Genome-centric metagenomics insights into functional divergence and horizontal gene transfer of denitrifying bacteria in anammox consortia.

Denitrifying bacteria with high abundances in anammox communities play crucial roles in achieving stable anammox-based systems. Despite the relative constant composition of denitrifying bacteria, their functional diversity remains to be explored in anammox communities. Herein, a total of 77 high-quality metagenome-assembled genomes (MAGs) of denitrifying bacteria were recovered from the anammox community in a full-scale swine wastewater treatment plant. Among these microbes, a total of 26 MAGs were affiliated with the seven dominant denitrifying genera that have total abundances higher than 1%. A meta-analysis of these species suggested that external organics reduced the abundances of genus Ignavibacterium and species MAG.305 of UTPRO2 in anammox communities. Comparative genome analysis revealed functional divergence across different denitrifying bacteria, largely owing to their distinct capabilities for carbohydrate (including endogenous and exogenous) utilization and vitamin (e.g., pantothenate and thiamine) biosynthesis. Serval microbes in this system contained fewer genes encoding biotin, pantothenate and methionine biosynthesis compared with their related species from other habitats. In addition, the genes encoding energy production and conversion (73 genes) and inorganic ion transport (53 genes) putatively transferred from other species to denitrifying bacteria, while these denitrifying bacteria (especially genera UTPRO2 and SCN-69-89) likely donated the genes encoding nutrients (e.g., inorganic ion and amino acid) transporter (64 genes) for other members to utilize new metabolites. Collectively, these findings highlighted the functional divergence of these denitrifying bacteria and speculated that the genetic interactions within anammox communities through horizontal gene transfer may be one of the reasons for their functional divergence.

State-Space-Based Framework for Predicting Microbial Interaction Variability in Wastewater Treatment Plants.

Substantial attempts have been made to control microbial communities for environmental integrity, biosystem performance, and human health. However, it is difficult to manipulate microbial communities in practice due to the varying and nonlinear nature of interspecific interaction networks. Here, we develop a manifold-based framework to investigate the patterns of microbial interaction variability in wastewater treatment plants using manifold geometric properties and design a simple control strategy to manipulate the microbes in nonlinear communities. We validate our framework using the readily available and nonsequential microbiome profiles of wastewater treatment plants. Our results show that some microbes in the activated sludge and anammox communities display deterministic rival or cooperative relationships and constitute a stable subnetwork within the whole nonlinear community network. We further use a simulation to demonstrate that these microbes can be used to drive a microbe in a target direction regardless of the community dynamics. Overall, our framework can provide a time-efficient solution to select effective control inputs for reliable manipulation in varying microbial networks, opening up new possibilities across a range of biological fields, including wastewater treatment plants.

The counteraction of anammox community to long-term nitrite stress: Crucial roles of rare subcommunity.

Understanding the temporal dynamics and recovery of anammox community under nitrite stress is critical for successful application of anammox-related processes. Here, the response behaviors of anammox community were investigated to characterize the reactor performance and ecological function under varied levels of nitrite stress (changing from 0, 50, 100, 200 to 0 mg-N/L) across a large temporal scale (588 days). The nitrogen removal rates decreased from 0.51 ± 0.02 to 0.16 ± 0.04 kg-N/(m3·d) under nitrite stress from 0 to 200 mg-N/L, while it was recovered to 0.29 ± 0.06 kg-N/(m3·d) as nitrite stress terminated. A strong community succession was driven by the initial nitrite stress of 50 mg-N/L, while the community dissimilarity mainly resulted from the increased beta diversity of rare subcommunity. Meanwhile, the rare subcommunity with high functional redundancy likely warranted the functional resilience of anammox community across the nitrite stress gradients. Moreover, the increased positive interactions between anammox bacteria and side populations supported the resilience of anammox after discontinuing nitrite stress, which facilitated the recovery of nitrogen removal efficiency. This study deciphers the interspecies interactions and functional redundancy of rare subcommunity in shaping the robustness and resilience of anammox-related processes when treating nitrite fluctuated wastewater.

Varied interspecies interactions between anammox and denitrifying bacteria enhanced nitrogen removal in a single-stage simultaneous anammox and denitrification system.

The simultaneous anammox and denitrification (SAD) system has received growing interest for the enhanced nitrogen removal, while the ecological traits of microbial community including spatial distribution characteristics, assembly processes and interspecies interactions have not been fully unraveled. The present study applied metagenomics and ecological analysis methods to gain the ecological traits of microbial communities in the SAD system across different organic substrate loadings. Results showed that organic matter significantly affected the bioreactor performance, and the optimal total nitrogen removal efficiency reached 93.4 ± 0.7% under the COD concentrations of 180 ± 18.2 mg/L. Functional organisms including Candidatus Brocadia (3.9%), Denitratisoma (1.6%), Dokdonella (4.4%) and Thauera (4.6%) obviously enriched under the optimal organic loading conditions. Moreover, microbial communities were significantly governed by deterministic process under high organic concentrations, and the denitrifying organisms displayed important ecological roles in the communities. Although anammox bacteria obviously enriched at the middle of bioreactor, it possessed the highest expression activities at both bottom and middle sites. Denitrifying bacteria that enriched at the bottom sites strongly achieved nitrate reduction and provided nitrite for anammox bacteria, while these organisms trended to compete nitrite with anammox bacteria at the middle site. These findings highlight the importance of microbial ecology in the SAD systems, which may expand our understanding of the synergistic patterns between anammox and denitrifying bacteria.

Biochemical characteristics and membrane fouling behaviors of soluble microbial products during the lifecycle of Escherichia coli.

The complexity of production process and chemical compositions of soluble microbial products (SMPs) largely limits the understanding of membrane fouling in membrane bioreactors (MBRs). Herein, we used a model single-strain Escherichia coli to better understand the chemical natures of SMPs and their roles in membrane fouling. The effects of carbon source and growth phase on the chemical compositions of SMPs were identified at both the compound and molecular levels by using advanced techniques including excitation emission matrix and parallel factor analysis (EEM-PARAFAC), size exclusion chromatography coupled with organic carbon detection (LC-OCD), and untargeted ultra-performance liquid chromatography - Q-Exactive - mass spectrometry (UPLC-Q-Exactive-MS). Subsequently, the roles of SMPs in the propensity of membrane fouling during ultrafiltration (UF) were studied. The results showed that the chemical compositions and fouling potentials of SMPs were carbon source- and growth phase-dependent. In the exponential phase, SMPs mainly consisted of utilization-associated products (UAPs) and remaining substrates. As the microorganism progressed into the stationary and senescent phases, UAPs and biomass-associated products (BAPs) were the main components, respectively. The SMP contents generated in glucose medium were higher than those generated in acetate medium, and higher abundances of humic fluorescent components were observed in glucose-fed SMPs. Van Krevelen diagrams of the UPLC-MS results revealed that acetate-fed SMPs contained more carboxylic-rich alicyclic molecules, peptides-like, aromatic, and carbohydrates-like components than glucose-fed SMPs in the stationary and senescent phases. These components played a significant role in irreversible membrane fouling, as evidenced in UF experiments. Standard blocking and cake filtration were the main fouling mechanisms for the filtration of SMPs collected in the exponential and stationary/senescent phases, respectively. Our findings highlight linkages between SMP compositions and membrane fouling at both the compound and molecular levels and suggest that both the carbon source and growth phase strongly determine the production potential, chemical nature, and fouling behavior of SMPs.

Metabolome responses of Enterococcus faecium to acid shock and nitrite stress.

Enterococcus faecium is gaining increasing interest due to its virulence and tolerance to a range of stresses (e.g., acid shock and nitrite stress in human stomach). The chemical taxonomy and basic structural features of cellular metabolite can provide us a deeper understanding of bacterial tolerance at molecular level. Here, we used hierarchical classification and molecular composition analysis to investigate the metabolome responses of E. faecium to acid shock and nitrite stress. Our results showed that considerable high biodegradable compounds (e.g., dipeptides) were produced by E. faecium under acid shock, while nitrite stress induced the accumulations of some low biodegradable compounds (e.g., organoheterocyclic compounds and benzenoids). Complete genome analysis and metabolic pathway profiling suggested that E. faecium produced high biodegradable metabolites responsible for the proton-translocation and biofilm formation, which increase its tolerance to acid shock. Yet, the presence of low biodegradable metabolites due to the nitrite exposure could disturb the bacterial productions of surface proteins, and thus inhibiting biofilm formation. Our approach uncovered the hidden interactions between intracellular metabolites and exogenous stress, and will improve the understanding of host-microbe interactions.

Impacts of diel temperature variations on nitrogen removal and metacommunity of anammox biofilm reactors.

The influence of diel temperature variations (DTVs) on nitrogen removal and bacterial communities was investigated in two parallel anammox reactors (i.e., control and DTV reactors). The control reactor was operated at a constant temperature of 30 °C, whereas the DTV reactor was operated in a temperature fluctuation mode with a cycle of 12/12 h of high/low temperatures. Nine water temperature variations for the day/night periods were set from 30/30 °C (i.e., Δ0 °C) to 38/22 °C (i.e., Δ16 °C). An increase in DTVs from Δ8 °C (34/26 °C) to Δ16 °C (38/22 °C) caused a significant decline in reactor performance and a shift in bacterial diversity. Compared to the control reactor, for instance, nitrogen removal efficiency decreased (P < 0.05) when temperature fluctuations exceeded Δ8 °C in the DTV reactor with a decreasing ΔNO3-/ΔNH4+ ratio (from 0.21 ±â€¯0.15 to 0.16 ±â€¯0.04). The results of 16S rRNA gene sequencing showed that the initial disturbance of temperature variations led to increased levels of bacterial diversity (i.e., alpha diversity) and decreased community levels of anammox consortia whereas they slightly recovered at the end of each DTV phase. Notably, Candidatus Jettenia was more sensitive to strong water temperature fluctuations, with the lower relative abundance at Δ14 °C (17.11 ±â€¯5.01%) and Δ16 °C (17.83 ±â€¯7.22%) than at Δ4 °C (39.82 ±â€¯0.01%). In contrast, Ca. Brocadia and Ca. Kuenenia had higher relative abundance at Δ14 °C (i.e., 0.24 ±â€¯0.07% and 0.09 ± 0.02%, respectively) and Δ16 °C (i.e., 0.28 ±â€¯0.05% and 0.12 ± 0.03%, respectively) compared to that at Δ4 °C (i.e., 0.15 ±â€¯0.04% and 0.04 ± 0.01%, respectively). Nitrifiers (i.e., unidentified_Nitrospiraceae and Nitrosomonas) and denitrifiers (i.e., Denitratisoma) were also capable of tolerating high temperature perturbations. Overall this study furthers our knowledge of responses of the microbial ecology of anammox bacteria to DTVs in anammox processes, which could aid us in optimizing anammox-related wastewater treatment systems and in understanding the nitrogen cycles of natural ecosystems.

Changes in nitrogen removal and microbiota of anammox biofilm reactors under tetracycline stress at environmentally and industrially relevant concentrations.

Anammox-related processes are often applied for the wastewater treatment which contains both ammonium and antibiotics. Herein, the long-term effects of tetracycline (TC), at environmentally and industrially relevant concentrations, on the performance, anammox activity and microbial community of anammox reactors were investigated for 518 days. The control reactor (without TC exposure) was stable for nitrogen removal during the long-term operation (a nitrogen removal rate of 0.56 ±â€¯0.05 kg-N·m-3·d-1). In the TC-added reactor, the nitrogen removal efficiency increased slightly at low TC levels (1-100 µg/L), whereas poor anammox performance occurred at high TC concentration (1000 µg/L). Furthermore, the concentrations of extracellular polymeric substances (EPS) were much higher at 10 µg/L than those in the control reactor (P < 0.01), whereas rapidly decreased at 1000 µg-TC/L. Furthermore, the reactor performance was highly consistent with the variations of the heme c contents. Consistently, exposure to TC changed the abundance of anammox bacteria, e.g., an increase in Candidatus Jettenia abundance occurred from 2.20 ±â€¯0.97% (0-10 µg/L) to 12.13 ±â€¯1.66% (100 µg/L). Similarly, the genus Denitratisoma, the most predominant denitrification bacteria, also had a higher abundance at a TC concentration of 100 µg/L (15.60 ±â€¯6.42%) than other TC concentrations (5.40 ±â€¯2.50% and 7.65 ±â€¯0.55% at concentrations of 10 and 1000 µg/L, respectively). The results can explain why the exposure of anammox bacteria to a lower TC concentration (100 µg/L) resulted in a better nitrogen removal rate. In contrast, exposure to a high TC level (1000 µg/L) led to a decline in the abundance of anammox bacteria and denitrifiers (1.53 ±â€¯0.64% and 8.18 ±â€¯0.63%, respectively) but an increased abundance in the nitrifier population (8.07 ±â€¯1.21%; P < 0.01). Therefore, this study can aid in the design and operation of anammox-based processes treating sewage and industrial wastewater.

Linking Exoproteome Function and Structure to Anammox Biofilm Development.

Extracellular proteins are of paramount importance in the cell-cell interactions of anammox biofilms. However, the inherent aggregation mechanisms of anammox have largely remained elusive. Herein, using a quartz sand extraction protocol and follow-up iTRAQ-based quantitative proteomics, we identified 367 extracellular proteins from initial colonizers, mature biofilm, and detached biofilm. The extracellular proteins were mainly membrane-associated. Most of the recovered proteins (226, 72.5%) originated from the phylum Planctomycetes. In summary, 215 and 190 of the 367 proteins recovered were up- and/or downregulated at least 1.2-fold during the biofilm formation and detachment periods, respectively. These differentially expressed proteins were dominantly involved in metal ion binding, which was regarded as strong evidence for their abilities to enhance ionic bridges in extracellular polymeric substances (EPS). Scanning electron microscopy-energy-dispersive X-ray spectroscopy (SEM-EDX) analysis of the biofilms further showed substantial levels of calcium and iron minerals. Critically, representative Sec-dependent secretory proteins affiliated with coccoid Planctomycetes, rod-shaped Proteobacteria, and filamentous Chloroflexi (11, 4, and 2 with differential expression, respectively) were found to have typical and abundant inner ß-sheet structures, wherein hydrophobic moieties can promote anammox aggregation. Overall, these findings highlight links between differentially expressed protein functions and morphologic traits of anammox consortia during biofilm development.

Increased salinity triggers significant changes in the functional proteins of ANAMMOX bacteria within a biofilm community.

Anaerobic ammonium oxidation (ANAMMOX) processes can potentially be influenced by salinity related to variable salinity in water environment. Here, we used 16S rRNA sequencing analysis combining with iTRAQ-based quantitative proteomic approach to reveal the response of microbial community and functional proteins to salinity, which was increased from 0 to 20 g L-1 with a step of 5 g L-1 (designed as S5, S10, S15 and S20) compared to control reactor (without salinity stress desined as S0). The 16S rRNA sequencing analysis showed that a high salinity (20 g L-1, S20) decreased the abundance of genus Candidatus Jettenia but increased that of Candidatus Kuenenia. A total of 1609 differentially expressed proteins were acquired in the three comparison groups (S5:S0, S20:S0 and S20:S5). Of these, 39 proteins co-occurred in the three salt-exposed samples. Hydrazine dehydrogenase (HDH; Q1PW30) and nitrate reductase (Q1PZD8) were up-regulated more than 3-folds in the exposure of 20 g-NaCl/L. The functional enrichment analysis further showed that some proteins responsible for ion binding, catalysis and oxidation-reduction reaction were up-regulated, which explained the physiological resilience of ANAMMOX bacteria under salinity stress. Additionally, ANAMMOX bacteria responded to salinity by modulating the electron transport systems, indicating that the cells retained a high potential for proton pumping, as well as the ATP production. Furthermore, the over-expression of HDH which associated with ANAMMOX metabolism, was potentially related to the increased abundance of halophilic Candidatus Kuenenia. These findings provide a comprehensive baseline for understanding the roles of salinity stresses in shaping the functional proteins of ANAMMOX bacteria.

Differential ultraviolet-visible absorbance spectra for characterizing metal ions binding onto extracellular polymeric substances in different mixed microbial cultures.

Ultraviolet-visible (UV-vis) absorbance spectra was adopted to quantify the binding of major metal ions (e.g., Na(I), Ca((II)), Fe(III), Cu(II), and Pb(II)) on extracellular polymeric substances (EPSs) extracted from different mixed cultures. The results showed that the differential absorbance spectra (DAS) provided discernible features for revealing the changes in optical properties of EPSs induced by metals, i.e., the intensity of DAS increased largely with incrementally increased metal concentrations (Fe(III), Cu(II), and Pb(II)). It can be assumed attributable to the changes in the conformations and inter-chromophores of the EPS biomolecules. In addition, the changes in spectral parameters of DSlope325-375 (spectral slope in the range of wavelengths 325-375 nm) and DA300 (differential absorbance at 300 nm) were found to be closely related to the amounts of metals bound onto all extracted EPSs, particularly for Fe(III) and Cu(II). The decreased SR (the ratio of slope275-295 to slope350-400) of the EPS solutions after dosage of metals suggested increased molecular weight or size of the EPS biomolecules. Deconvolution of the DAS yielded six Gaussian bands, which were present in all of the EPS samples with various metals. Moreover, the relative contributions of different Gaussian bands in the DAS were determined by the nature of EPS-metal ions interactions good correlated with the covalent-bonding index. This study concluded that DAS and selected spectral parameters (DA300, DSlope325-375 and SR) can be used to successfully characterize the binding of metals onto EPS at environmentally relevant concentrations.