Performance characteristics of WWTPs
Three main technologies are used in the investigated WWTPs. The simplest aeration process is implemented at WWTPs 1 and 7, where wastewater is supplied either at the beginning of the bioreactor corridor (CAS process, WWTP7), or along the entire aeration tank (SF process, WWTP1). The WWTP1 removed more than 98% of organics (according to the BOD data) and about 90% of ammonium, while the purification efficiency of WWTP7 was lower (95% removal of organics and 70% of ammonium) (Table 1). Interestingly, although these units were not designed to remove phosphorus, the WWTP7 removed more than 70% of phosphorus, while in WWTP1 its removal was inefficient. The nitrification/ denitrification technology, realized at WWTP2, enabled removal of more than 98% of organics and more than 99% of ammonium, while phosphorous was not removed (Table 1). Six WWTPs operated by the UCT technology enabled efficient purification of the wastewater from both organics (> 98%), ammonium (> 99%) and phosphorous (> 90%) (Table 1). The concentrations of N-NO3 in the effluent were much lower than N-NH4 in the influent indicating efficient denitrification in all WWTPs (Table 1).
Diversity of microbial communities of AS
Between 23,167 and 84,634 16S rRNA gene sequences were obtained for 27 analysed AS samples (9 WWTPs, 3 replicas) and clustered into 14,690 OTUs at the level of 97% identity. Neighbour-joining trees based of the UniFrac analysis (Fig. 2) and Jaccard similarity (Supplementary Fig. S1) of OTU datasets revealed that replicate samples formed distinct branches for all WWTPs except for WWTP8 and WWTP9 which use identical bioreactors and treatment technologies. Therefore, for subsequent analysis, for each of the 9 WWTPs, three replicates were combined into one dataset. Both UniFrac and Jaccard trees revealed clustering of samples according to the technology used (Fig. 2 and Supplementary Fig. S2).
Neighbor joining tree illustrating weighted UniFrac distances between microbial communities of AS samples from 9 WWTPs (three replications). Sample IDs are shown in brackets after the WWTP number.
The number of species-level OTUs present in AS at individual WWTPs ranged between 3860 and 4868, these values are typical for large-scale wastewater treatment plants10. Overall, the diversity of microbial communities did not significantly vary between WWTPs employing different technologies, while the evenness of microbial communities in plants operated by the UCT process was slightly lower than in the others (Supplementary Table S2).
Microbial community patterns at the phylum level
Taxonomic assignment of OTUs revealed the presence of 53 phylum-level lineages of Bacteria and Archaea, recognized in the Genome Taxonomy Database (GTDB)37. However, top 11 phyla comprising on average more than 1% of all the 16S rRNA gene sequences together accounted for more than 90% of the community (Fig. 3 and Supplementary Table S3).
Bacterial and archaeal community composition in AS samples according to the results of 16S rRNA gene sequencing. The composition is displayed at the phylum level. Average values for three replicas are shown for each WWTP.
Archaea represented less than 2% of sequences in all samples and were assigned to the phyla Nanoarchaeota, Halobacterota, Euryarchaeota and Thermoplasmatota; each was detected in all samples. Besides members of the phylum Nanoarchaeota, known to comprise partner-dependent parasites or symbionts with small genome size and limited metabolic capacities38, most of other Archaea represented known methanogenic lineages of the families Methanobacteriaceae and Methanosaetaceae.
Bacterial communities were dominated by the Proteobacteria (on average 27.8% of 16S rRNA gene reads), mostly of classes gamma (23.5%) and alpha (4.3%). Other major groups were the Bacteroidota (15.7%), Actinobacteriota (12.5%), Chloroflexi (6.6%), Myxococcota (5.9%), Firmicutes (5.6%), Patescibacteria (5.5%), Verrucomicrobiota (4.5%), Bdellovibrionota (3.9%), Nitrospirota (2.7%), and Planctomycetota (1.3%). The relative abundances of these lineages in different samples differ by no more than several times, with the exception of Nitrospirota, the share of which is minimal (0.07%) in WWTP3 and maximal (7.1%) in WWTP2. All other bacterial phyla accounted for less than 2% of 16S rRNA gene reads in all samples except for the Campylobacterota representing about 3.5% of the community in WWTP1.
The main microbial drivers of wastewaters treatment
The biological wastewater treatment includes several microbial-driven processes, such as mineralization of organics by heterotrophs, oxidation of ammonia to nitrite and finally to nitrate, denitrificartion with the production of N2 gas, enhanced biological phosphorus removal etc. In this section we analyze the presence of the microbial groups which could be involved in these processes. The activities of microbial processes depend on the absolute concentrations of microorganisms, but given that the concentration of AS in all bioreactors was similar, we can consider the efficiency of processes in relation to the relative abundance of the corresponding functional group in the community. Data on the shares of the discussed groups of microorganisms are shown in Table 2.
Oxidation of ammonia and denitrification
Oxidation of ammonia to nitrate via nitrite is usually accomplished by two groups of microorganisms, although complete oxidation of ammonia to nitrate by Nitrospira sp. (comammox process) has been also reported39. Among known ammonia oxidizers (AOM) only members of the family Nitrosomonadaceae, mostly of the genus Nitrosomonas, were detected. The relative abundance of Nitrosomonadaceae clearly correlated with efficiency of ammonia removal: it ranged from 0.8% to 2.9% in all WWTPs enabling good removal of ammonia (> 99%) and was below 0.4% in WWTPs 1 and 7 where ammonia removal was less efficient.
Two genera of nitrite-oxidizing bacteria (NOB) were identified, Nitrospira and Candidatus Nitrotoga. The relative abundance of Nitrospirae sp. was the highest (7.06%) in WWTP2, which uses nitrification–denitrification process and much lower in WWTPs 1 and 7 (0.78% and 0.08%). WWTPs using the UCT process in terms of Nitrospirae content were clearly divided into two groups. AS samples of WWTPs 6, 8 and 9 harbored between 3.9% and 6.5% of Nitrospirae sp., while its relative abundance was less than 0.5% in WWTPs 3, 4 and 5. However, microbial communities of these three bioreactors contained 0.26 to 0.46% of Ca. Nitrotoga, nearly absent in other WWTPs. This recently described Ca. Nitrotoga species can be functionally and sometimes dominant important nitrite oxidizers in WWTPs due to their relatively higher resistance to free nitrous acid and free ammonia than other NOB and the presence of complete pathways for hydrogen and sulfite oxidation, suggesting that alternative energy metabolisms enable these bacteria to survive nitrite depletion40,41,42.
Regardless of the used purification technology and the efficiency of ammonia removal, the concentration of N-NO3 in the effluent was in the range from 7 to 10.5 mg/L, which corresponds to 15–20% of N–NH4 in the influent and indicates effective denitrification and removal of nitrogen in the gaseous form. The presence of numerous heterotrophic denitrifying bacterial is consistent with this observation.
Biological phosphorus removal
The next main issue of wastewater treatment is the removal of phosphorus. This process is carried out by microorganisms of the group of phosphate-accumulating organisms (PAO) capable of intracellular accumulation of polyphosphates under cyclic growth conditions, with alternated presence and absence of electron acceptors43,44,45,46.
Typical PAO, most often found in WWTPs, are members of the candidate species Candidatus Accumulibacter phosphatis (family Rhodocyclaceae, Gammaproteobacteria)47,48,49,50. During the anaerobic phase, Ca. Accumulibacter phosphatis takes up volatile fatty acids present in the wastewater and stores the carbon from these substrates intracellularly as polyhydroxyalkanoates51. At the same time, intracellular polyphosphate is degraded to form ATP, releasing phosphate into the medium. During the aerobic phase, stored polyhydroxyalkanoates are used for energy production while phosphate is taken up from the medium and accumulated as polyphosphate. Ca. Accumulibacter phosphatis were found in all AS samples. Its relative abundance was the lowest (0.12%) in WWTP2 employing the nitrification/ denitrification process and poorly removing phosphorus. In other plants Ca. Accumulibacter phosphatis accounted for 0.43 to 1.05% of the communities and its share does not correlate with the phosphorus removal efficiency or the type of treatment process.
Several other bacteria, despite the difference in their metabolism from Ca. Accumulibacter phosphatis, are considered as likely PAO46, including members of Proteobacteria (Dechloromonas52,53, Zooglea54,55, Thauera56, Thiothrix57, Ca. Accumulimonas58, Malikia59), Actinobacteria (Tetrasphaera60,61, Microlunatus62, Candidatus Microthrix63), Gemmatimonadetes (Gemmatimonas64) and Melainabacteria (Candidatus Obscuribacter phosphatis65). Among these bacteria, members of the genera Tetrasphaera, Zoogloea, Thiothrix, Dechloromonas, Ca. Obscuribacter, Gemmatimonas, and Ca. Microthrix were detected.
The most abundant potential PAO, Ca. Microthrix sp., (average share 6.8%) was presented in all WWTPs and its share was higher in WWTPs employing the UCT process than in other types of bioreactors. Earlier it has been shown that Candidatus Microthrix parvicella contained of large polyphosphate granules and this microbial group might have been responsible for phosphorus removal during the sludge bulking period when Ca. Accumulibacter phosphatis was excluded from the system63. Dechloromonas sp., denitrifying bacteria capable of acetate uptake and polyphosphate storage, accounted for 1.5% to 5.8% of AS microbiomes in bioreactors operating under aerobic and UCT processes, while in WWTP2 it accounted for only 0.6%. Members of the genera Zoogloea and Thiothrix, aerobic bacteria capable of denitrification, often occur in ASs57,66,67. Both genera were more abundant in WWTPs employing aerobic process. Ca. Obscuribacter was most frequent in the WWTP6 (about 1.9%) and accounted for less than 1% in other samples. Members of the genus Tetrasphaera, known to be capable of aerobic polyphosphate accumulation under condition of assimilating glucose and/or amino acids anaerobically in advance68 were found in all WWTPs. Their relative abundance was minimal in WWTPs 8 and 9 (about 0.2%) and reached 1.35% in WWTP1. Although nitrate-reducing bacteria of the genus Thauera are primary known for their ability to perform anaerobic degradation of aromatic and other refractory compounds69, recently Thauera sp. strain SND5 was found to be a phosphate-accumulating organism56. The relative abundance of Thauera sp. ranged from 0.2 to 2.2% and was maximal in WWTP8.
Glycogen accumulating organisms (GAOs) can accumulate glycogen and polyhydroxyalkanoates inside cells, but do not have ability to intracellular accumulation of polyphosphates. They are commonly found together with PAO in EBPR bioreactors where their predominance may lead to EBPR failure due to the competition for carbon source with PAO70. Known GAO belongs to the Gammaproteobacteria (Candidatus Competibacter phosphatis) and Alphaproteobacteria (Defluvicoccus sp.)71,72. Ca. Competibacter accounted for up to 8.5% of the communities in UCT WWTPs, but were less numerous (< 1%) in WWTPs 2 and 7. The relative abundance of Defluvicoccus sp. was less than 0.1% in all samples. Thus, in WWTPs using the UCT process, a high relative abundance of GAO did not adversely affected the efficiency of phosphorus removal.
Microbial community composition and efficiency of wastewater purification
All five WWTPs using the UCT process ensured effective removal of not only organic matter, but also nitrogen and phosphorus, which is consistent with the high content of nitrifying and phosphate accumulating bacteria.
WWTP2, using the nitrification–denitrification process, effectively removed nitrogen, and the relative abundance of nitrifiers in this AS community was the highest. However, there was almost no phosphorus removal, and the share of PAO was the lowest. The low efficiency of phosphorus removal and the low abundance of PAO in WWTP2 are associated with the absence of anaerobic zones in bioreactors, as well as the consumption of easily degradable organic matter by denitrifying microorganisms.
Contrary to expectations, the two WWTPs, using a simple aeration process, provided the poorest removal of both organics and ammonia, while the nitrate content in the effluent was approximately the same as in other installations. However, the total nitrogen content in the form of nitrate and ammonium in the effluent was much lower than in the influent, indicating that nitrogen was removed during denitrification. Probably, under conditions of insufficient aeration, formation of local anoxic zones within microbial flocs facilitates the denitrification performed by heterotrophic bacteria73, while part of the ammonia and organic matter remain non-oxidized and enters the effluent water.
Interestingly, relatively efficient phosphorus removal was observed in WWTP7 but not in WWTP1. These two bioreactors differ in that in WWTP7 the organic-rich influent is fed to the beginning of the bioreactor, while in WWTP1 it is distributed along the entire length. It is likely that in WWTP7 local organic-rich anaerobic zones are formed within AS flocks near the point of influent supply, while in WWTP1 the concentration of organic substances is everywhere lower. In such organic-rich zones denitrifying PAO of the genera Zoogloea and Thiothrix can proliferate and their shares were maximal in WWTP7.
Other abundant community members
Analysis of the compositions of microbial communities revealed 16 genus-level lineages, the average share of which across nine WWTPs exceeds 1% (Supplementary Table S4). In total, these 16 genera accounted for 28 to 51% (on average about 36%) of analyzed microbiomes. The most abundant genus, Ca. Microthrix, was the most numerous in AS samples of WWTPs 4, 6, 8 and 9 (11—13%) using the UCT process, while in other samples its relative abundance was much lower (1.2—3.4%). Besides its potential role in phosphorus removal, Ca. Microthrix is known as a slowly growing filamentous bacterium causing bulking and foaming in activated sludge systems thus seriously affecting the stable operation of WWTPs74. The growth of cultured species of this genus, M. parvicella, is susceptible to both environmental and operational parameters, such as temperature, oxygen concentration, type of substrate, electron acceptor, organic loading, and sludge retention time (reviewed in75). Probably, enrichment of Ca. Microthrix in the AS of the UCT WWTPs was associated with low oxygen concentrations in the anaerobic and anoxic zones of these bioreactors, favorable for the growth of these bacteria7. The dominant Ca. Microthrix phylotype was identified as Ca Microthrix subdominans, a species typically present along with Ca. M. parvicella, although usually in lower abundances76.
Besides Ca. Microthrix and above mentioned Ca. Competibacter, Dechloromonas, Nitrospira, Nitrosomonas and Thauera, the list of most abundant genera includes 3 cultured (Trichococcus, Haliangium, and Chitinivorax) and 7 uncultured lineages, defined in the Silva database as OM27_clade (Bdellovibrionota, Bdellovibrionaceae), Candidatus Nomurabacteria (Patescibacteria), AKYH767 (Bacteroidota, Sphingobacteriales), a member of the family Moraxellaceae (Gammaproteobacteria) defined as “Agitococcus lubricus group’, mle1-27 (Myxococcota), env.OPS_17 (Bacteroidota, Sphingobacteriales), and OLB12 (Bacteroidota, Microscillaceae).
Members of the genera Trichococcus, Haliangium, and Chitinivorax are often found in AS of wastewater treatment facilities. Trichococcus sp. are filamentous bacteria that can degrade a wide range of carbohydrates77, while Chitinivorax sp. are devoted to the hydrolysis of chitin78. Similarly to Dechloromonas sp., members of the Haliangium are active denitrifiers in the AS communities79.
All uncultured genus-level lineages were previously detected in bioreactors treating wastewater80,81,82,83, but their functional roles remain elusive. The most abundant uncultured OM27_clade, with an average share of about 3%, was hypothesized to be predatory bacteria84. Members of the candidate genus OLB12 are aerobic heterotrophs abundant in the anammox granules collected from partial-nitritation anammox reactor85. The only cultured member of “Agitococcus lubricus group’, A. lubricus, is a lipolytic aerobic heterotroph found in freshwater bodies86. The relative abundance of this lineage reached 4% in WWTP2, while it was below 0.2% in all AS samples currently described in the MiDAS database14.
Network analysis
Various functional characteristics of microorganisms are closely related to the differentiation of ecological niches, and network analysis allows one to deduce patterns of interactions that develop within the WWTP ecosystem. Network analysis indicated possible cooperation (co-occurrence) and mutual exclusion among diverse microorganisms in WWTPs (Fig. 4).
Network of co-occurring abundant microbial OTUs of AS based on correlation analysis. A connection stands for a strong (Spearman’s rho > 0.6) and significant (adjusted p value < 0.001) correlation. OTUs are colored according to phylum. Co-presence and mutual exclusion of OTUs are shown in white and red lines, respectively.
Of 104 OTUs, with abundances more than 0.5% in at least one of the AS samples, 83 had statistically significant connections with other OTUs the maximum number of which reached 18 (Supplementary Table S5). OTUs with a high number of connections (“degree”) are considered as hub members, supposed to be key players in sustaining the overall community87. The largest number of connections (18) was observed for OTU assigned to the genus Haliangium, whose members, together with Dechloromonas sp., are active denitrifiers in WWTPs communities79. Notably, bacteria participating in ammonia and phosphorous removal were among the hub members, namely, Nitrosomonas, Nitrospira, Ca. Competibacter, Dechloromonas, and Thauera. Positive connection was detected between denitrifiers (Thauera Otu26 and Haliangium Otu95) and bacteria involved in the oxidation of ammonia (Nitrosomonas Otu18 and Nitrospira Otu2). This link is consistent with the fact that the source of nitrate is mainly microbial oxidation of ammonium, rather than influent sewage water. Interestingly, we did not detected mutual exclusion between Ca. Accumulibacter and Ca. Competibacter.
The nest largest number of connections was detected for Otu105, assigned to the candidate genus Sumerlaea (candidate phylum Sumerlaeota, previously known as BRC1). Members of this genus were predicted to be metabolically versatile bacteria capable to utilize various carbohydrates, including chitin, performing fermentation as well as complete oxidation of organic substrates through aerobic and anaerobic (with nitrate and sulfur compounds) respiration88. Co-presence of Sumerlaea and denitrifiers (Thauera and Haliangium) suggest that Sumerlaea sp. could be involved in degradation of organic substrates linked to dissimilatory nitrate reduction. Interestingly, no connection to other community members was found for the most abundant species, Ca. Microthrix (Otu1). Ca. Microthrix has been considered a specialized consumer of long-chain fatty acids that have been confirmed to be the key carbon and energy sources for its growth both under aerobic, anoxic and anaerobic conditions75,89. Probably due to such metabolic specialization Ca. Microthrix has limited interactions with other community members.
Microbial communities of Moscow WWTPs on a global scale
Variations in community composition are key points for understanding community functioning. To understand how the composition of microbial communities of AS varied across different geographic locations, we compared taxonomic structures of our microbiomes with that reported in a worldwide study performed by a Global Water Microbiome Consortium10. That study showed that although the taxonomic community structures observed on different continents were not clearly separated at the OTU level in two dimensional ordinations, it was significantly different between any two continents10, and although the type of treatment process exerted significant effects on microbial community structures, it was overwhelmed by continental geographical separation10.
Despite its high diversity, AS microbiome has a small global core bacterial community (28 OTUs) that is strongly linked to WWTP performance and accounted for an average 12.4% of the 16S rRNA gene sequences in AS samples10. 26 of these OTUs were present in Moscow WWTPs and accumulated from 11.3 to 23.5% of 16S rRNA gene reads (Supplementary Table S6).
Clustering based on the Bray–Curtis dissimilarity showed that all samples from Moscow WWTPs clustered together and formed a distinct branch not embedded into European or any other large groups, but forming a sister lineage to a subsets of Asian, South American and North American clusters (Fig. 5). These data indicate that influent characteristics, related to cultural, social and environmental factors in each region, could be more important than a plant operating conditions. Similar observations have been previously reported for the microbial communities of AS from WWTPs in Korea and Vietnam2. For example, due to the relatively low cost of water for household consumption, wastewater in Moscow has a relatively low content of organic matter compared to the values typical for most large cities (COD of 600–900 mg/L). It should be noted that the primers used in our work for the 16S rRNA gene profiling differed from those used by the GWMC consortium (515F/806R), which may affect the observed shares of particular microbial taxa. However, both primer pairs (515F/806R and 341F/806R) enabled good coverage of overall microbial diversity and provided similar estimates of the relative abundancies of most species in various environments90,91.
Neighbor joining tree illustrating clustering based on the Bray–Curtis dissimilarity between microbial communities of AS samples from 9 WWTPs obtained in this study and ones reported by a Global Water Microbiome Consortium (http://gwmc.ou.edu/). Samples are colored according to geographic location.
