Annotation of the results obtained in 2023
The feasibility of using vermiculite, zeolite, expanded clay, sandy loam and various clays to create a biogeochemical barrier in groundwater contaminated with ammonium and nitrates using the anammox community has been studied. The smallest biofilm thickness was observed on bentonite clays and sands with a clay content of more than 15%. The most developed surface is found in bentonite clays and expanded clay. The largest biofilm area was observed on kaolin clays, expanded clay, sand with 5% clay and vermiculite. For kaolin clays, zeolite and expanded clay, a direct correlation was found between the total biofilm area and the rate of ammonium consumption. Thus, anammox bacteria form active biofilms that provide high rates of ammonium nitrogen consumption. The formation of biofilms was influenced by the morphology and mineral composition of the studied materials, as well as their ability to sorb ammonium ions, thereby reducing the nitrogen load. Combined and separate images of biofilms of groundwater microorganisms accumulated in the bioreactor and biofilms from the surface of rock materials were obtained. The technique of staining and double hybridization made it possible to carry out hybridization even on native rock samples; the localization of dyes inside the biofilm was revealed. Only cy-5 dye revealed individual cells outside the total biomass of anammox bacteria. This method made it possible to make assumptions regarding the spatial coordination of individual cells, subject to their labeling with specialized dyes conjugated to hybridization probes. In incubation experiments using natural coals and biochars, it was found that the biotic consumption of ammonium and nitrite nitrogen was maximum when anthracite coals, coconut biochar and activated carbon were added. The maximum rate of nitrogen consumption (total nitrite and ammonium) at 10°C was 3.0-4.9 mg N/day, and at 30°C – 8.1-11.37 mg N/day; while in the control without the addition of coals, the maximum rate of nitrogen consumption was 0.4 and 1.2 mg N/day. A significant increase in the respiratory activity of biofilms on the surface of coals was revealed. The mechanism for accelerating nitrogen consumption by the anammox community with the addition of coals and biochars is explained by the immobilization of cells due to the porous and adsorption structure of coals, the ability of the anammox community to transfer electrons by the electrically conductive surface of coal, the functional role of EPS in biofilms as a mediator for electron transfer, the protective function of biofilms on coals from unfavorable factors. It was shown for the first time that natural coals, predominantly of anthracite nature, can stimulate N-cycle processes, in particular anammox, along with biochars. The reduction of nitrate nitrogen in contaminated groundwater was maximum when using lactic acid (97.9%), whey (91.3%) and molasses (92.3%) as Corg, and the first to reveal the potential of biogas from digesters for treatment of polluted aquifer. The removal of ammonium nitrogen occurred to the greatest extent when lactic acid was added (65%). The maximum negative modulus values of the redox potential were achieved, which is an important factor for the reduction of uranyl ions and the stabilization of uranium in the form of poorly soluble biogenic solid mineral phases of reduced forms of uranium, as well as the formation of other mineral phases - phosphates, sulfides of associated heavy metals. Study of two groundwater (more and less contaminated) near the surface storage facility for radioactive waste at the Chepetsk Mechanical Plant (Glazov, Udmurtia, Russia) showed the presence of a microbial community in which N-cycle microorganisms abound and anammox bacteria dominate. Further metagenomic analysis resulted in retrieval of metagenome-assembled genomes of 4 distinct anammox bacteria: a new genus Ca. Frigussubterria, a new species in Ca. Kuenenia and two strains of a new species Ca. Scalindua. Genomic analysis has identified important genes involved in anammox metabolism. Both strains of Ca. Scalindua chemeplantae had high copy numbers of genes encoding the cold shock proteins CspA/B, which can also function as an antifreeze protein (CspB). It is noteworthy that strains of Ca. Scalindua chemeplantae P6-7 and S40-8 had much higher copy numbers of CspA/B proteins than in all previously analyzed genomes of nine different anammox bacterial species. Genes for urea utilization, reduction of insoluble Fe2O3 or MnO2, assimilation sulfate reduction, and presumably arsenate respiration were discovered. The results obtained enrich our knowledge of the functional and phylogenetic diversity of anammox bacteria. According to the PCR results, the number of anammox bacteria in the community of a flow bioreactor cultivated at 15°C turned out to be relatively low, which may indicate unfavorable growth conditions for this group of microorganisms and the possible beginning of succession in the community, in which denitrifiers displace anammox bacteria, competing with them for nitrite under anaerobic conditions. In the community, denitrifiers were found that had both types of nitrite reductases (nirK and nirS), and the number of the latter was lower, which may have been due to a lack of copper in the bioreactor medium, which is important for the synthesis of the corresponding nitrite reductase. Interestingly, the number of first-stage nitrifiers also turned out to be significant, indicating that even under such unfavorable conditions (lack of aeration) there are microniches suitable for this group in the bioreactor community. In enrichment cultures, by profiling the 16S rRNA gene, the phylogenetic affiliation of anammox, nitrifying, comammox, and denitrifying microorganisms was assessed. Analysis of the microbial diversity of the enrichment culture of ammonium-oxidizing bacteria (AOB) showed the dominance of Pseudomonadaceae and Rhodanobacteracea. “Canonical” nitrifiers were not found either in the microbial communities of enrichment cultures or in the groundwater. However, in the enrichment culture of nitrite-oxidizing bacteria (NOB), ammonium-oxidizing archaea (AOA) Crenarchaeota were present (0.5% of phylotypes belonged to the family Nitrososphaeraceae - Ca. Nitrocosmicus. Methanogenic archaea (Methanobacteriaceae) and iron-oxidizing archaea were also found. In enrichment cultures Anammox, AOB and heterotrophic denitrification (DB) were dominated by heterotrophic nitrifying bacteria, also capable of aerobic denitrification, which belonged to the families Pseudomonadaceae, Rhodanobacteraceae, Xanthomonadaceae, Alcaligenaceae.The microbial community of the flow bioreactor cultivated at 15 °C included various phylotypes of microorganisms involved in nitrogen transformation. AOB were represented by autotrophic (Nitrosomonas) and heterotrophic (Shinella) genera. Potential denitrifying bacteria included representatives of the families Rhodocyclaceae, Comammonadaceae, Burkholderiaceae and Xanthobacteraceae. Nitrogen-fixing microorganisms included representatives of the family Beijerinckiaceae, order Rhizobiale. Using the IVICODAK program, the flow reactor community shows the pathways of anammox, denitrification and nitrification, which is confirmed by qPCR and FISH methods. It was concluded that batch culture is not suitable for the enrichment of anammox bacteria, while flow-through culture allows the creation of conditions for the growth of metabolically and phylogenetically versatile microbial communities, which ensures their stable functioning over time and under changing environmental conditions.

 

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Annotation of the results obtained in 2022
Groundwater in the area of sludge storage near Chepetsky Mechanical Plant (CHMZ) (Glazov, Udmurtia, Russia) was characterized by a rather low temperature (7.8-8.5 C) and a significant excess of nitrate, sulfate and ammonium ions, while the MPC standards for ammonium were exceeded on average by 3.5-4 orders. In a number of wells, an excess of the content of a number of technogenic metals, including uranium, zinc, nickel, and manganese, was found. The content of ammonium and nitrate had a significant effect on the number of different functional groups of bacteria in the groundwater. The number of aerobic heterotrophic bacteria in the presence of high concentrations of ammonium and nitrate was minimal (10 cells/ml), with a decrease in nitrate concentration it increased by 3 orders of magnitude. It reached its maximum values (10^6 cells/ml) in the zone with the minimum content of ammonium and nitrate. The number of fermenting, sulfate-reducing and denitrifying bacteria also negatively correlated with nitrogen load. It has been suggested that the main limiting factor in the development of these microorganisms is the content of the nitrate ion, and not ammonium. Nitrifying bacteria of two stages of nitrification were found in minimal amounts (10 cells/mL) in all samples, except for samples with the maximum ammonium content, which may indicate its possibly inhibitory role for this group. Anammox bacteria were found in all wells, with a maximum representation in groundwater with an ammonium content of 80 to 216 mg/l. At the same time, according to the results of FISH analysis of suspensions, they formed microcolonies from several cells with a diameter of 5–25 µm, which indicates the initial stage of microcolony development. Sulfate reducers were found in all samples with a sufficiently high abundance - 10^3-10^4 cells/ml. Enrichment cultures of denitrifying and nitrifying bacteria were obtained from groundwater samples with a consumption rate of 14 mg ammonium/l/day and 34 mg nitrate/l/day. According to high-throughput sequencing and metagenomic analysis, the composition of the groundwater community is diverse and includes representatives of various physiological groups, primarily metabolizing nitrogen and sulfur compounds, as well as heavy metals. The dominant microorganisms include anammox bacteria of the family Scalinduaceae, which live in various, mainly natural habitats, such as sea water and sediments, fresh water, including species active at low temperatures, which can be used for bioremediation of cold polluted ecosystems. The community also includes microorganisms capable of heterotrophic nitrification (Devosiaceae and Aeromonadaceae), while “classical” autotrophic nitrifiers were not found. Denitrifying microorganisms in the community are represented by bacteria of the families Pseudomonadaceae, Devosiaceae, Aeromonadaceae. Interestingly, according to metagenomics data, the community includes representatives of the genus Paenibacillus, in whose genome nitrogen fixation genes were found. Potential participants in the sulfur cycle include Pseudomonadaceae, Aeromonadaceae, and Actinomarinales. The discovered representatives of the genus Pantoea (family Erwiniaceae) are predominantly mesophilic facultative anaerobes capable of growing by combining the oxidation of acetate or H2 with the reduction of various electron acceptors, including Fe(III), Mn(IV), Cr(VI), As(V). Representatives of the genus Paenibacillus (family Paenibacillaceae) belong to heterotrophic aerobic or facultative anaerobic bacteria, some representatives are capable of reducing metals (for example, Fe (III)) and immobilizing uranium. To obtain enrichment cultures of psychrotrophic bacteria of the nitrogen cycle (mainly anammox bacteria) in a (semi-)continuous flow mode, a 0.5-liter bioreactor was developed and started. The reactor was packed with membrane-like non-woven fabric made of polypropylene, which previously showed a high intensity of biofouling, which correlated with high activity of consumption of ammonium and nitrite in batch culture. The bioreactor was operated at 15°C and was fed with a synthetic mineral medium containing ammonium and nitrite salts and kept under argon atmosphere to maintain anaerobic conditions. During 285 days of flow culture, the nitrogen loading rate was changed in 4 stages by changing both the concentration of substrates and the flow rate. During operation of the reactor, a high efficiency of ammonium and nitrite removal (up to 100%) was noted, however, the removal of total nitrogen decreased as the nitrogen load increased and amounted to 55% at the late stage of cultivation. This was associated with an increased formation of nitrate, which did not correspond to the canonical stoichiometry of the anammox process, and was presumably explained by a change in the metabolism of anammox bacteria under the influence of low temperature, which requires further careful study, since is of particular practical importance for technologies of wastewater treatment from nitrogen at low temperatures. According to the FISH analysis, the cell number of nitrifiers of the first and second stages increased throughout the experiment. The cell number of nitrospira, among which there may be representatives of comammox-nitrospira, was insignificant. The cell number of anammox bacteria was also insignificant, but remained at a certain level. Thus, an enrichment culture containing anammox bacteria, nitrifiers of the first and second stages, including those potentially capable of carrying out the comammox process, was obtained in a continuous flow bioreactor. Our findings open up new knowledge about the metabolic capabilities of nitrogen cycle bacteria and potential approaches to sustainable bioremediation of extremely nitrogen-polluted cold ecosystems.

 

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