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Article

Genomic Analysis of the Uncultured AKYH767 Lineage from a Wastewater Treatment Plant Predicts a Facultatively Anaerobic Heterotrophic Lifestyle and the Ability to Degrade Aromatic Compounds

by
Shahjahon Begmatov
,
Alexey V. Beletsky
,
Andrey V. Mardanov
and
Nikolai V. Ravin
*
Institute of Bioengineering, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow 119071, Russia
*
Author to whom correspondence should be addressed.
Water 2025, 17(7), 1061; https://doi.org/10.3390/w17071061
Submission received: 21 February 2025 / Revised: 27 March 2025 / Accepted: 1 April 2025 / Published: 3 April 2025
(This article belongs to the Special Issue Advances in the Application of Molecular Biology to Biological Water Treatment Processes)
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Abstract

:
Microbial communities in wastewater treatment plants (WWTPs) play a crucial role in the decontamination of polluted water. An uncultured order-level lineage AKYH767 of the phylum Bacteroidota has been consistently detected in microbial consortia of activated sludge at WWTPs worldwide, but its functional role remains elusive. Representatives of AKYH767 were also detected in soils and freshwater bodies, which may be their natural reservoirs. Here, we obtained ten high-quality metagenome-assembled genomes, including one closed circular genome, of AKYH767 bacteria from metagenomes of the wastewater and activated sludge and used genomic data to uncover the metabolic potential of these bacteria and to predict their functional role. The cells of the AKYH767 bacteria were inferred to be rod-shaped and non-motile. Genome-based metabolic reconstruction predicted the Embden–Meyerhof pathway, the non-oxidative stage of the pentose phosphate pathway, and the complete tricarboxylic acid cycle. A facultatively anaerobic chemoheterotrophic lifestyle with the capacity to oxidize low organic substrates through aerobic respiration was suggested. Under anaerobic conditions AKYH767 bacteria can perform different steps of denitrification. They have limited capacities to hydrolyze carbohydrates and proteinaceous substrates but can utilize fatty acids. A peculiar property of AKYH767 bacteria is the presence of the phenylacetyl-CoA pathway for the utilization of phenylacetate, and about half of the genomes encoded the benzoate degradation pathway. Apparently, in bioreactors at WWTPs, the AKYH767 bacteria could be involved in the denitrification and biodegradation of aromatic compounds. Based on phylogenetic and genomic analyses, the novel AKYH767 bacterium is proposed to be classified as Candidatus Pollutiaquabacter aromativorans, within the candidate order Pollutiaquabacterales.

1. Introduction

Worldwide, freshwater consumption is growing at about 1% per year, and one of the global problems is water pollution, which poses a serious threat to natural ecosystems and human health. Organic and inorganic compounds and microbiological contaminants produced by agriculture, industry, and households can pollute aquatic environments. Sources of water pollution include population growth, industrial activity, radioactive waste, chemical fertilizers, pesticides, urbanization, and other anthropogenic factors [1]. Biological wastewater treatment, along with physical and chemical methods, is the main approach to wastewater purification. Many studies have shown that the microbial communities of wastewater treatment plants (WWTPs) play an important role in biological wastewater treatment, and studies on the ecophysiology of these microorganisms, the dynamics, and the diversity of these microbial consortia are ongoing using both traditional microbiological approaches and modern molecular techniques. Despite the expansion of such research, most WWTP microorganisms remain uncultivable, and the functional role of many of them is unknown [2,3]. Today, the application of metagenomics methods in biological wastewater treatment studies makes it possible to assess microbial communities, identify microorganisms involved in the decomposition of organic matter, the removal of phosphorus and nitrogen, and the study of antibiotic resistance genes [4].
Metagenomic analysis enabled to assemble genome sequences of microorganisms, which could be involved in the removal of the main contaminants from wastewater. For instance, it was shown that Chloroflexota species from activated sludge (AS) could play a crucial role in organic matter degradation and nitrogen removal [5,6]. Another vivid example is the metagenome-assembled genome (MAG) of the bacterium Candidatus Jettenia ecosi, which was retrieved from an anammox bioreactor [7]. The number of metagenomic studies aimed at identifying and characterizing new microorganisms that can participate in wastewater treatment is increasing every year.
In a previous study, we analyzed microbial communities of activated sludge at nine large-scale WWTPs in Moscow (Russia) operating with different technologies using 16S rRNA gene profiling [8]. This study uncovered the presence of several abundant uncultivated lineages of bacteria and archaea [8]. One of them was uncultivated lineage AKYH767 (a family within the order Sphingobacteriales of the phylum Bacteroidota, according to the SILVA database), which accounted for 0.5–6.7% of the microbial community of activated sludge. Members of this order were also found in the untreated sewage (up to 0.04% of 16S rRNA gene sequences) but appeared to be more numerous in the activated sludge (0.4–1%) and purified effluent (up to 0.5%) at two other large-scale WWTPs in Moscow [9].
According to the results of the large-scale study of microbial consortia of activated sludge samples from around the world, performed by the Global Water Microbiome Consortium, the AKYH767 lineage was widespread and accounted for 0.1 to 21% of the microbiomes of about 1200 activated sludge samples from 269 WWTPs in 23 countries on six continents [10]. This lineage was also abundant in bioreactors performing partial denitrification/anammox processes [11], in anaerobic-anoxic-aerobic bioreactors [12], and was found to be associated with enhanced ammonia nitrogen reduction from swine farm wastewater [13]. However, the possible functional and ecological role of the AKYH767 lineage in wastewater treatment environments remains unknown.
The AKYH767 lineage was first discovered in 1995 by sequencing a 16S rRNA gene amplicon from activated sludge at a sequencing batch bioreactor (GenBank X84525) [14]. During the next 30 years, 16S rRNA gene sequences of AKYH767 were detected in different environments, including activated sludge, soils, waters, and sediments. Currently, the SILVA database (v. 138.2) [15] contains 4422 16S rRNA gene sequences assigned to AKYH767, obtained mostly from activated sludge, wastewater, and bioreactor-associated samples (about 44% of the total), soils (20%), and freshwater bodies (about 5%). In the genome-based taxonomic system, this lineage, designated AKYH767-A, is classified as a candidate order in the class Bacteroidia of the phylum Bacteroidota in the latest release R220 of the Genome Taxonomy Database (GTDB). At present, GTDB contains 184 high-quality genomes of AKYH767-A, and more than half of these were recovered from activated sludge and related sources. However, these genomes were not analyzed, and the metabolic potential of the AKYH767 lineage has not been reported. Given the prevalence of the AKYH767 lineage in activated sludge from wastewater treatment plants, we aimed to investigate its metabolic capabilities and potential functional role in wastewater treatment using genome-centric metagenomic analysis. In this work, we obtained the genomes of AKYH767 members from wastewater and activated sludge at WWTPs in Moscow, reconstructed their metabolic pathways, and predicted their role in wastewater purification.

2. Materials and Methods

2.1. Characteristics of WWTPs, Sampling Sources, and Metagenome Sequencing

The WWTP complex “Lyuberetskiy” of JSC “Mosvodokanal” with a capacity of ~2 million m3/day performs wastewater treatment in Moscow. One of the studied WWTPs (LOS) is operated using an anaerobic/anoxic/aerobic process. Another WWTP (NLOS2) uses nitrification/denitrification technology. Both WWTPs efficiently removed organic matter, nitrogen, and phosphates from wastewater [9].
Previously we reported 16S rRNA gene profiling and metagenomic analysis of microbial communities of untreated wastewater (influent), activated sludge (AS) samples, and purified effluents from two WWTPs [9]. Metagenomic DNA sequencing using the Illumina HiSeq2500 platform yielded 150–260 million read pairs (2 × 150 bp) per sample. The obtained reads were assembled into contigs using program SPAdes v.3.15.4 [16].

2.2. Assembly and Taxonomic Identification of MAGs

Programs MetaBAT v.2.2.15 [17], MaxBin v.2.2.7 [18], and CONCOCT v.1.1.0 [19] were used for binning of contigs into MAGs. The obtained MAGs were merged into an optimized set employing DAS Tool v.1.1.4 [20]. The completeness and possible contamination of the MAGs were predicted using CheckM2 v.1.0.2 [21]. Taxonomic assignment of the obtained MAGs was performed using the GTDB-Tk v.2.0.0 [22] and Genome Taxonomy Database (GTDB) [23].

2.3. Metagenome Sequencing Using the Oxford Nanopore Technique and Assembly of the Complete Genome of the AKYH767 Bacterium

Metagenomic DNA of purified effluent from NLOS2 WWTP was additionally sequenced on a MinION instrument (Oxford Nanopore Technologies, Oxford, UK) using the 1D Genomic DNA ligation sequencing kit LSK-109 and FLO-MIN106D cell. A total of 12.1 Gb (3,298,575 reads) of Nanopore sequences were obtained. The MinION reads were assembled using Flye v. 2.9.5 [24] with the –nano-raw and –meta parameters. The sequences of the assembled contigs were polished with Illumina reads using Pypolca v. 0.3.1 [25]. A single MAG consisting of 9 contigs was obtained using SemiBin2 v.1.5.1 [26] and assigned to the AKYH767 lineage using GTDB-Tk v.2.0.0. The combination of this assembly and Illumina data allowed obtaining a single circular contig.

2.4. Annotation of MAGs, Phylogenetic Analysis, and Metabolic Reconstruction

Annotation of the genomes was performed employing the NCBI Prokaryotic Genome Annotation Pipeline [27] and the RAST server [28], followed by manual correction of the annotation of key metabolic functions by comparing the predicted protein sequences with the NCBI databases. Barrnap v.0.9 (https://github.com/tseemann/barrnap accessed on 21 February 2025) was used to identify rRNA genes. The average nucleotide identity (ANI) and average amino acid identity (AAI) between the genomes were determined using appropriate scripts from the Enveomics Collection [29]. GTDB-Tk v.2.0.0 was used to identify 120 single-copy marker genes in the analyzed genomes and to make multiple alignments of concatenated amino acid sequences. PhyML v. 3.3 [30] was used to generate the maximum likelihood phylogenetic tree. The Bayesian method was used to quantify the branch-support values.
The presence and completeness of metabolic pathways in the genomes were evaluated using the Distilled and Refined Annotation of Metabolism (DRAM) tool [31]. The KEGG functional annotation of genes was performed using the KofamScan v.1.3.0 tool [32]; best hits with a 1 × 10−10 e-value threshold were used, followed by manual curation. All software was used with default parameters unless otherwise specially mentioned.

3. Results

3.1. Genomic Features of the AKYH767 Lineage

A total of 204 high-quality MAGs with more than 90% completeness and less than 5% contamination were assembled from metagenomes of the studied wastewater and AS samples. Ten MAGs were assigned to the candidate order AKYH767-A in the GTDB (Table 1). The NLOS2-E-001 genome is the first reported complete circular genome of the AKYH767 lineage, since the only known complete genome initially assigned to AKYH767 [33] according to the current GTDB classification (release 220) actually represented another uncultured order, CAMBQF01.
The sizes of AKYH767 genomes ranged from 2,764,887 to 3,820,763 bp, the G+C content was between 37 and 54%, and the coding density was 90–92%, which slightly exceeded the average value (87%) for bacterial genomes (Table 1).
Inspection of the obtained AKYH767 genomes revealed that all of them lacked genes encoding the flagellar components, chemotaxis, and type IV pili, suggesting that these bacteria are not motile. The finding of genes for the rod-shape determining proteins MreBCD, RodA, and the peptidoglycan D,D-transpeptidase RodA [34] indicates that AKYH767 bacteria are rod-shaped.

3.2. Phylogenetic Placement of AKYH767 Genomes

The taxonomic placement of AKYH767 MAGs in the GTDB showed that four of them belong to the candidate family OLB10 and six represented the 2013-40CM-41-45 family. To further characterize the phylogenetic position of the obtained MAGs, a maximum likelihood phylogenetic tree based on 120 conserved genes was constructed for the obtained MAG and representative genomes from all other genus-level lineages (1 genome per genus) of the AKYH767-A order (Figure 1).
MAGs LOS-E-003, LOS-AS-003, NLOS2-AS-012, and INF-064 cluster with the MAG (GCA_016699635.1) from the genus OLB10, assembled from the metagenome of the activated sludge sample [35] (Figure 1). Since the pairwise ANI values between all these MAGs were above 99%, they all represented different strains of the same species, designated OLB10 sp016699635 in the GTDB. The second cluster includes four obtained MAGs (NLOS2-E-001, LOS-E-007, LOS-AS-011, and NLOS2-E-027) and the genome of the member of the genus CAINVI01 and the species CAINVI01 sp016713765 (GCA_016713765.1), assembled from the metagenome of the same sample, as the GCA_016699635.1 genome. Since the pairwise ANI values between all these genomes exceed 98.5%, they all belong to the species CAINVI01 sp016713765. The remaining two MAGs, NLOS2-AS-005 and NLOS2-E-013, clustered with the representative genome of the genus JADKIH01 (GCA_016719005.1). NLOS2-AS-005 and NLOS2-E-013 are closely related (99.7% ANI) and share about 89% AAI with the GCA_016719005.1 genome, therefore representing a novel species of this genus. The candidate genera OLB10 and JADKIH01 are currently represented only by MAGs obtained from wastewater-related environments, while CAINVI01 also includes MAGs from freshwater lakes.
All genera and families defined in the GTDB within the AKYH767-A order formed distinct branches (Figure 1). Interestingly, the basic phylogenetic lineages of this order (the JAHEYN01 and JABDAW01 families) were represented only by genomes obtained from water bodies and soils. In the other two families, bacteria from the WWTPs were present in different genera, which probably indicate multiple adaptations of AKYH767 bacteria to such artificial environments.

3.3. Central Metabolic Pathways

To reconstruct the metabolic pathways of the AKYH767 lineage, we analyzed 9 assembled MAGs. An overview of metabolic reconstruction is shown in Figure 2. All genomes encoded the Embden-Meyerhof pathway of glycolysis, whereas the Entner–Doudoroff pathway was absent. Only the non-oxidative branch, the pentose phosphate pathway, was present. Pyruvate produced by glycolysis could be converted to acetyl-CoA through the activity of the pyruvate dehydrogenase complex. All genes of the tricarboxylic acid (TCA) cycle, which could enable complete oxidation of organic substrates, were identified in the genomes. The absence of citrate lyase suggests that the cycle can operate only in the oxidative direction. The key enzymes of other autotrophic carbon fixation pathways (the Wood-Ljungdahl pathway, the 3-hydroxypropionate bicycle, the hydroxypropionate-hydroxybutyrate cycle, and the dicarboxylate-hydroxybutyrate cycle) were also absent.
The AKYH767 genomes encoded three enzymes that link the TCA cycle and glycolysis. The phosphoenolpyruvate carboxykinase converts oxaloacetate into phosphoenolpyruvate and CO2, while NAD+ malic enzyme decarboxylates malate into pyruvate and CO2. A third enzyme, pyruvate carboxylase, that can perform irreversible carboxylation of pyruvate to form oxaloacetate, was encoded in the genomes of the genera 2013-40CM-41-4 and CAINVI01 but was absent from the OLB10 genomes.
All main enzymes of the electron transfer chain for oxidative phosphorylation were predicted in the AKYH767 bacteria, including NADH dehydrogenase, succinate dehydrogenase, and terminal reductases for aerobic and anaerobic respiration (Figure 2). The generated transmembrane proton gradient may be used by the F0F1-type ATPase to produce ATP. All genomes encode two types of cytochrome c oxidases of the b(o/a)3 and cbb3 types. Genes encoding heme o and heme a synthases were identified as well. The quinol bd oxidase was not found. The cbb3-type cytochrome c oxidases are known to have a high affinity for oxygen and enable respiration under lower levels of oxygen [36], while b(o/a)3 is a low-affinity enzyme typically found in aerobes. The presence of superoxide dismutase and catalase-peroxidase indicates the ability of the AKYH767 bacteria to grow under aerobic conditions. These two enzymes are involved in protecting against reactive oxygen species in aerobes. Catalase-peroxidases are bifunctional enzymes induced in response to oxidative stress [37].
All analyzed AKYH767 genomes encode membrane-bound NAD(P)+ transhydrogenase. These enzymes catalyze the electron transfer reaction between NAD(H) and NADP(H) coupled to the translocation of a proton across the membrane [38]. The resulting NADPH can be used in biosynthesis reactions.
A genome analysis revealed capacities of the AKYH767 bacteria for anaerobic respiration with nitrogen compounds. All genomes encoded membrane-linked nitric oxide reductases that perform the reduction of nitric oxide (NO) to nitrous oxide (N2O), and nitrous oxide reductases perform the last step in denitrification, the reduction of N2O to gaseous N2. The OLB10 genomes additionally encode copper-containing nitrite reductase (CuNIR), which performs the reduction of nitrite to NO, while 2013-40CM-41-4 and CAINVI01 genomes lacked this enzyme. Genes for dissimilatory nitrate reductases were not found in any of the AKYH767 genomes, as well as reductases for dissimilatory reduction of sulfate, other sulfur compounds, arsenate, and iron.

3.4. Possible Growth Substrates

Analysis of AKYH767 genomes revealed a limited set of enzymes for the hydrolysis and utilization of carbohydrates. Enzymes for the utilization of cellulose and xylan were not found. On the contrary, all analyzed genomes encoded chitinases of the GH18 family [39]. These enzymes were predicted to carry N-terminal secretion signal peptides, suggesting that they could perform extracellular hydrolysis of chitin. Beta-N-acetylhexosaminidases that perform the hydrolysis of terminal, non-reducing β-N-acetylglucosamine residues from chito-oligosaccharides [40] were also identified. Interestingly, genes coding for enzymes involved in further intracellular metabolism of N-acetylglucosamine were not annotated, namely, N-acetylglucosamine kinase, glucosamine-6-phosphate deaminase, and N-acetylglucosamine-6-phosphate deacetylase. It is possible that their functions could be performed by related enzymes, as it was proposed for the glucokinase of the chitinolytic bacterium Chitinivibrio alkaliphilus [41].
Bacteria utilize different mechanisms for the uptake of sugars, and the genomes of AKYH767 bacteria contained genes for the ABC-2 type transport system (TC:1.B.18 and TC:3.A.1), MFS transporters of the FHS family (TC: 2.A.1.7), the solute:Na+ symporter of the SSS family (TC:2.A.21), and the polysaccharide transporter of the PST family (TC:2.A.66.2). These transporters could be responsible for the transport of arabinose, galactose/glucose, maltose, fucose, xylose, and mannose [42,43]. Some of these sugars may possibly be used as substrates by AKYH767 bacteria.
Analysis of annotation amino acid biosynthesis pathways via GapMind [44] showed that the analyzed AKYH767 bacteria have complete biosynthesis pathways for asparagine, glutamine, glycine, lysine, methionine, serine, threonine, cysteine, and chorismate. The chorismate is the common precursor for the biosynthesis of the aromatic amino acids, phenylalanine, tyrosine, and tryptophan [45]. These data suggest that AKYH767-A bacteria can probably synthesize amino acids and do not need to acquire amino acids from the environment. This is consistent with the absence of a large number of amino acid and peptide transporter genes, noting only the presence of an oligopeptide transporter of the OPT family.
All analyzed AKYH767 genomes were predicted to encode the fatty acid β-oxidation pathway, including long-chain fatty acid-CoA ligase, acyl-CoA dehydrogenase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase.
Bacteria use different strategies for the degradation of aromatic compounds. These strategies are O2-dependent ring cleavage of dihydroxylated aromatic compounds, O2-dependent ring epoxidation of CoA thioesters, ATP-dependent ring reduction of CoA thioesters, and ATP-independent ring reduction of CoA thioesters [46]. Analysis of the obtained AKYH767 genomes showed that they encode the phenylacetyl-CoA pathway for the aerobic degradation of phenylacetate (Figure 2). Many other aromatic compounds, such as phenylalanine, phenylethylamine, and the environmental pollutants styrene and ethylbenzene, are also metabolized via the phenylacetate degradation pathway [47]. The pathway starts with the transformation of phenylacetate to phenylacetyl-CoA by phenylacetate-CoA ligase (PaaK). The key enzyme of this pathway, the multisubunit ring-1,2-phenylacetyl-CoA epoxidase (PaaABCD), performing O2-dependent ring epoxidation of CoA thioesters [48], is encoded in all genomes. The reactive non-aromatic epoxide is then isomerized to an oxepin-CoA by ring-1,2-epoxyphenylacetyl-CoA isomerase (PaaG). The latter is the substrate of hydrolytic ring cleavage followed by aldehyde oxidation to the corresponding carboxylic acid. This step is performed by the bifunctional oxepin-CoA hydrolase/3-oxo-5,6-dehydrosuberyl-CoA semialdehyde dehydrogenase (PaaZ), encoded in all AKYH767 genomes. The finding of these genes shows that the bacterium possesses the phenylacetyl-CoA pathway degradation pathway. Subsequent β-oxidation-like reactions resulted in the production of acetyl-CoA and succinyl-CoA [48].
In half of the analyzed genomes of AKYH767 bacteria, a locus encoding the benzoate degradation pathway is contained (Figure 2). Benzoyl-CoA is the most important intermediate for the utilization of aromatic compounds since many of them, including chloro-, nitro-, and aminobenzoates, aromatic hydrocarbons, and phenols, are converted to benzoyl-CoA before ring reduction and cleavage. Benzoyl-CoA reductase is a key enzyme for the biodegradation of aromatic compounds [49]. It catalyzes the ATP-dependent ring reduction in benzoyl-CoA to 1,5-dienoyl-CoA using a ferredoxin as the electron donor and is composed of four subunits encoded by the bcrCBAD operon. The produced 1,5-dienoyl-CoA is then hydrated by dienoyl-CoA hydratase (Dch) to form 6-hydroxycylohex-1-en-1-carbonyl-CoA. The following steps are performed by 6-hydroxycylohex-1-en-1-carbonyl-CoA dehydrogenase (Had) and the ring hydrolyzing enzyme, 6-oxocyclohex-1-ene-1-carbonyl-CoA hydrolase (Oah), to yield 3-hydroxypimelyl-CoA. The latter could be metabolized via the beta-oxidation pathway encoded at the same locus. The benzoate degradation locus also contains a gene annotated as fatty-acid-CoA ligase that could actually function as a benzoate-CoA ligase.

3.5. Description of New Taxa

The genome of the NLOS2-E-001 bacterium meets the criteria suggested for the description of new taxa of uncultivated microorganisms [50], and we propose the following taxonomic names for the novel genus and species of NLOS2-E-001.
Description of the novel genus Candidatus Pollutiaquabacter (Pol.lu.ti.a.qua bac.ter. L. masc. perf. part. pollutus, polluted; L. fem. n. aqua, water; N.L. masc. n. bacter, rod; N.L. masc. n. Pollutiaquabacter, a rod microbe from a polluted aquatic environment).
Description of the novel species Candidatus Pollutiaquabacter aromativorans (a.ro.ma′ti.vo.rans. L. n. aroma-atis spice; L. part. adj. vorans devouring; N.L. part. adj. aromativorans degrading aromatic compounds).
Not cultivated. Inferred to be a rod-shaped, non-motile, facultatively anaerobic organotroph that obtains energy by fermentation, aerobic respiration, or denitrification, and is capable of using aromatic compounds, fatty acids, and some sugars as growth substrates. Represented by the complete genome NLOS2-E-001 (GenBank acc. no. CP185379) obtained from the metagenome of wastewater in Moscow, Russia.
Based on this, and because of the appearance of a representative with a known complete genome sequence, we propose the following names for the order and family:
  • Candidatus Pollutiaquabacterales ord. nov.
  • Candidatus Pollutiaquabacteraceae fam. nov.
Candidatus Pollutiaquabacter, Candidatus Pollutiaquabacteraceae, and Candidatus Pollutiaquabacterales correspond to the genus CAINVI01, family 2013-40CM-41-45, and order AKYH767-A in the GTDB, respectively.

4. Discussion

The main function of wastewater treatment plants is the removal of organic contaminants, nitrogen, and phosphorus. Microbial communities of activated sludge play a key role in these processes. Bacteria of the AKYH767 lineage were found mainly in activated sludge of WWTP, as well as in freshwater bodies and soils, which may be their natural source. This assumption is consistent with the fact that the basic phylogenetic lineages of the order AKYH767 (families JAHEYN01 and JABDAW01) are represented by genomes only from water bodies and soils (Figure 1). Therefore, it can be assumed that AKYH767 bacteria can play some significant role in the processes of biological wastewater treatment.
Activated sludge bacteria can oxidize organic matter through aerobic respiration or denitrification, oxidize ammonium to nitrate in the aerobic zone of the bioreactor, and then reduce it to gaseous nitrogen in the anaerobic zone. Phosphorus-accumulating organisms (PAOs) can also perform biological phosphorus removal. Genome analysis predicted that AKYH767 bacteria are facultative anaerobic heterotrophs capable of the complete oxidation of organic matter. They have a limited capacity to hydrolyze complex polysaccharides and proteinaceous substrates and are mostly devoted to fatty acids and low-molecular-weight compounds. An unexpected exception is the presence of chitinase genes. Natural sources of chitin are shells of crustaceans, insects, and cell walls of fungi. Chitin and its derivatives are present in freshwater environments and soils and probably in municipal wastewater and can be used as substrates for AKYH767 bacteria.
Aromatic compounds, particularly oil-derived aromatic substances such as polycyclic aromatic hydrocarbons, are ubiquitous in wastewater [51,52]. WWTPs have become a main contributor of these substances present in freshwater systems [53]. Microbial communities of activated sludge can biodegrade most aromatic organic compounds and efficiently remove them from wastewater [54,55,56]. Analysis of the metabolic capabilities of the AKYH767 lineage predicted that these bacteria could biodegrade these hazardous and highly toxic materials. Although the ability to degrade aromatic compounds at WWTPs is not unique to the AKYH767 lineage and was found in various bacteria found in AS (e.g., Pseudomonas, Rhodococcus, Thauera, etc.), this trait can be further exploited for targeted cultivation of the AKYH767 lineage.
The genomes of AKYH767 bacteria do not encode enzymes for the oxidation of ammonium and nitrite, as well as for the reduction of nitrate to form nitrite. However, they can perform subsequent stages of denitrification. All of them have genetic potential for the reduction of nitric oxide to nitrous oxide and subsequently to gaseous nitrogen. Some AKYH767 bacteria possess nitrite reductase and could reduce nitrite to NO. Therefore, they can participate in denitrification, which is consistent with observations of AKYH767 abundance in bioreactors performing partial denitrification/anammox processes [11].
Assessment of the phosphate-accumulating potential of AKYH767 revealed that the genomes of these bacteria contain some genes involved in the intracellular accumulation of polyphosphates, namely, polyphosphate kinase, exopolyphosphatase, inorganic phosphate transporter (PiT family), and the high-affinity PstSCAB transporter [57]. These genes are typical but not exclusive to organisms that show PAO activity. However, cation/acetate symporter (ActP) facilitating acetate uptake during the anaerobic stage, polyphosphate glucokinase, and polyphosphate-AMP phosphotransferase were not detected. Therefore, it is likely that AKYH767 bacteria are not PAO, and the found genes of phosphorous cycling are involved in purine metabolism and oxidative phosphorylation.
Thus, bacteria of the AKYH767 lineage apparently participate in the process of removing pollutants from wastewater, primarily low-molecular-weight organic matter such as aromatic compounds, but do not perform any unique function. This is consistent with the observation that AKYH767, although widespread, are minor components of the microbiome of activated sludge. In natural ecosystems, they can play the role of scavengers and participate in the biodegradation of low-molecular-weight organic compounds in environments with various oxygen contents, as indicated by the presence of cytochrome c oxidases with different affinities for oxygen, as well as some enzymes of the denitrification pathway. One of the natural substrates for AKYH767 bacteria can be chitin and its derivatives, which are widespread in water bodies and soils. Interestingly, AKYH767 members from WWTPs do not form a distinct phylogenetic branch in this order but are found in different genera. Probably, such distribution reflects multiple independent events of recruitment of AKYH767 bacteria from natural sources to activated sludge of WWTP facilities.

5. Conclusions

The results of this study provide the first insight into the biology and ecological role of uncultured lineage AKYH767, typically found in bioreactors of wastewater treatment plants. Genome analysis predicted that AKYH767 bacteria are facultative anaerobic heterotrophs capable of complete oxidation of organic matter. Metabolic pathways reconstruction revealed the presence of Embden–Meyerhof glycolytic pathway and gluconeogenesis, the non-oxidative branch of the pentose phosphate pathway, and the complete TCA cycle. The genomes of AKYH767 bacteria encode the electron transfer chain for aerobic respiration ending with cytochrome c oxidases. Under anaerobic conditions, they can perform different steps of denitrification. The AKYH767 bacteria have limited capacities to hydrolyze carbohydrates and proteinaceous substrates but can utilize fatty acids. A peculiar property of AKYH767 bacteria is the presence of pathways for the aerobic degradation of aromatic compounds, in particular phenylacetate and benzoate. Apparently, in the wastewater treatment plants, AKYH767 bacteria could be involved in the biodegradation of aromatic compounds and perform some stages of denitrification. Confirmation of these hypotheses will require the use of experimental approaches such as metatranscriptomics and the biochemical characterization of the relevant enzymes, and finally, the cultivation of AKYH767 bacteria.

Author Contributions

Conceptualization, N.V.R.; investigation, S.B., A.V.B. and A.V.M.; data curation, N.V.R.; writing—original draft preparation, S.B. and N.V.R.; writing—review and editing, S.B. and N.V.R.; funding acquisition, S.B. and N.V.R.; supervision, N.V.R. All authors have read and agreed to the published version of the manuscript.

Funding

This study was partly supported by the Russian Science Foundation (Project No. 24-74-10045 to SB).

Data Availability Statement

The genome sequences of AKYH767 bacteria are openly available in NCBI GenBank database within the BioProject PRJNA1165690. The complete genome sequence of Candidatus Pollutiaquabacter aromativorans strain NLOS2-E-001 has been deposited in the NCBI GenBank database under the accession number CP185379.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Figure 1. Genome-based phylogeny of the AKYH767. Taxonomic lineages according to the GTDB are shown (o, order; f, family; and g, genus). The level of support for internal branches was calculated using the Bayesian test. The genome sequence of a member of the order 2-12-FULL-35-15 (GCA_001769385) was used as an outgroup. Circles with colored sectors show the sources from which the genomes were obtained.
Figure 1. Genome-based phylogeny of the AKYH767. Taxonomic lineages according to the GTDB are shown (o, order; f, family; and g, genus). The level of support for internal branches was calculated using the Bayesian test. The genome sequence of a member of the order 2-12-FULL-35-15 (GCA_001769385) was used as an outgroup. Circles with colored sectors show the sources from which the genomes were obtained.
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Water 17 01061 g002
Figure 2. Overview of the main metabolic pathways of the AKYH767 bacteria. Enzymes: GK, glucokinase; PGI, glucose-6-phosphate isomerase; PFK, phosphofructokinase; FBA, fructose-bisphosphate aldolase; TIM, triose phosphate isomerase; GPDH, glyceraldehyde 3-phosphate dehydrogenase; PGK, phosphoglycerate kinase; PGM, phosphoglycerate mutase; PYK, pyruvate kinase; FBP, fructose-1,6-bisphosphatase; Rpe, ribulose-5-phosphate epimerase; Rpi, ribulose-5-phosphate isomerase; Tal, transaldolase; Tkt, transketolase; PDH, pyruvate dehydrogenase; ACS, acetyl-CoA synthetase (ADP-forming); ACD, acetyl-CoA synthetase; GltA, citrate synthase; Acn, aconitase; Icd, isocitrate dehydrogenase; OOR, 2-oxoglutarate ferredoxin oxidoreductase; SCS, succinyl-CoA ligase; Fum, fumarate hydratase; Mdh, malate dehydrogenase; Mae, malic enzyme; PC, pyruvate carboxylase; PEPCK, phosphoenolpyruvate carboxykinase; COX, cytochrome c oxidase; PPase, pyrophosphatase; FadD, fatty acid-CoA ligase; CuNIR, copper-containing nitrite reductase; Nor, nitric oxide reductases; Nos, nitrous oxide reductase; FadE, acyl-CoA dehydrogenase; ECH, enoyl-CoA hydratase; HADH, 3-hydroxyacyl-CoA dehydrogenase; FadA, 3-ketoacyl-CoA thiolase; Bcl, benzoate-CoA ligase; BcrCBAD, benzoyl-CoA reductase; Dch, dienoyl-CoA hydratase; Had, 6-hydroxycyclohex-1-ene-1-carbonyl-CoA dehydrogenase; Oah, 6-oxocyclohex-1-ene-1-carbonyl-CoA hydrolase; PaaK, phenylacetate-CoA ligase; PaaABCD, 1,2-phenylacetyl-CoA epoxidase; PaaG, 1,2-epoxy-phenylacetyl-CoA isomerase; PaaZ, oxepin-CoA hydrolase/3-oxo-5,6-dehydrosuberyl-CoA semialdehyde dehydrogenase. Organic compounds: G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; F-1,6BP, fructose 1,6-bisphosphate; GAP, glyceraldehyde-3-phosphate; 1,3BPG, 1,3-bisphosphoglycerate; 3PG, 3-phosphoglycerate; 2PG, 2-phosphoglycerate; PEP, phosphoenolpyruvate; N+, NAD(P)+; NH, NAD(P)H; CoA, coenzyme A; PA-CoA, phenylacetyl-CoA; Ep-PA-CoA, epoxy-PA-CoA; 3-oxo-5,6-dhsb-CoA, 3-oxo-5,6-dehydrosuberyl-CoA; c-1,5-d-1-carbonyl-CoA, cyclohexa-1,5-diene-1-carbonyl-CoA; 6-h-1-e-1-carbonyl-CoA, 6-hydroxycyclohex-1-ene-1-carbonyl-CoA; 6-o-1-e-1-carbonyl-CoA, 6-oxocyclohex-1-ene-1-carbonyl-CoA; GlcNAc, N-acetylglucosamine. Other abbreviations: CM, cytoplasmic membrane; OM, outer membrane; CW, cell wall.
Figure 2. Overview of the main metabolic pathways of the AKYH767 bacteria. Enzymes: GK, glucokinase; PGI, glucose-6-phosphate isomerase; PFK, phosphofructokinase; FBA, fructose-bisphosphate aldolase; TIM, triose phosphate isomerase; GPDH, glyceraldehyde 3-phosphate dehydrogenase; PGK, phosphoglycerate kinase; PGM, phosphoglycerate mutase; PYK, pyruvate kinase; FBP, fructose-1,6-bisphosphatase; Rpe, ribulose-5-phosphate epimerase; Rpi, ribulose-5-phosphate isomerase; Tal, transaldolase; Tkt, transketolase; PDH, pyruvate dehydrogenase; ACS, acetyl-CoA synthetase (ADP-forming); ACD, acetyl-CoA synthetase; GltA, citrate synthase; Acn, aconitase; Icd, isocitrate dehydrogenase; OOR, 2-oxoglutarate ferredoxin oxidoreductase; SCS, succinyl-CoA ligase; Fum, fumarate hydratase; Mdh, malate dehydrogenase; Mae, malic enzyme; PC, pyruvate carboxylase; PEPCK, phosphoenolpyruvate carboxykinase; COX, cytochrome c oxidase; PPase, pyrophosphatase; FadD, fatty acid-CoA ligase; CuNIR, copper-containing nitrite reductase; Nor, nitric oxide reductases; Nos, nitrous oxide reductase; FadE, acyl-CoA dehydrogenase; ECH, enoyl-CoA hydratase; HADH, 3-hydroxyacyl-CoA dehydrogenase; FadA, 3-ketoacyl-CoA thiolase; Bcl, benzoate-CoA ligase; BcrCBAD, benzoyl-CoA reductase; Dch, dienoyl-CoA hydratase; Had, 6-hydroxycyclohex-1-ene-1-carbonyl-CoA dehydrogenase; Oah, 6-oxocyclohex-1-ene-1-carbonyl-CoA hydrolase; PaaK, phenylacetate-CoA ligase; PaaABCD, 1,2-phenylacetyl-CoA epoxidase; PaaG, 1,2-epoxy-phenylacetyl-CoA isomerase; PaaZ, oxepin-CoA hydrolase/3-oxo-5,6-dehydrosuberyl-CoA semialdehyde dehydrogenase. Organic compounds: G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; F-1,6BP, fructose 1,6-bisphosphate; GAP, glyceraldehyde-3-phosphate; 1,3BPG, 1,3-bisphosphoglycerate; 3PG, 3-phosphoglycerate; 2PG, 2-phosphoglycerate; PEP, phosphoenolpyruvate; N+, NAD(P)+; NH, NAD(P)H; CoA, coenzyme A; PA-CoA, phenylacetyl-CoA; Ep-PA-CoA, epoxy-PA-CoA; 3-oxo-5,6-dhsb-CoA, 3-oxo-5,6-dehydrosuberyl-CoA; c-1,5-d-1-carbonyl-CoA, cyclohexa-1,5-diene-1-carbonyl-CoA; 6-h-1-e-1-carbonyl-CoA, 6-hydroxycyclohex-1-ene-1-carbonyl-CoA; 6-o-1-e-1-carbonyl-CoA, 6-oxocyclohex-1-ene-1-carbonyl-CoA; GlcNAc, N-acetylglucosamine. Other abbreviations: CM, cytoplasmic membrane; OM, outer membrane; CW, cell wall.
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Table 1. Main characteristics of AKYH767 genomes obtained in this work.
Table 1. Main characteristics of AKYH767 genomes obtained in this work.
MAG IDSourceCompleteness, %Contamination, %Coding Density, %ContigsContig N50 (nt)MAG Length, MbGC Content, %Protein-Coding GenestRNA GenesrRNA Operons *
NLOS2-E-001Effluent, NLOS21000.110.90713,719,8303.72543005423
LOS-E-007Effluent, LOS90.240.710.92825617,4132.82542436422
LOS-AS-011AS, LOS96.811.910.92638715,5553.44542927452
NLOS2-E-027Effluent, NLOS293.131.960.92440715,2203.55543040493
LOS-E-003Effluent, LOS99.940.480.9277088,6743.08372585641
LOS-AS-003AS, LOS99.920.460.92560122,9213.12372664692
NLOS2-AS-012AS, NLOS21000.40.92513337,3413.11372681601
INF-064Influent, NLOS297.291.460.92831811,7952.76372567441
NLOS2-E-013Effluent, NLOS296.371.020.90232117,0203.53393023332
NLOS2-AS-005AS, NLOS297.360.220.90221130,1603.82393166383
Note: * predicted by Barrnap v.0.9.
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Begmatov, S.; Beletsky, A.V.; Mardanov, A.V.; Ravin, N.V. Genomic Analysis of the Uncultured AKYH767 Lineage from a Wastewater Treatment Plant Predicts a Facultatively Anaerobic Heterotrophic Lifestyle and the Ability to Degrade Aromatic Compounds. Water 2025, 17, 1061. https://doi.org/10.3390/w17071061

AMA Style

Begmatov S, Beletsky AV, Mardanov AV, Ravin NV. Genomic Analysis of the Uncultured AKYH767 Lineage from a Wastewater Treatment Plant Predicts a Facultatively Anaerobic Heterotrophic Lifestyle and the Ability to Degrade Aromatic Compounds. Water. 2025; 17(7):1061. https://doi.org/10.3390/w17071061

Chicago/Turabian Style

Begmatov, Shahjahon, Alexey V. Beletsky, Andrey V. Mardanov, and Nikolai V. Ravin. 2025. "Genomic Analysis of the Uncultured AKYH767 Lineage from a Wastewater Treatment Plant Predicts a Facultatively Anaerobic Heterotrophic Lifestyle and the Ability to Degrade Aromatic Compounds" Water 17, no. 7: 1061. https://doi.org/10.3390/w17071061

APA Style

Begmatov, S., Beletsky, A. V., Mardanov, A. V., & Ravin, N. V. (2025). Genomic Analysis of the Uncultured AKYH767 Lineage from a Wastewater Treatment Plant Predicts a Facultatively Anaerobic Heterotrophic Lifestyle and the Ability to Degrade Aromatic Compounds. Water, 17(7), 1061. https://doi.org/10.3390/w17071061

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