Time-series metagenomics reveals changing protistan ecology of a temperate dimictic lake.

BACKGROUND: Protists, single-celled eukaryotic organisms, are critical to food web ecology, contributing to primary productivity and connecting small bacteria and archaea to higher trophic levels. Lake Mendota is a large, eutrophic natural lake that is a Long-Term Ecological Research site and among the world's best-studied freshwater systems. Metagenomic samples have been collected and shotgun sequenced from Lake Mendota for the last 20 years. Here, we analyze this comprehensive time series to infer changes to the structure and function of the protistan community and to hypothesize about their interactions with bacteria. RESULTS: Based on small subunit rRNA genes extracted from the metagenomes and metagenome-assembled genomes of microeukaryotes, we identify shifts in the eukaryotic phytoplankton community over time, which we predict to be a consequence of reduced zooplankton grazing pressures after the invasion of a invasive predator (the spiny water flea) to the lake. The metagenomic data also reveal the presence of the spiny water flea and the zebra mussel, a second invasive species to Lake Mendota, prior to their visual identification during routine monitoring. Furthermore, we use species co-occurrence and co-abundance analysis to connect the protistan community with bacterial taxa. Correlation analysis suggests that protists and bacteria may interact or respond similarly to environmental conditions. Cryptophytes declined in the second decade of the timeseries, while many alveolate groups (e.g., ciliates and dinoflagellates) and diatoms increased in abundance, changes that have implications for food web efficiency in Lake Mendota. CONCLUSIONS: We demonstrate that metagenomic sequence-based community analysis can complement existing efforts to monitor protists in Lake Mendota based on microscopy-based count surveys. We observed patterns of seasonal abundance in microeukaryotes in Lake Mendota that corroborated expectations from other systems, including high abundance of cryptophytes in winter and diatoms in fall and spring, but with much higher resolution than previous surveys. Our study identified long-term changes in the abundance of eukaryotic microbes and provided context for the known establishment of an invasive species that catalyzes a trophic cascade involving protists. Our findings are important for decoding potential long-term consequences of human interventions, including invasive species introduction. Video Abstract.

Using a standardized sound set to help characterize misophonia: The International Affective Digitized Sounds.

Misophonia is a condition characterized by negative affect, intolerance, and functional impairment in response to particular repetitive sounds usually made by others (e.g., chewing, sniffing, pen tapping) and associated stimuli. To date, researchers have largely studied misophonia using self-report measures. As the field is quickly expanding, assessment approaches need to advance to include more objective measures capable of differentiating those with and without misophonia. Although several studies have used sounds as experimental stimuli, few have used standardized stimuli sets with demonstrated reliability or validity. To conduct rigorous research in an effort to better understand misophonia, it is important to have an easily accessible, standardized set of acoustic stimuli for use across studies. Accordingly, in the present study, the International Affective Digitized Sounds (IADS-2), developed by Bradley and Lang (Bradley MM et al., 2007), were used to determine whether participants with misophonia responded to certain standardized sounds differently than a control group. Participants were 377 adults (132 participants with misophonia and 245 controls) recruited from an online platform to complete several questionnaires and respond to four probes (arousal, valence, similarity to personally-relevant aversive sounds, and sound avoidance) in response to normed pleasant, unpleasant, and neutral IADS-2 sounds. Findings indicated that compared to controls, participants with high misophonia symptoms rated pleasant and neutral sounds as significantly more (a) arousing and similar to trigger sounds in their everyday life, (b) unpleasant and (c) likely to be avoided in everyday life. For future scientific and clinical innovation, we include a ranked list of IADS-2 stimuli differentiating responses in those with and without misophonia, which we call the IADS-M.

Bacterial ecology and evolution converge on seasonal and decadal scales.

Ecology and evolution are distinct theories, but the short lifespans and large population sizes of microbes allow evolution to unfold along contemporary ecological time scales. To document this in a natural system, we collected a two-decade, 471-metagenome time series from a single site in a freshwater lake, which we refer to as the TYMEFLIES dataset. This massive sampling and sequencing effort resulted in the reconstruction of 30,389 metagenomic-assembled genomes (MAGs) over 50% complete, which dereplicated into 2,855 distinct genomes (>96% nucleotide sequence identity). We found both ecological and evolutionary processes occurred at seasonal time scales. There were recurring annual patterns at the species level in abundances, nucleotide diversities (π), and single nucleotide variant (SNV) profiles for the majority of all taxa. During annual blooms, we observed both higher and lower nucleotide diversity, indicating that both ecological differentiation and competition drove evolutionary dynamics. Overlayed upon seasonal patterns, we observed long-term change in 20% of the species' SNV profiles including gradual changes, step changes, and disturbances followed by resilience. Most abrupt changes occurred in a single species, suggesting evolutionary drivers are highly specific. Nevertheless, seven members of the abundant Nanopelagicaceae family experienced abrupt change in 2012, an unusually hot and dry year. This shift coincided with increased numbers of genes under selection involved in amino acid and nucleic acid metabolism, suggesting fundamental organic nitrogen compounds drive strain differentiation in the most globally abundant freshwater family. Overall, we observed seasonal and decadal trends in both interspecific ecological and intraspecific evolutionary processes. The convergence of microbial ecology and evolution on the same time scales demonstrates that understanding microbiomes requires a new unified approach that views ecology and evolution as a single continuum.

Refinement of the "Candidatus Accumulibacter" genus based on metagenomic analysis of biological nutrient removal (BNR) pilot-scale plants operated with reduced aeration.

Members of the "Candidatus Accumulibacter" genus are widely studied as key polyphosphate-accumulating organisms (PAOs) in biological nutrient removal (BNR) facilities performing enhanced biological phosphorus removal (EBPR). This diverse lineage includes 18 "Ca. Accumulibacter" species, which have been proposed based on the phylogenetic divergence of the polyphosphate kinase 1 (ppk1) gene and genome-scale comparisons of metagenome-assembled genomes (MAGs). Phylogenetic classification based on the 16S rRNA genetic marker has been difficult to attain because most "Ca. Accumulibacter" MAGs are incomplete and often do not include the rRNA operon. Here, we investigate the "Ca. Accumulibacter" diversity in pilot-scale treatment trains performing BNR under low dissolved oxygen (DO) conditions using genome-resolved metagenomics. Using long-read sequencing, we recovered medium- and high-quality MAGs for 5 of the 18 "Ca. Accumulibacter" species, all with rRNA operons assembled, which allowed a reassessment of the 16S rRNA-based phylogeny of this genus and an analysis of phylogeny based on the 23S rRNA gene. In addition, we recovered a cluster of MAGs that based on 16S rRNA, 23S rRNA, ppk1, and genome-scale phylogenetic analyses do not belong to any of the currently recognized "Ca. Accumulibacter" species for which we propose the new species designation "Ca. Accumulibacter jenkinsii" sp. nov. Relative abundance evaluations of the genus across all pilot plant operations revealed that regardless of the operational mode, "Ca. A. necessarius" and "Ca. A. propinquus" accounted for more than 40% of the "Ca. Accumulibacter" community, whereas the newly proposed "Ca. A. jenkinsii" accounted for about 5% of the "Ca. Accumulibacter" community.IMPORTANCEOne of the main drivers of energy use and operational costs in activated sludge processes is the amount of oxygen provided to enable biological phosphorus and nitrogen removal. Wastewater treatment facilities are increasingly considering reduced aeration to decrease energy consumption, and whereas successful BNR has been demonstrated in systems with minimal aeration, an adequate understanding of the microbial communities that facilitate nutrient removal under these conditions is still lacking. In this study, we used genome-resolved metagenomics to evaluate the diversity of the "Candidatus Accumulibacter" genus in pilot-scale plants operating with minimal aeration. We identified the "Ca. Accumulibacter" species enriched under these conditions, including one novel species for which we propose "Ca. Accumulibacter jenkinsii" sp. nov. as its designation. Furthermore, the MAGs obtained for five additional "Ca. Accumulibacter" species further refine the phylogeny of the "Ca. Accumulibacter" genus and provide new insight into its diversity within unconventional biological nutrient removal systems.

Laboratory measurements underestimate persistence of the aquatic herbicide fluridone in lakes.

Fluridone is an aquatic herbicide commonly used to treat invasive freshwater plant species such as Eurasian watermilfoil, hydrilla, and curly-leaf pondweed. However, required exposures times are very long and often exceed 100 days. Thus, understanding the mechanisms that determine the fate of fluridone in lakes is critical for supporting effective herbicide treatments and minimizing impacts to non-target species. We use a combination of laboratory and field studies to quantify fluridone photodegradation, as well as sorption and microbial degradation in water and sediment microcosms. Laboratory irradiation studies demonstrate that fluridone is susceptible to direct photodegradation with negligible indirect photodegradation, with predicted half-lives in sunlight ranging from 2.3 days (1 cm path length) to 118 days (integrated over 1 meter). Biodegradation is attributable to microbes in sediment with an observed half-life of 57 days. Lastly, fluridone sorbs to sediments (Koc = 340 ± 28 L kg-1); sorption accounts for 16% of fluridone loss in the microcosm experiments. While the laboratory results indicate that all three loss pathways can influence fluridone fate, these controlled studies oversimplify herbicide behavior due to their inability to replicate field conditions. Fluridone concentration measurements in a lake following commercial application demonstrate a half-life of >150 days, indicating that the herbicide is very persistent in water. This study illustrates why caution should be used when relying on laboratory studies to predict the fate of pesticides and other polar organic compounds in the environment.

Quantifying the Role of Simultaneous Transformation Pathways in the Fate of the Novel Aquatic Herbicide Florpyrauxifen-Benzyl.

Predicting the fate of organic compounds in the environment is challenging due to the inability of laboratory studies to replicate field conditions. We used the intentionally applied aquatic herbicide florpyrauxifen-benzyl (FPB) as a model compound to investigate the contribution of multiple transformation pathways to organic compound fate in lakes. FPB persisted in five Wisconsin lakes for 5-7 days with an in-lake half-life of <2 days. FPB formed four transformation products, with the bioactive product florpyrauxifen persisting up to 30 days post-treatment. Parallel laboratory experiments showed that FPB degrades to florpyrauxifen via base-promoted hydrolysis. Hydroxy-FPB and hydroxy-florpyrauxifen were identified as biodegradation products, while dechloro-FPB was identified as a photoproduct. Material balance calculations using both laboratory rates and field product concentrations demonstrated that hydrolysis (∼47% of loss), biodegradation (∼20%), sorption (∼13%), and photodegradation (∼4%) occurred on similar timescales. Furthermore, the combined results demonstrated that abiotic and plant-catalyzed hydrolysis of FPB to florpyrauxifen, followed by biodegradation of florpyrauxifen to hydroxy-florpyrauxifen, was the dominant transformation pathway in lakes. This study demonstrates how combined field and laboratory studies can be used to elucidate the role of simultaneous and interacting pathways in the fate of organic compounds in aquatic environments.

Metabolically diverse microorganisms mediate methylmercury formation under nitrate-reducing conditions in a dynamic hydroelectric reservoir.

Brownlee Reservoir is a mercury (Hg)-impaired hydroelectric reservoir that exhibits dynamic hydrological and geochemical conditions and is located within the Hells Canyon Complex in Idaho, USA. Methylmercury (MeHg) contamination in fish is a concern in the reservoir. While MeHg production has historically been attributed to sulfate-reducing bacteria and methanogenic archaea, microorganisms carrying the hgcA gene are taxonomically and metabolically diverse and the major biogeochemical cycles driving mercury (Hg) methylation are not well understood. In this study, Hg speciation and redox-active compounds were measured throughout Brownlee Reservoir across the stratified period in four consecutive years (2016-2019) to identify the location where and redox conditions under which MeHg is produced. Metagenomic sequencing was performed on a subset of samples to characterize the microbial community with hgcA and identify possible links between biogeochemical cycles and MeHg production. Biogeochemical profiles suggested in situ water column Hg methylation was the major source of MeHg. These profiles, combined with genome-resolved metagenomics focused on hgcA-carrying microbes, indicated that MeHg production occurs in this system under nitrate- or manganese-reducing conditions, which were previously thought to preclude Hg-methylation. Using this multidisciplinary approach, we identified the cascading effects of interannual variability in hydrology on the redox status, microbial metabolic strategies, abundance and metabolic diversity of Hg methylators, and ultimately MeHg concentrations throughout the reservoir. This work expands the known conditions conducive to producing MeHg and suggests that the Hg-methylation mitigation efforts by nitrate or manganese amendment may be unsuccessful in some locations.

Physiological and genomic evidence of cysteine degradation and aerobic hydrogen sulfide production in freshwater bacteria.

The sulfur-containing amino acid cysteine is abundant in the environment, including in freshwater lakes. Biological cysteine degradation can result in hydrogen sulfide (H2S), a toxic and ecologically relevant compound that is a central player in biogeochemical cycling in aquatic environments. Here, we investigated the ecological significance of cysteine in oxic freshwater, using isolated cultures, controlled experiments, and multiomics. We screened bacterial isolates enriched from natural lake water for their ability to produce H2S when provided cysteine. We identified 29 isolates (Bacteroidota, Proteobacteria, and Actinobacteria) that produced H2S. To understand the genomic and genetic basis for cysteine degradation and H2S production, we further characterized three isolates using whole-genome sequencing (using a combination of short-read and long-read sequencing) and tracked cysteine and H2S levels over their growth ranges: Stenotrophomonas maltophilia (Gammaproteobacteria), S. bentonitica (Gammaproteobacteria), and Chryseobacterium piscium (Bacteroidota). Cysteine decreased and H2S increased, and all three genomes had genes involved in cysteine degradation. Finally, to assess the presence of these organisms and genes in the environment, we surveyed a 5-year time series of metagenomic data from the same isolation source (Lake Mendota, Madison, WI, USA) and identified their presence throughout the time series. Overall, our study shows that diverse isolated bacterial strains can use cysteine and produce H2S under oxic conditions, and we show evidence using metagenomic data that this process may occur more broadly in natural freshwater lakes. Future considerations of sulfur cycling and biogeochemistry in oxic environments should account for H2S production from the degradation of organosulfur compounds. IMPORTANCE Hydrogen sulfide (H2S), a naturally occurring gas with both biological and abiotic origins, can be toxic to living organisms. In aquatic environments, H2S production typically originates from anoxic (lacking oxygen) environments, such as sediments, or the bottom layers of thermally stratified lakes. However, the degradation of sulfur-containing amino acids such as cysteine, which all cells and life forms rely on, can be a source of ammonia and H2S in the environment. Unlike other approaches for biological H2S production such as dissimilatory sulfate reduction, cysteine degradation can occur in the presence of oxygen. Yet, little is known about how cysteine degradation influences sulfur availability and cycling in freshwater lakes. In our study, we identified diverse bacteria from a freshwater lake that can produce H2S in the presence of O2. Our study highlights the ecological importance of oxic H2S production in natural ecosystems and necessitates a change in our outlook on sulfur biogeochemistry.

Species invasions shift microbial phenology in a two-decade freshwater time series.

Invasive species impart abrupt changes on ecosystems, but their impacts on microbial communities are often overlooked. We paired a 20 y freshwater microbial community time series with zooplankton and phytoplankton counts, rich environmental data, and a 6 y cyanotoxin time series. We observed strong microbial phenological patterns that were disrupted by the invasions of spiny water flea (Bythotrephes cederströmii) and zebra mussels (Dreissena polymorpha). First, we detected shifts in Cyanobacteria phenology. After the spiny water flea invasion, Cyanobacteria dominance crept earlier into clearwater; and after the zebra mussel invasion, Cyanobacteria abundance crept even earlier into the diatom-dominated spring. During summer, the spiny water flea invasion sparked a cascade of shifting diversity where zooplankton diversity decreased and Cyanobacteria diversity increased. Second, we detected shifts in cyanotoxin phenology. After the zebra mussel invasion, microcystin increased in early summer and the duration of toxin production increased by over a month. Third, we observed shifts in heterotrophic bacteria phenology. The Bacteroidota phylum and members of the acI Nanopelagicales lineage were differentially more abundant. The proportion of the bacterial community that changed differed by season; spring and clearwater communities changed most following the spiny water flea invasion that lessened clearwater intensity, while summer communities changed least following the zebra mussel invasion despite the shifts in Cyanobacteria diversity and toxicity. A modeling framework identified the invasions as primary drivers of the observed phenological changes. These long-term invasion-mediated shifts in microbial phenology demonstrate the interconnectedness of microbes with the broader food web and their susceptibility to long-term environmental change.

Environmental formation of methylmercury is controlled by synergy of inorganic mercury bioavailability and microbial mercury-methylation capacity.

Methylmercury (MeHg) production is controlled by the bioavailability of inorganic divalent mercury (Hg(II)i ) and Hg-methylation capacity of the microbial community (conferred by the hgcAB gene cluster). However, the relative importance of these factors and their interaction in the environment remain poorly understood. Here, metagenomic sequencing and a full-factorial MeHg formation experiment were conducted across a wetland sulfate gradient with different microbial communities and pore water chemistries. From this experiment, the relative importance of each factor on MeHg formation was isolated. Hg(II)i bioavailability correlated with the dissolved organic matter composition, while the microbial Hg-methylation capacity correlated with the abundance of hgcA genes. MeHg formation responded synergistically to both factors. Notably, hgcA sequences were from diverse taxonomic groups, none of which contained genes for dissimilatory sulfate reduction. This work expands our understanding of the geochemical and microbial constraints on MeHg formation in situ and provides an experimental framework for further mechanistic studies.

Diversity, distribution, and expression of opsin genes in freshwater lakes.

Microbial rhodopsins are widely distributed in aquatic environments and may significantly contribute to phototrophy and energy budgets in global oceans. However, the study of freshwater rhodopsins has been largely limited. Here, we explored the diversity, ecological distribution, and expression of opsin genes that encode the apoproteins of type I rhodopsins in humic and clearwater lakes with contrasting physicochemical and optical characteristics. Using metagenomes and metagenome-assembled genomes, we recovered opsin genes from a wide range of taxa, mostly predicted to encode green light-absorbing proton pumps. Viral opsin and novel bacterial opsin clades were recovered. Opsin genes occurred more frequently in taxa from clearwater than from humic water, and opsins in some taxa have nontypical ion-pumping motifs that might be associated with physicochemical conditions of these two freshwater types. Analyses of the surface layer of 33 freshwater systems revealed an inverse correlation between opsin gene abundance and lake dissolved organic carbon (DOC). In humic water with high terrestrial DOC and light-absorbing humic substances, opsin gene abundance was low and dramatically declined within the first few meters, whereas the abundance remained relatively high along the bulk water column in clearwater lakes with low DOC, suggesting opsin gene distribution is influenced by lake optical properties and DOC. Gene expression analysis confirmed the significance of rhodopsin-based phototrophy in clearwater lakes and revealed different diel expressional patterns among major phyla. Overall, our analyses revealed freshwater opsin diversity, distribution and expression patterns, and suggested the significance of rhodopsin-based phototrophy in freshwater energy budgets, especially in clearwater lakes.

A consensus protocol for the recovery of mercury methylation genes from metagenomes.

Mercury (Hg) methylation genes (hgcAB) mediate the formation of the toxic methylmercury and have been identified from diverse environments, including freshwater and marine ecosystems, Arctic permafrost, forest and paddy soils, coal-ash amended sediments, chlor-alkali plants discharges and geothermal springs. Here we present the first attempt at a standardized protocol for the detection, identification and quantification of hgc genes from metagenomes. Our Hg-cycling microorganisms in aquatic and terrestrial ecosystems (Hg-MATE) database, a catalogue of hgc genes, provides the most accurate information to date on the taxonomic identity and functional/metabolic attributes of microorganisms responsible for Hg methylation in the environment. Furthermore, we introduce "marky-coco", a ready-to-use bioinformatic pipeline based on de novo single-metagenome assembly, for easy and accurate characterization of hgc genes from environmental samples. We compared the recovery of hgc genes from environmental metagenomes using the marky-coco pipeline with an approach based on coassembly of multiple metagenomes. Our data show similar efficiency in both approaches for most environments except those with high diversity (i.e., paddy soils) for which a coassembly approach was preferred. Finally, we discuss the definition of true hgc genes and methods to normalize hgc gene counts from metagenomes.

Environmental predictors of electroactive bacterioplankton in small boreal lakes.

Extracellular electron transfer (EET) by electroactive bacteria in anoxic soils and sediments is an intensively researched subject, but EET's function in planktonic ecology has been less considered. Following the discovery of an unexpectedly high prevalence of EET genes in a bog lake's bacterioplankton, we hypothesized that the redox capacities of dissolved organic matter (DOM) enrich for electroactive bacteria by mediating redox chemistry. We developed the bioinformatics pipeline FEET (Find EET) to identify and summarize predicted EET protein-encoding genes from metagenomics data. We then applied FEET to 36 bog and thermokarst lakes and correlated gene occurrence with environmental data to test our predictions. Our results provide indirect evidence that DOM may participate in bacterioplankton EET. We found a similarly high prevalence of genes encoding putative EET proteins in most of these lakes, where oxidative EET strongly correlated with DOM. Numerous novel clusters of multiheme cytochromes that may enable EET were identified. Taxa previously not considered EET-capable were found to carry EET genes. We propose that EET and DOM interactions are of ecologically important to bacterioplankton in small boreal lakes, and that EET, particularly by methylotrophs and anoxygenic phototrophs, should be further studied and incorporated into methane emission models of melting permafrost.

Synthesizing Laboratory and Field Experiments to Quantify Dominant Transformation Mechanisms of 2,4-Dichlorophenoxyacetic Acid (2,4-D) in Aquatic Environments.

Laboratory studies used to assess the environmental fate of organic chemicals such as pesticides fail to replicate environmental conditions, resulting in large errors in predicted transformation rates. We combine laboratory and field data to identify the dominant loss processes of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) in lakes for the first time. Microbial and photochemical degradation are individually assessed using laboratory-based microcosms and irradiation studies, respectively. Field campaigns are conducted in six lakes to quantify 2,4-D loss following large-scale herbicide treatments. Irradiation studies show that 2,4-D undergoes direct photodegradation, but modeling efforts demonstrated that this process is negligible under environmental conditions. Microcosms constructed using field inocula show that sediment microbial communities are responsible for degradation of 2,4-D in lakes. Attempts to quantify transformation products are unsuccessful in both laboratory and field studies, suggesting that their persistence is not a major concern. The synthesis of laboratory and field experiments is used to demonstrate best practices in designing laboratory persistence studies and in using those results to mechanistically predict contaminant fate in complex aquatic environments.

Reevaluation of the Phylogenetic Diversity and Global Distribution of the Genus "Candidatus Accumulibacter".

"Candidatus Accumulibacter" was the first microorganism identified as a polyphosphate-accumulating organism (PAO) important for phosphorus removal from wastewater. Members of this genus are diverse, and the current phylogeny and taxonomic framework appear complicated, with most publicly available genomes classified as "Candidatus Accumulibacter phosphatis," despite notable phylogenetic divergence. The ppk1 marker gene allows for a finer-scale differentiation into different "types" and "clades"; nevertheless, taxonomic assignments remain inconsistent across studies. Therefore, a comprehensive reevaluation is needed to establish a common understanding of this genus, in terms of both naming and basic conserved physiological traits. Here, we provide this reassessment using a comparison of genome, ppk1, and 16S rRNA gene-based approaches from comprehensive data sets. We identified 15 novel species, along with "Candidatus Accumulibacter phosphatis," "Candidatus Accumulibacter delftensis," and "Candidatus Accumulibacter aalborgensis." To compare the species in situ, we designed new species-specific fluorescence in situ hybridization (FISH) probes and revealed their morphology and arrangement in activated sludge. Based on the MiDAS global survey, "Ca. Accumulibacter" species were widespread in wastewater treatment plants (WWTPs) with phosphorus removal, indicating process design as a major driver for their abundance. Genome mining for PAO-related pathways and FISH-Raman microspectroscopy confirmed the potential for PAO metabolism in all "Ca. Accumulibacter" species, with detection in situ of the typical PAO storage polymers. Genome annotation further revealed differences in the nitrate/nitrite reduction pathways. This provides insights into the niche differentiation of these lineages, potentially explaining their coexistence in the same ecosystem while contributing to overall phosphorus and nitrogen removal. IMPORTANCE "Candidatus Accumulibacter" is the most studied PAO, with a primary role in biological nutrient removal. However, the species-level taxonomy of this lineage is convoluted due to the use of different phylogenetic markers or genome sequencing approaches. Here, we redefined the phylogeny of these organisms, proposing a comprehensive approach which could be used to address the classification of other diverse and uncultivated lineages. Using genome-resolved phylogeny, compared to phylogeny based on the 16S rRNA gene and other phylogenetic markers, we obtained a higher-resolution taxonomy and established a common understanding of this genus. Furthermore, genome mining of genes and pathways of interest, validated in situ by application of a new set of FISH probes and Raman microspectroscopy, provided additional high-resolution metabolic insights into these organisms.

Pilot-scale comparison of biological nutrient removal (BNR) using intermittent and continuous ammonia-based low dissolved oxygen aeration control systems.

Sensor driven aeration control strategies have recently been developed as a means to efficiently carry out biological nutrient removal (BNR) and reduce aeration costs in wastewater treatment plants. Under load-based aeration control, often implemented as ammonia-based aeration control (ABAC), airflow is regulated to meet desired effluent standards without specifically setting dissolved oxygen (DO) targets. Another approach to reduce aeration requirements is to constantly maintain low DO conditions and allow the microbial community to adapt to the low-DO environment. In this study, we compared the performance of two pilot-scale BNR treatment trains that simultaneously used ABAC and low-DO operation to evaluate the combination of these two strategies. One pilot plant was operated with continuous ABAC while the other one used intermittent ABAC. Both processes achieved greater than 90% total Kjehldal nitrogen (TKN) removal, 60% total nitrogen removal, and nearly 90% total phosphorus removal. Increasing the solids retention time (SRT) during the period of cold (∼12 °C) water temperatures helped maintain ammonia removal performance under low-DO conditions. However, both processes experienced poor solids settling characteristics during winter. While settling was recovered under warmer temperatures, improving settling quality remains a challenge under low-DO operation.

Prospects for multi-omics in the microbial ecology of water engineering.

Advances in high-throughput sequencing technologies and bioinformatics approaches over almost the last three decades have substantially increased our ability to explore microorganisms and their functions - including those that have yet to be cultivated in pure isolation. Genome-resolved metagenomic approaches have enabled linking powerful functional predictions to specific taxonomical groups with increasing fidelity. Additionally, related developments in both whole community gene expression surveys and metabolite profiling have permitted for direct surveys of community-scale functions in specific environmental settings. These advances have allowed for a shift in microbiome science away from descriptive studies and towards mechanistic and predictive frameworks for designing and harnessing microbial communities for desired beneficial outcomes. Water engineers, microbiologists, and microbial ecologists studying activated sludge, anaerobic digestion, and drinking water distribution systems have applied various (meta)omics techniques for connecting microbial community dynamics and physiologies to overall process parameters and system performance. However, the rapid pace at which new omics-based approaches are developed can appear daunting to those looking to apply these state-of-the-art practices for the first time. Here, we review how modern genome-resolved metagenomic approaches have been applied to a variety of water engineering applications from lab-scale bioreactors to full-scale systems. We describe integrated omics analysis across engineered water systems and the foundations for pairing these insights with modeling approaches. Lastly, we summarize emerging omics-based technologies that we believe will be powerful tools for water engineering applications. Overall, we provide a framework for microbial ecologists specializing in water engineering to apply cutting-edge omics approaches to their research questions to achieve novel functional insights. Successful adoption of predictive frameworks in engineered water systems could enable more economically and environmentally sustainable bioprocesses as demand for water and energy resources increases.

Investigating the Chemolithoautotrophic and Formate Metabolism of Nitrospira moscoviensis by Constraint-Based Metabolic Modeling and 13C-Tracer Analysis.

Nitrite-oxidizing bacteria belonging to the genus Nitrospira mediate a key step in nitrification and play important roles in the biogeochemical nitrogen cycle and wastewater treatment. While these organisms have recently been shown to exhibit metabolic flexibility beyond their chemolithoautotrophic lifestyle, including the use of simple organic compounds to fuel their energy metabolism, the metabolic networks controlling their autotrophic and mixotrophic growth remain poorly understood. Here, we reconstructed a genome-scale metabolic model for Nitrospira moscoviensis (iNmo686) and used flux balance analysis to evaluate the metabolic networks controlling autotrophic and formatotrophic growth on nitrite and formate, respectively. Subsequently, proteomic analysis and [13C]bicarbonate and [13C]formate tracer experiments coupled to metabolomic analysis were performed to experimentally validate model predictions. Our findings corroborate that N. moscoviensis uses the reductive tricarboxylic acid cycle for CO2 fixation, and we also show that N. moscoviensis can indirectly use formate as a carbon source by oxidizing it first to CO2 followed by reassimilation, rather than direct incorporation via the reductive glycine pathway. Our study offers the first measurements of Nitrospira's in vivo central carbon metabolism and provides a quantitative tool that can be used for understanding and predicting their metabolic processes. IMPORTANCE Nitrospira spp. are globally abundant nitrifying bacteria in soil and aquatic ecosystems and in wastewater treatment plants, where they control the oxidation of nitrite to nitrate. Despite their critical contribution to nitrogen cycling across diverse environments, detailed understanding of their metabolic network and prediction of their function under different environmental conditions remains a major challenge. Here, we provide the first constraint-based metabolic model of Nitrospira moscoviensis representing the ubiquitous Nitrospira lineage II and subsequently validate this model using proteomics and 13C-tracers combined with intracellular metabolomic analysis. The resulting genome-scale model will serve as a knowledge base of Nitrospira metabolism and lays the foundation for quantitative systems biology studies of these globally important nitrite-oxidizing bacteria.

Metabolic Differentiation of Co-occurring Accumulibacter Clades Revealed through Genome-Resolved Metatranscriptomics.

Natural microbial communities consist of closely related taxa that may exhibit phenotypic differences and inhabit distinct niches. However, connecting genetic diversity to ecological properties remains a challenge in microbial ecology due to the lack of pure cultures across the microbial tree of life. "Candidatus Accumulibacter phosphatis" (Accumulibacter) is a polyphosphate-accumulating organism that contributes to the enhanced biological phosphorus removal (EBPR) biotechnological process for removing excess phosphorus from wastewater and preventing eutrophication from downstream receiving waters. Distinct Accumulibacter clades often coexist in full-scale wastewater treatment plants and laboratory-scale enrichment bioreactors and have been hypothesized to inhabit distinct ecological niches. However, since individual strains of the Accumulibacter lineage have not been isolated in pure culture to date, these predictions have been made solely on genome-based comparisons and enrichments with varying strain compositions. Here, we used genome-resolved metagenomics and metatranscriptomics to explore the activity of coexisting Accumulibacter strains in an engineered bioreactor environment. We obtained four high-quality genomes of Accumulibacter strains that were present in the bioreactor ecosystem, one of which is a completely contiguous draft genome scaffolded with long Nanopore reads. We identified core and accessory genes to investigate how gene expression patterns differed among the dominating strains. Using this approach, we were able to identify putative pathways and functions that may confer distinct functions to Accumulibacter strains and provide key functional insights into this biotechnologically significant microbial lineage. IMPORTANCE "Candidatus Accumulibacter phosphatis" is a model polyphosphate-accumulating organism that has been studied using genome-resolved metagenomics, metatranscriptomics, and metaproteomics to understand the EBPR process. Within the Accumulibacter lineage, several similar but diverging clades are defined by the shared sequence identity of the polyphosphate kinase (ppk1) locus. These clades are predicted to have key functional differences in acetate uptake rates, phage defense mechanisms, and nitrogen-cycling capabilities. However, such hypotheses have largely been made based on gene content comparisons of sequenced Accumulibacter genomes, some of which were obtained from different systems. Here, we performed time series genome-resolved metatranscriptomics to explore gene expression patterns of coexisting Accumulibacter clades in the same bioreactor ecosystem. Our work provides an approach for elucidating ecologically relevant functions based on gene expression patterns between closely related microbial populations.