeplantae had a high copy number of genes encoding the cold shock proteins CspA/B, which can also function as an antifreeze protein (CspB). Ca. Kuenenia glazoviensis and Ca. Frigussubterria udmurtiae were abundant in less N-polluted site, while Ca. Scalindua chemeplantae inhabited both sites. Genes for urea utilization, reduction of insoluble Fe2O3 or MnO2, assimilatory sulfate reduction, reactive oxygen detoxification, nitrate reduction to ammonium, and putatively arsenate respiration were found. These findings enrich knowledge of the functional and phylogenetic diversity of anammox bacteria and improve understanding of the nitrogen cycle in polluted aquifers.

Didecyldimethylammonium chloride (DDAC) and polyhexamethylene guanidine (PHMG) exhibit high antimicrobial activity and are widely used as biocidal agents in chemical toilet additives for the management of fecal sludge (FS). Disposal of such biocide-treated FS to a wastewater treatment plant (WWTP) is a major environmental problem. It is possible to reduce environmental damage through the use of biocidal agents, which easily decompose after performing their main biocidal functions. In this work, it is proposed to use the fact of a gradual increase in pH of FS from the initial 7.5 to 9.0-10.0 due to the decomposition of urea. Six biocidal compounds were selected that are capable of rapidly degrading in an alkaline environment and one that naturally degrades upon prolonged incubation. Four of them: bronopol (30 mg/L), DBNPA (500 mg/L), Sharomix (500 mg/L), and sodium percarbonate (6000 mg/L) have shown promise for environmentally friendly management of FS. In selected dosage, they successfully reduced microbial activity under both aerobic and anaerobic conditions and are cost-effective. After 10 days of incubation, degradation of the biocide occurred as measured by biological oxygen demand (BOD5) in biocide-treated FS. Such FS can be discharged to WWTP without severe damage to the activated sludge process, the need for dilution and additional procedures to neutralize toxicity.

The effect of C-type natriuretic peptide in a concentration closer to the normal level in human blood plasma was studied on the mono-species and dual-species biofilms of the skin commensal bacteria Cutibacterium acnes HL043PA2 and Staphylococcus epidermidis ATCC14990. Despite the marginal effect of the hormone on cutibacteria in mono-species biofilms, the presence of staphylococci in the community resulted in a global shift of the CNP effect, which appeared to increase the competitive properties of C. acnes, its proliferation and the metabolic activity of the community. S. epidermidis was mostly inhibited in the presence of CNP. Both bacteria had a significant impact on the gene expression levels revealed by RNA-seq. CNP did not affect the gene expression levels in mono-species cutibacterial biofilms; however, in the presence of staphylococci, five genes were differentially expressed in the presence of the hormone, including two ribosomal proteins and metal ABC transporter permease. In staphylococci, the Na-translocating system protein MpsB NADH-quinone oxidoreductase subunit L was downregulated in the dual-species biofilms in the presence of CNP, while in mono-species biofilms, two proteins of unknown function were downregulated. Hypothetically, at least one of the CNP mechanisms of action is via the competition for zinc, at least on cutibacteria.

Anammox bacteria related to Candidatus Scalindua were recently discovered in a cold (7.5 °C) aquifer near sludge repositories containing solid wastes of uranium and processed polymetallic concentrate. Groundwater has a very high level of nitrate and ammonia pollution (up to 10 and 0.5 g/L, respectively) and a very low content of organic carbon (2.5 mg/L). To assess the potential for bioremediation of polluted groundwater in situ, enrichment cultures of anammox, nitrifying, and denitrifying bacteria were obtained and analyzed. Fed-batch enrichment of anammox bacteria was not successful. Stable removal of ammonium and nitrite (up to 100%) was achieved in a continuous-flow reactor packed with a nonwoven fabric at 15 °C, and enrichment in anammox bacteria was confirmed by FISH and qPCR assays. The relatively low total N removal efficiency (up to 55%) was due to nonstoichiometric nitrate buildup. This phenomenon can be explained by a shift in the metabolism of anammox bacteria towards the production of more nitrates and less N2 at low temperatures compared to the canonical stoichiometry. In addition, the too high an estimate of specific anammox activity suggests that N cycle microbial groups other than anammox bacteria may have contributed significantly to N removal. Stable nitrite production was observed in the denitrifying enrichment culture, while no “conventional” nitrifiers were found in the corresponding enrichment cultures. Xanthomonadaceae was a common taxon for all microbial communities, indicating its exclusive role in this ecosystem. This study opens up new knowledge about the metabolic capabilities of N cycle bacteria and potential approaches for sustainable bioremediation of heavily N-polluted cold ecosystems.

A primary study of the quality of wastewater treatment and the composition of the microbial community of the activated sludge of the treatment plant and the storage pond of a pig farm is carried out. Activated sludge is widely represented by nitrogen-cycle microorganisms, including denitrifying and nitrifying bacteria (Nitrosomonas (4%), Nitrospira (8%), as well as, to a lesser extent, anammox microorganisms (Candidatus Kuenenia (0.2%)). Phosphorus is removed by phosphate-accumulating bacteria, primarily Thauera (5%) and Candidatus Accumulibacter (1%). Two-stage aerobic treatment has a very high degree of chemical oxygen demand (97%) and ammonium (99.7%) removal. At the same time, a rather high concentration of nitrates (569 mg/L) and phosphates (75 mg/L) in the treated water probably caused the dramatic development of purple sulfur bacteria of the genus Thiolamprovum in the storage pond, coloring the water a red-burgundy color ("borscht" color). To improve the quality of treatment, reduce energy costs, obtain an energy carrier in the form of biogas, as well as solve the problem of water bloom in the storage pond, a scheme for anaerobic-anoxic-aerobic wastewater treatment of the pig farm is proposed.

— Investigation of the interactions between Staphylococcus aureus 209P and Kytococcus schroeteri H01 in binary biofilms revealed the sensitivity of these bacteria to the atrial natriuretic peptide (ANP) in a concentration of 6.5 × 10 –10 M. In the presence of ANP, affinity of both bacteria to hexadecane decreased significantly, as was determined by MATS (microbial adhesion to solvents) experiments. Their ability to aggregate with each other and on the surface also altered in the presence of the hormone, affecting mainly the initial adhesion stage in biofilm formation. Plating of the binary communities revealed that they consisted mostly of staphylococci, while K. schroeteri constituted the minor part of the cell biomass. They, however, affected cell aggregation and the ANP effect on the community. K. schroeteri played a dualistic role, both facilitating S. aureus adhesion and acting as its antagonist in the biofilm. Moreover, in binary communities K. schroeteri was responsible for the shift of ANP action on S. aureus from neutral/stimulatory to inhibitory.

The importance of the impact of human hormones on commensal microbiota and microbial biofilms is established in lots of studies. In the present investigation, we continued and extended the research of epinephrine effects on the skin commensal Micrococcus luteus C01 and its biofilms, and also the matrix changes during the biofilm growth. Epinephrine in concentration 4.9 × 10–9 M which is close to normal blood plasma level increased the amount of polysaccharides and extracellular DNA in the matrix, changed extensively its protein, lipid and polysaccharide composition. The Ef-Tu factor was one of the most abundant proteins in the matrix and its amount increased in the presence of the hormone. One of the glucose-mannose polysaccharide was absent in the matrix in presence of epinephrine after 24 h of incubation. The matrix phospholipids were also eradicated by the addition of the hormone. Hence, epinephrine has a great impact on the M. luteus biofilms and their matrix composition, and this fact opens wide perspectives for the future research.

Antibiotics guard us against bacterial infections and are among the most commonly used medicines. The immediate consequence of their large-scale production and prescription is the development of antibiotic resistance. Therefore, rapid detection of antibiotic susceptibility is required for efficient antimicrobial therapy. One of the promising methods for rapid antibiotic susceptibility testing is Raman spectroscopy. Raman spectroscopy combines fast and contactless acquisition of spectra with good selectivity towards bacterial cells. The antibiotic-induced changes in bacterial cell physiology are detected as distinct features in Raman spectra and can be associated with antibiotic susceptibility. Therefore, the Raman-based approach may be beneficial in designing therapy against multidrug-resistant infections. The surface-enhanced Raman spectroscopy (SERS) and resonance Raman spectroscopy (RRS) additionally provide excellent sensitivity. In this review, we present an analysis of the Raman spectroscopy–based optical biosensing approaches aimed at antibiotic susceptibility testing.

The impact of steroid hormones, and particularly estradiol, on human microbiota could be recognized as a substantial part of human-microbiota interactions. However, an area that remains poorly investigated is that of the skin and vaginal microbial communities and biofilms, which contain non-pathogenic bacteria of phyla Firmicutes and Actinobacteria, especially probiotic bacteria of the genus Lactobacillus and the widespread, safe skin genus, Micrococcus. Experiments with Lactobacillus paracasei AK508 and Micrococcus luteus C01 biofilms on PTFE cubes showed dose-dependent effects of estradiol at concentrations of 0.22 nM and 22 nM. The hormone mostly inhibits L. paracasei growth and stimulates M. luteus. The presented studies of colony-forming unit (CFU) amountsand cell aggregation in biofilms on glass fiber filters showed the same general tendencies. Estradiol generally increased the aggregation of cells in monospecies communities and potentially changed the synthesis of antibacterial metabolites in L. paracasei. The balance between two bacteria in mixed-species biofilms depended on the initial adhesion stage, and when this stage was reduced, micrococci were more resistant to the antagonistic action of L. paracasei. Moreover, in mixed-species biofilms, the effect of estradiol on lactobacilli altered from inhibition to stimulation, potentially due to the presence of M. luteus. At the same time, ethanol as a solvent for estradiol at the concentration 0.6% acted mostly as an antagonist of the hormone and had an opposite effect on bacteria; nevertheless, the overlapping of ethanol and estradiol effects was shown to be minimal. The data obtained prove the complexity of microbial interactions and the regulatory effect of estradiol on commensal bacteria biofilms.

The complex pollution of aquifers by reduced and oxidized nitrogen compounds is currently considered one of the urgent environmental problems that require non-standard solutions. This work was a laboratory-scale trial to show the feasibility of using various mineral carriers to create a permeable in situ barrier in cold (10 °C) aquifers with extremely high nitrogen pollution and inhabited by the Candidatus Scalindua-dominated indigenous anammox community. It has been established that for the removal of ammonium and nitrite in situ due to the predominant contribution of the anammox process, quartz, kaolin clays of the Kantatsky and Kamalinsky deposits, bentonite clay of the Berezovsky deposit, and zeolite of the Kholinsky deposit can be used as components of the permeable barrier. Biofouling of natural loams from a contaminated aquifer can also occur under favorable conditions. It has been suggested that the anammox activity is determined by a number of factors, including the presence of the essential trace elements in the carrier and the surface morphology. However, one of the most important factors is competition with other microbial groups that can develop on the surface of the carrier at a faster rate. For this reason, carriers with a high specific surface area and containing the necessary microelements were overgrown with the most rapidly growing microorganisms. Bioaugmentation with a “warm” anammox community from a laboratory reactor dominated by Ca. Kuenenia improved nitrogen removal rates and biofilm formation on most of the mineral carriers, including bentonite clay of the Dinozavrovoye deposit, as well as loamy rock and zeolite-containing tripoli, in addition to carriers that perform best with the indigenous anammox community. The feasibility of coupled partial denitrification–anammox and the adaptation of a “warm” anammox community to low temperatures and hazardous components contained in polluted groundwater prior to bioaugmentation should be the scope of future research to enhance the anammox process in cold, nitrate-rich aquifers.

The composting of a mixture of excess sludge from biological treatment facilities for wastewater from dairy production and vegetable and wood waste in a ratio of 3 : 3 : 4 (by volume) with a high amount of dry substance (27.5%) at extremely high temperatures (75–82°C) made it possible to isolate active mesophilic and thermophilic methanogenic consortia. The dynamics of methane formation upon the liquid-phase fermentation of the studied mixture was studied. Accelerated methanogenesis and the highest methane yield (31.7 ± 2.9 mM L –1 ) were observed with culturing at 55°C. Analysis of the volatile fatty-acid production with liquid-phase fermentation showed that their content was low (<10 mM L –1 ). This amount could not inhibit methanogenesis, regardless of the applied substrates and temperature conditions. The 16S rRNA sequencing of the obtained consortia showed the presence of methanogenic archaea Methanosarcina thermophila , Methanothermobacter thermoautotrophicus , Candidatus ‘Methanogranum caenicola’, and Methanofollis ethanolicus. The consortia also included archaea that represent putative new taxa that are phylogenetically close to Candidatus ‘Methanoplasma termitum’ and Methanomassiliicoccus luminyensis . Of the mentioned methanogenic archaea, the latter four were found in composting organic waste for the first time. The obtained consortia can conduct active methanogenesis and can be applied as an inoculum in the anaerobic treatment of organic wastes, including composting wastes, in order to intensify and increase the biogas yield.

The work investigated the characteristics of the dark fermentation (DF) process of a number of simple (starch, sunflower oil, peptone, both separately and mixed) and complex (dog food, pig feed, sewage sludge) substrates using a mixed culture of microorganisms, with a controlled pH (5.5), at 55 degrees C. Peptone and sunflower oil were characterized by the lowest production of H2, namely 5.0 and 2.3 ml H2/g COD, respectively. The specific hydrogen yield from starch was 1.55 mol H2/mol hexose. The addition of peptone and sunflower oil to starch reduced the specific yield of hydrogen from starch by 23%. A large difference in hydrogen production was observed during DF of complex substrates. The specific hydrogen yield from dog food was 46.5 ml H2/g COD or 143.4 ml H2/g carbohydrates; from pig feed - 32.1 ml H2/g COD or 91.6 ml H2/g carbohydrates; and from sewage sludge 9.3 ml H2/g COD or 98.0 ml H2/g carbohydrates. Possible relationships between the biopolymer composition of substrates and characteristics of the DF process were analyzed using Spearman's rank correlation coefficients. The concentration of carbohydrates, as well as the ratio of carbohydrates/proteins and carbohydrates/fats, were the main factors influencing the high specific yield of H2, its content in biogas, as well as the ratio of H2/ soluble metabolites. The concentration of proteins had a statistically significant positive effect on the accumulation of acetate and succinate, and carbohydrates - on the accumulation of caproate. (c) 2021 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.

The first evidence of the atrial natriuretic peptide (ANP) effect on mono-species and dual-species biofilms of skin commensals Cutibacterium acnes and Staphylococcus epidermidis was obtained in different model systems. Elucidation of the mechanism of action of hormones on the microbial communities of human skin is an important physiological and medical aspect. Under anaerobic conditions, ANP at a concentration of 6.5 × 10−10 M inhibits the growth of S. epidermidis biofilms and stimulates the growth of C. acnes biofilms, and a lesser effect has been demonstrated on planktonic cultures. In biofilms, ANP stimulates aggregation in C. acnes and aggregate dispersion of S. epidermidis, while in S. epidermidis, ANP also stimulates the metabolic activity of cells. Analysis of dual-species biofilms has shown the dominance of S. epidermidis, while ANP increases the ratio of C. acnes biomass in the community. ANP decreases the growth rate of S. epidermidis biofilms and increases that of C. acnes. The effect of ANP is not dependent on the surface type and probably affects other targets in microbial cells. Thus, the potential regulatory effect of human ANP on skin microbe dual-species communities has been shown, and its potential has been demonstrated to change microbiota homeostasis on the skin.

In this study, the effect of epinephrine on the biofilm formation of Micrococcus luteus C01 isolated from human skin was investigated in depth for the first time. This hormone has a complex effect on biofilms in various systems. In a system with polytetrafluoroethylene (PTFE) cubes, treatment with epinephrine at a physiological concentration of 4.9 x 10(-9) M increased the total amount of 72-h biofilm biomass stained with crystal violet and increased the metabolic activity of biofilms, but at higher and lower concentrations, the treatment had no significant effect. On glass fiber filters, treatment with the hormone decreased the number of colony forming units (CFUs) and changed the aggregation but did not affect the metabolic activity of biofilm cells. In glass bottom plates examined by confocal microscopy, epinephrine notably inhibited the growth of biofilms. RNA-seq analysis and RT-PCR demonstrated reproducible upregulation of genes encoding Fe-S cluster assembly factors and cyanide detoxification sulfurtransferase, whereas genes encoding the co-chaperone GroES, the LysE superfamily of lysine exporters, short-chain alcohol dehydrogenase and the potential c-di-GMP phosphotransferase were downregulated. Our results suggest that epinephrine may stimulate matrix synthesis in M. luteus biofilms, thereby increasing the activity of NAD(H) oxidoreductases. Potential c-di-GMP pathway proteins are essential in these processes.

The effect of norepinephrine (“NE”) on Gram-negative bacteria is well characterized; however, little is known about the impact of NE on cutaneous Gram-positive skin residents, especially staphylococci. In this study, the impact of NE on monospecies and dual-species biofilms of Staphylococcus epidermidis and S. aureus model strains was investigated for the first time. Biofilms were grown in two different models (on polytetrafluoroethylene (“PTFE”) cubes and glass microfiber filters (“GMFFs”)) and additionally kinetic measurements of bacterial growth was performed. We have shown that NE can affect the biofilm formation of both species with a strong dependence on aerobic or anaerobic culture conditions in different models. It was shown that S. epidermidis suppresses S. aureus growth in dual-species biofilms and that NE can accelerate this process, contributing to the competitive behavior of staphylococci.

This is the study of efficiency of anaerobic reject water treatment and of changes in the relative abundance of the major microbial groups involved in nitrogen removal at increase in nitrogen and COD (chemical oxygen demand) load. A two-reactor laboratory system carried out anammox and partial nitrification, with the biomass immobilized on a brush carrier and water recycling. Nitrogen concentration in the water varied from 50 to 250 mg N-NH4/L, while COD varied from 50 to 1650 mg О2/L. Efficiency of the process was assessed by monitoring the concentrations of N-NH4, N-NO2, N-NO3, COD, dissolved oxygen, and pH in the inflowing and treated sludge liquor. The efficiency of nitrogen removal was 75‒90%. Active development of anammox bacteria and first step nitrifiers was observed in the anammox reactor, while almost no second step nitrifiers were present. Elevated content of organic matter in sludge liquor was shown to cause a significant decrease in the water purification degree due to lower abundance of anammox bacteria and the first step nitrifying Nitrosomonas species, while the abundance of the second step nitrifying Nitrospira species increased. The major microbial groups involved in nitrogen removal were better represented in biofilms than in free-floating sludge (flocs and granules). While the size of the granules and flocs did not change at increased load, their color changed from gray-red to a darker one.

The microbial community of a laboratory-scale bioreactor based on the anammox process was investigated by using metagenomic approaches and fluorescent in situ hybridization (FISH). The bioreactor was initially inoculated with activated sludge from the denitrifying bioreactor of a municipal wastewater treatment station. By constantly increasing the ammonium and nitrite load, a microbial community containing the novel species of anammox bacteria “Candidatus Jettenia ecosi” developed in the bioreactor after 5 years when the maximal daily nitrogen removal rate reached 8.5 g/L. Sequencing of the metagenome of anammox granules and the binning of the contigs obtained, allowed a high quality draft genome of the dominant anammox bacterium, “Candidatus Jettenia ecosi” to be assembled. Annotation of the 3.9 Mbp long genome revealed 3970 putative protein-coding genes, 45 tRNA genes, and genes for 16S/23S rRNAs. Analysis of the genome of “Candidatus Jettenia ecosi” revealed genes involved in anammox metabolism, including nitrite and ammonium transporters, copper-containing nitrite reductase, a nitrate reductase complex, hydrazine synthase, and hydrazine dehydrogenase. Autotrophic carbon fixation could be accomplished through the Wood Ljungdahl pathway. The composition of the community was investigated through a search of 16S rRNA sequences in the metagenome and FISH analysis of the anammox granules. The presence of the members of Ignavibacteriae, Betaproteobacteria, Chloroflexi and other microbial lineages reflected the complexity of the microbial processes in the studied bioreactor performed by anammox Planctomycetes, fermentative bacteria, and denitrifiers.

In skin, Cutibacterium acnes (former Propionibacterium acnes) can behave as an opportunistic pathogen, depending on the strain and environmental conditions. Acneic strains of C. acnes form biofilms inside skin-gland hollows, inducing inflammation and skin disorders. The essential exogenous products of C. acnes accumulate in the extracellular matrix of the biofilm, conferring essential bacterial functions to this structure. However, little is known about the actual composition of the biofilm matrix of C. acnes. Here, we developed a new technique for the extraction of the biofilm matrix of Gram-positive bacteria without the use of chemical or enzymatic digestion, known to be a source of artifacts. Our method is based on the physical separation of the cells and matrix of sonicated biofilms by ultracentrifugation through a CsCl gradient. Biofilms were grown on the surface of cellulose acetate filters, and the biomass was collected without contamination by the growth medium. The biofilm matrix of the acneic C. acnes RT5 strain appears to consist mainly of polysaccharides. The following is the ratio of the main matrix components: 62.6% polysaccharides, 9.6% proteins, 4.0% DNA, and 23.8% other compounds (porphyrins precursors and other). The chemical structure of the major polysaccharide was determined using a nuclear magnetic resonance technique, the formula being →6)-α-D-Galp-(1→4)-β-D-ManpNAc3NAcA-(1→6)-α-D-Glcp-(1→4)-β-D-ManpNAc3NAcA-(1→3)-β-GalpNAc-(1→. We detected 447 proteins in the matrix, of which the most abundant were the chaperonin GroL, the elongation factors EF-Tu and EF-G, several enzymes of glycolysis, and proteins of unknown function. The matrix also contained more than 20 hydrolases of various substrata, pathogenicity factors, and many intracellular proteins and enzymes. We also performed surface-enhanced Raman spectroscopy analysis of the C. acnes RT5 matrix for the first time, providing the surface-enhanced Raman scattering (SERS) profiles of the C. acnes RT5 biofilm matrix and biofilm biomass. The difference between the matrix and biofilm biomass spectra showed successful matrix extraction rather than simply the presence of cell debris after sonication. These data show the complexity of the biofilm matrix composition and should be essential for the development of new anti-C. acnes biofilms and potential antibiofilm drugs.

The article was translated by the authors.