Mass-immigration determines the assembly of activated sludge microbial communities.

The assembly of bacterial communities in wastewater treatment plants (WWTPs) is affected by immigration via wastewater streams, but the impact and extent of bacterial immigrants are still unknown. Here, we quantify the effect of immigration at the species level in 11 Danish full-scale activated sludge (AS) plants. All plants have different source communities but have very similar process design, defining the same overall environmental growth conditions. The AS community composition in each plant was strongly reflected by the corresponding influent wastewater (IWW) microbial composition. Most species in AS across the plants were detected and quantified in the corresponding IWW, allowing us to identify their fate in the AS: growing, disappearing, or surviving. Most of the abundant species in IWW disappeared in AS, so their presence in the AS biomass was only due to continuous mass-immigration. In AS, most of the abundant growing species were present in the IWW at very low abundances. We predicted the AS species abundances from their abundance in IWW by using a partial least square regression model. Some species in AS were predicted by their own abundance in IWW, while others by multiple species abundances. Detailed analyses of functional guilds revealed different prediction patterns for different species. We show, in contrast to the present understanding, that the AS microbial communities were strongly controlled by the IWW source community and could be quantitatively predicted by taking into account immigration. This highlights a need to revise the way we understand, design, and manage the microbial communities in WWTPs.

Toward a Universal Unit for Quantification of Antibiotic Resistance Genes in Environmental Samples.

Surveillance of antibiotic resistance genes (ARGs) has been increasingly conducted in environmental sectors to complement the surveys in human and animal sectors under the "One-Health" framework. However, there are substantial challenges in comparing and synthesizing the results of multiple studies that employ different test methods and approaches in bioinformatic analysis. In this article, we consider the commonly used quantification units (ARG copy per cell, ARG copy per genome, ARG density, ARG copy per 16S rRNA gene, RPKM, coverage, PPM, etc.) for profiling ARGs and suggest a universal unit (ARG copy per cell) for reporting such biological measurements of samples and improving the comparability of different surveillance efforts.

Improved description of terrestrial habitat types by including microbial communities as indicators.

Soils host diverse communities of microorganisms essential for ecosystem functions and soil health. Despite their importance, microorganisms are not covered by legislation protecting biodiversity or habitats, such as the Habitats Directive. Advances in molecular methods have caused breakthroughs in microbial community analysis, and recent studies have shown that parts of the communities are habitat-specific. If distinct microbial communities are present in the habitat types defined in the Habitats Directive, the Directive may be improved by including these communities. Thus, monitoring and reporting of biodiversity and conservation status of habitat types could be based not only on plant communities but also on microbial communities. In the present study, bacterial and plant communities were examined in six habitat types defined in the Habitats Directive by conducting botanical surveys and collecting soil samples for amplicon sequencing across 19 sites in Denmark. Furthermore, selected physico-chemical properties expected to differ between habitat types and explain variations in community composition of bacteria and vegetation were analysed (pH, electrical conductivity (EC), soil texture, soil water repellency, soil organic carbon content (OC), inorganic nitrogen, and in-situ water content (SWC)). Despite some variations within the same habitat type and overlaps between habitat types, habitat-specific communities were observed for both bacterial and plant communities, but no correlation was observed between the alpha diversity of vegetation and bacteria. PERMANOVA analysis was used to evaluate the variables best able to explain variation in the community composition of vegetation and bacteria. Habitat type alone could explain 46% and 47% of the variation in bacterial and plant communities, respectively. Excluding habitat type as a variable, the best model (pH, SWC, OC, fine silt, and Shannon's diversity index for vegetation) could explain 37% of the variation for bacteria. For vegetation, the best model (pH, EC, ammonium content and Shannon's diversity index for bacteria) could explain 25% of the variation. Based on these results, bacterial communities could be included in the Habitats Directive to improve the monitoring, as microorganisms are more sensitive to changes in the environment compared to vegetation, which the current monitoring is based on.

Quantification of Biologically and Chemically Bound Phosphorus in Activated Sludge from Full-Scale Plants with Biological P-Removal.

Phosphorus (P) is present in activated sludge from wastewater treatment plants in the form of metal salt precipitates, extracellular polymeric substances, or bound into the biomass, for example, as intracellular polyphosphate (poly-P). Several methods for a reliable quantification of the different P-fractions have recently been developed, and this study combines them to obtain a comprehensive P mass-balance of activated sludge from four enhanced biological phosphate removal (EBPR) plants. Chemical characterization by ICP-OES and sequential P fractionation showed that chemically bound P constituted 38-69% of total P, most likely in the form of Fe, Mg, or Al minerals. Raman microspectroscopy, solution state 31P NMR, and 31P MAS NMR spectroscopy applied before and after anaerobic P-release experiments, were used to quantify poly-P, which constituted 22-54% of total P and was found in approximately 25% of all bacterial cells. Raman microspectroscopy in combination with fluorescence in situ hybridization was used to quantify poly-P in known polyphosphate-accumulating organisms (PAO) (Tetrasphaera, Candidatus Accumulibacter, and Dechloromonas) and other microorganisms known to possess high level of poly-P, such as the filamentous Ca. Microthrix. Interestingly, only 1-13% of total P was stored by unidentified PAO, highlighting that most PAOs in the full-scale EBPR plants investigated are known.

Bacteria from the Genus Arcobacter Are Abundant in Effluent from Wastewater Treatment Plants.

Pathogenic bacteria in wastewater are generally considered to be efficiently removed in biological wastewater treatment plants. This understanding is almost solely based on culture-based control measures, and here we show, by applying culture-independent methods, that the removal of species in the genus Arcobacter was less effective than for many other abundant genera in the influent wastewater. Arcobacter was one of the most abundant genera in influent wastewater at 14 municipal wastewater treatment plants and was also abundant in the "clean" effluent from all the plants, reaching up to 30% of all bacteria as analyzed by 16S rRNA gene amplicon sequencing. Metagenomic analyses, culturing, genome sequencing of Arcobacter isolates, and visualization by fluorescent in situ hybridization (FISH) confirmed the presence of the human-pathogenic Arcobacter cryaerophilus and A. butzleri in both influent and effluent. The main reason for the high relative abundance in the effluent was probably that Arcobacter cells, compared to those of other abundant genera in the influent, did not flocculate and attach well to the activated sludge flocs, leaving a relatively large fraction dispersed in the water phase. The study shows there is an urgent need for new standardized culture-independent measurements of pathogens in effluent wastewaters, e.g., amplicon sequencing, and an investigation of the problem on a global scale to quantify the risk for humans and livestock.IMPORTANCE The genus Arcobacter was unexpectedly abundant in the effluent from 14 Danish wastewater treatment plants treating municipal wastewater, and the species included the human-pathogenic A. cryaerophilus and A. butzleri Recent studies have shown that Arcobacter is common in wastewater worldwide, so the study indicates that discharge of members of the genus Arcobacter may be a global problem, and further studies are needed to quantify the risk and potentially minimize the discharge. The study also shows that culture-based analyses are insufficient for proper effluent quality control, and new standardized culture-independent measurements of effluent quality encompassing most pathogens should be considered.

Proteogenomic Refinement of the Neomegalonema perideroedesT Genome Annotation.

Neomegalonema perideroedes (formerly Meganema perideroedes) str. G1 is the type strain and only described isolate of the genus Neomegalonema (formerly Meganema) which belongs to the Alphaproteobacteria. N. perideroedes is distinguished by the ability to accumulate high amounts of polyhydroxyalkanoates and has been associated with bulking problems in wastewater treatment plants due to its filamentous morphology. In 2013, its genome was sequenced as part of the Genomic Encyclopedia of Bacteria and Archaea (GEBA), which aims to improve the sequencing coverage of the poorly represented regions of the bacterial and archaeal branches of the tree of life. As N. perideroedes str. G1 is relatively distantly related to well described species-being the only sequenced member of its proposed family-the in silico prediction of genes by nucleotide homology to reference genes might be less reliable. Here, a proteomic dataset for the refinement of the N. perideroedes genome annotations is generated which clearly indicates the shortcomings of high-throughput in silico genome annotation.

Characterization of a thaumarchaeal symbiont that drives incomplete nitrification in the tropical sponge Ianthella basta.

Marine sponges represent one of the few eukaryotic groups that frequently harbour symbiotic members of the Thaumarchaeota, which are important chemoautotrophic ammonia-oxidizers in many environments. However, in most studies, direct demonstration of ammonia-oxidation by these archaea within sponges is lacking, and little is known about sponge-specific adaptations of ammonia-oxidizing archaea (AOA). Here, we characterized the thaumarchaeal symbiont of the marine sponge Ianthella basta using metaproteogenomics, fluorescence in situ hybridization, qPCR and isotope-based functional assays. 'Candidatus Nitrosospongia ianthellae' is only distantly related to cultured AOA. It is an abundant symbiont that is solely responsible for nitrite formation from ammonia in I. basta that surprisingly does not harbour nitrite-oxidizing microbes. Furthermore, this AOA is equipped with an expanded set of extracellular subtilisin-like proteases, a metalloprotease unique among archaea, as well as a putative branched-chain amino acid ABC transporter. This repertoire is strongly indicative of a mixotrophic lifestyle and is (with slight variations) also found in other sponge-associated, but not in free-living AOA. We predict that this feature as well as an expanded and unique set of secreted serpins (protease inhibitors), a unique array of eukaryotic-like proteins, and a DNA-phosporothioation system, represent important adaptations of AOA to life within these ancient filter-feeding animals.

The Composition and Implications of Polyphosphate-Metal in Enhanced Biological Phosphorus Removal Systems.

The individual cellular level and quantitative Polyphosphate (PolyP)-metal compositions in EBPR (enhanced biological phosphorus removal) systems have hardly been investigated and its potential link to EBPR performance therefore remain largely unknown. In this study, we applied scanning electron microscopy combined with energy dispersive X-ray spectroscopy (SEM/EDX) method that enabled detection and semiquantification of metal elemental compositions in intact intracellular PolyP granules in individual PAO (polyphosphate accumulating organism) cells. We, for the first time, revealed diverse and dynamic distributions of different metals ions in the PolyP-metal granules in different EBPR systems operated with the same influent metal composition but varying SRT of 5-30 days. We further demonstrated that the PolyP-metal composition diversity correlated with 16S rRNA gene based PAO phylogenetic diversity, suggesting the possible phylogeny-dependent PolyP-metal composition variation. The impact of PolyP metal composition in EBPR system, especially the Mg content in PolyP granules, was evidenced by the significant and strong positive correlation between PolyP-Mg content and the long-term stability of the four EBPR systems with varying SRTs. The PolyP-Mg content can therefore possibly serve as an indicator for EBPR performance monitoring. The results demonstrated that phenotyping techniques, such as PolyP-metal-based profiling, in compliment, or combined with genotyping techniques such as phylogenetic and functional gene sequencing, can provide more insights into the mechanisms and performance prediction of this important microbial ecosystem.

Toward Better Understanding of EBPR Systems via Linking Raman-Based Phenotypic Profiling with Phylogenetic Diversity.

This study reports a proof-of concept study to demonstrate the novel approach of phenotyping microbial communities in enhanced biological phosphorus removal (EBPR) systems using single cell Raman microspectroscopy and link it with phylogentic structures. We use hierarchical clustering analysis (HCA) of single-cell Raman spectral fingerprints and intracellular polymer signatures to separate and classify the functionally relevant populations in EBPR systems, namely polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs), as well as other microbial populations. We then investigated the link between Raman-based community phenotyping and 16S rRNA gene-based phylogenetic characterization of four lab-scale EBPR systems with varying solid retention time (SRT) to gain insights into possible genotype-function relationships. Combined and simultaneous phylogenetic and phenotypic evaluation of EBPR ecosystems revealed SRT-dependent phylogenetic and phenotypic characteristics of the PAOs and GAOs, and their association with EBPR performance. The phenotypic diversity and plasticity of PAO populations, which otherwise could not be obtained with phylogenetic analysis alone, showed complex but potentially crucial association with EBPR process stability.

Expanded metabolic versatility of ubiquitous nitrite-oxidizing bacteria from the genus Nitrospira.

Nitrospira are a diverse group of nitrite-oxidizing bacteria and among the environmentally most widespread nitrifiers. However, they remain scarcely studied and mostly uncultured. Based on genomic and experimental data from Nitrospira moscoviensis representing the ubiquitous Nitrospira lineage II, we identified ecophysiological traits that contribute to the ecological success of Nitrospira. Unexpectedly, N. moscoviensis possesses genes coding for a urease and cleaves urea to ammonia and CO2. Ureolysis was not observed yet in nitrite oxidizers and enables N. moscoviensis to supply ammonia oxidizers lacking urease with ammonia from urea, which is fully nitrified by this consortium through reciprocal feeding. The presence of highly similar urease genes in Nitrospira lenta from activated sludge, in metagenomes from soils and freshwater habitats, and of other ureases in marine nitrite oxidizers, suggests a wide distribution of this extended interaction between ammonia and nitrite oxidizers, which enables nitrite-oxidizing bacteria to indirectly use urea as a source of energy. A soluble formate dehydrogenase lends additional ecophysiological flexibility and allows N. moscoviensis to use formate, with or without concomitant nitrite oxidation, using oxygen, nitrate, or both compounds as terminal electron acceptors. Compared with Nitrospira defluvii from lineage I, N. moscoviensis shares the Nitrospira core metabolism but shows substantial genomic dissimilarity including genes for adaptations to elevated oxygen concentrations. Reciprocal feeding and metabolic versatility, including the participation in different nitrogen cycling processes, likely are key factors for the niche partitioning, the ubiquity, and the high diversity of Nitrospira in natural and engineered ecosystems.

Epigallocatechin Gallate Remodels Overexpressed Functional Amyloids in Pseudomonas aeruginosa and Increases Biofilm Susceptibility to Antibiotic Treatment.

Epigallocatechin-3-gallate (EGCG) is the major polyphenol in green tea. It has antimicrobial properties and disrupts the ordered structure of amyloid fibrils involved in human disease. The antimicrobial effect of EGCG against the opportunistic pathogen Pseudomonas aeruginosa has been shown to involve disruption of quorum sensing (QS). Functional amyloid fibrils in P. aeruginosa (Fap) are able to bind and retain quorum-sensing molecules, suggesting that EGCG interferes with QS through structural remodeling of amyloid fibrils. Here we show that EGCG inhibits the ability of Fap to form fibrils; instead, EGCG stabilizes protein oligomers. Existing fibrils are remodeled by EGCG into non-amyloid aggregates. This fibril remodeling increases the binding of pyocyanin, demonstrating a mechanism by which EGCG can affect the QS function of functional amyloid. EGCG reduced the amyloid-specific fluorescent thioflavin T signal in P. aeruginosa biofilms at concentrations known to exert an antimicrobial effect. Nanoindentation studies showed that EGCG reduced the stiffness of biofilm containing Fap fibrils but not in biofilm with little Fap. In a combination treatment with EGCG and tobramycin, EGCG had a moderate effect on the minimum bactericidal eradication concentration against wild-type P. aeruginosa biofilms, whereas EGCG had a more pronounced effect when Fap was overexpressed. Our results provide a direct molecular explanation for the ability of EGCG to disrupt P. aeruginosa QS and modify its biofilm and strengthens the case for EGCG as a candidate in multidrug treatment of persistent biofilm infections.

Enhancing metaproteomics--The value of models and defined environmental microbial systems.

Metaproteomics--the large-scale characterization of the entire protein complement of environmental microbiota at a given point in time--has provided new features to study complex microbial communities in order to unravel these "black boxes." New technical challenges arose that were not an issue for classical proteome analytics before that could be tackled by the application of different model systems. Here, we review different current and future model systems for metaproteome analysis. Following a short introduction to microbial communities and metaproteomics, we introduce model systems for clinical and biotechnological research questions including acid mine drainage, anaerobic digesters, and activated sludge. Model systems are useful to evaluate the challenges encountered within (but not limited to) metaproteomics, including species complexity and coverage, biomass availability, or reliable protein extraction. The implementation of model systems can be considered as a step forward to better understand microbial community responses and ecological functions of single member organisms. In the future, improvements are necessary to fully explore complex environmental systems by metaproteomics.

The Tubular Sheaths Encasing Methanosaeta thermophila Filaments Are Functional Amyloids.

Archaea are renowned for their ability to thrive in extreme environments, although they can be found in virtually all habitats. Their adaptive success is linked to their unique cell envelopes that are extremely resistant to chemical and thermal denaturation and that resist proteolysis by common proteases. Here we employ amyloid-specific conformation antibodies and biophysical techniques to show that the extracellular cell wall sheaths encasing the methanogenic archaea Methanosaeta thermophila PT are functional amyloids. Depolymerization of sheaths and subsequent MS/MS analyses revealed that the sheaths are composed of a single major sheath protein (MspA). The amyloidogenic nature of MspA was confirmed by in vitro amyloid formation of recombinant MspA under a wide range of environmental conditions. This is the first report of a functional amyloid from the archaeal domain of life. The amyloid nature explains the extreme resistance of the sheath, the elastic properties that allow diffusible substrates to penetrate through expandable hoop boundaries, and how the sheaths are able to split and elongate outside the cell. The archaeal sheath amyloids do not share homology with any of the currently known functional amyloids and clearly represent a new function of the amyloid protein fold.

Functional amyloids keep quorum-sensing molecules in check.

The mechanism by which extracellular metabolites, including redox mediators and quorum-sensing signaling molecules, traffic through the extracellular matrix of biofilms is poorly explored. We hypothesize that functional amyloids, abundant in natural biofilms and possessing hydrophobic domains, retain these metabolites. Using surface plasmon resonance, we demonstrate that the quorum-sensing (QS) molecules, 2-heptyl-3-hydroxy-4(1H)-quinolone and N-(3-oxododecanoyl)-l-homoserine lactone, and the redox mediator pyocyanin bind with transient affinity to functional amyloids from Pseudomonas (Fap). Their high hydrophobicity predisposes them to signal-amyloid interactions, but specific interactions also play a role. Transient interactions allow for rapid association and dissociation kinetics, which make the QS molecules bioavailable and at the same time secure within the extracellular matrix as a consequence of serial bindings. Retention of the QS molecules was confirmed using Pseudomonas aeruginosa PAO1-based 2-heptyl-3-hydroxy-4(1H)-quinolone and N-(3-oxododecanoyl)-l-homoserine lactone reporter assays, showing that Fap fibrils pretreated with the QS molecules activate the reporters even after sequential washes. Pyocyanin retention was validated by electrochemical analysis of pyocyanin-pretreated Fap fibrils subjected to the same washing process. Results suggest that QS molecule-amyloid interactions are probably important in the turbulent environments commonly encountered in natural habitats.

Detection of Pathogenic Biofilms with Bacterial Amyloid Targeting Fluorescent Probe, CDy11.

Bacterial biofilms are responsible for a wide range of persistent infections. In the clinic, diagnosis of biofilm-associated infections relies heavily on culturing methods, which fail to detect nonculturable bacteria. Identification of novel fluorescent probes for biofilm imaging will greatly facilitate diagnosis of pathogenic bacterial infection. Herein, we report a novel fluorescent probe, CDy11 (compound of designation yellow 11), which targets amyloid in the Pseudomonas aeruginosa biofilm matrix through a diversity oriented fluorescent library approach (DOFLA). CDy11 was further demonstrated for in vivo imaging of P. aeruginosa in implant and corneal infection mice models.

Identification of active denitrifiers in full-scale nutrient removal wastewater treatment systems.

Denitrification is essential to the removal of nitrogen from wastewater during treatment, yet an understanding of the diversity of the active denitrifying bacteria responsible in full-scale wastewater treatment plants (WWTPs) is lacking. In this study, stable-isotope probing (SIP) was applied in combination with microautoradiography (MAR)-fluorescence in situ hybridization (FISH) to identify previously unrecognized active denitrifying phylotypes in a full-scale WWTP with biological N and P removal. Acknowledging that different denitrifiers will have specific carbon source preferences, a fully (13)C-labelled complex substrate was used for SIP incubations, under nitrite-reducing conditions, in order to maximize the capture of the potentially metabolically diverse denitrifiers likely present. Members of the Rhodoferax, Dechloromonas, Sulfuritalea, Haliangium and Thermomonas were represented in the 16S rRNA gene clone libraries from DNA enriched in (13)C, with FISH probes optimized here for their in situ characterization. FISH and MAR confirmed that they were all active denitrifiers in the community. The combined approach of SIP and MAR-FISH represents an excellent approach for identifying and characterizing an un-described diversity of active denitrifiers in full-scale systems.

In vivo gene expression in a Staphylococcus aureus prosthetic joint infection characterized by RNA sequencing and metabolomics: a pilot study.

BACKGROUND: Staphylococcus aureus gene expression has been sparsely studied in deep-sited infections in humans. Here, we characterized the staphylococcal transcriptome in vivo and the joint fluid metabolome in a prosthetic joint infection with an acute presentation using deep RNA sequencing and nuclear magnetic resonance spectroscopy, respectively. We compared our findings with the genome, transcriptome and metabolome of the S. aureus joint fluid isolate grown in vitro. RESULT: From the transcriptome analysis we found increased expression of siderophore synthesis genes and multiple known virulence genes. The regulatory pattern of catabolic pathway genes indicated that the bacterial infection was sustained on amino acids, glycans and nucleosides. Upregulation of fermentation genes and the presence of ethanol in joint fluid indicated severe oxygen limitation in vivo. CONCLUSION: This single case study highlights the capacity of combined transcriptome and metabolome analyses for elucidating the pathogenesis of prosthetic infections of major clinical importance.

Comparing culture and molecular methods for the identification of microorganisms involved in necrotizing soft tissue infections.

BACKGROUND: Necrotizing soft tissue infections (NSTIs) are a group of infections affecting all soft tissues. NSTI involves necrosis of the afflicted tissue and is potentially life threatening due to major and rapid destruction of tissue, which often leads to septic shock and organ failure. The gold standard for identification of pathogens is culture; however molecular methods for identification of microorganisms may provide a more rapid result and may be able to identify additional microorganisms that are not detected by culture. METHODS: In this study, tissue samples (n = 20) obtained after debridement of 10 patients with NSTI were analyzed by standard culture, fluorescence in situ hybridization (FISH) and multiple molecular methods. The molecular methods included analysis of microbial diversity by 1) direct 16S and D2LSU rRNA gene Microseq 2) construction of near full-length 16S rRNA gene clone libraries with subsequent Sanger sequencing for most samples, 3) the Ibis T5000 biosensor and 4) 454-based pyrosequencing. Furthermore, quantitative PCR (qPCR) was used to verify and determine the relative abundance of Streptococcus pyogenes in samples. RESULTS: For 70 % of the surgical samples it was possible to identify microorganisms by culture. Some samples did not result in growth (presumably due to administration of antimicrobial therapy prior to sampling). The molecular methods identified microorganisms in 90 % of the samples, and frequently detected additional microorganisms when compared to culture. Although the molecular methods generally gave concordant results, our results indicate that Microseq may misidentify or overlook microorganisms that can be detected by other molecular methods. Half of the patients were found to be infected with S. pyogenes, but several atypical findings were also made including infection by a) Acinetobacter baumannii, b) Streptococcus pneumoniae, and c) fungi, mycoplasma and Fusobacterium necrophorum. CONCLUSION: The study emphasizes that many pathogens can be involved in NSTIs, and that no specific "NSTI causing" combination of species exists. This means that clinicians should be prepared to diagnose and treat any combination of microbial pathogens. Some of the tested molecular methods offer a faster turnaround time combined with a high specificity, which makes supplemental use of such methods attractive for identification of microorganisms, especially for fulminant life-threatening infections such as NSTI.

Label-free quantification reveals major proteomic changes in Pseudomonas putida F1 during the exponential growth phase.

The physiological adaptation to stationary growth by Pseudomonas putida F1, a model organism for the degradation of aromatic compounds, was investigated by proteome-wide label-free quantification.The data unveiled that entrance to the stationary phase did not involve an abrupt switch within the P. putida F1 proteome, but rather an ongoing adaptation that started already during the mid-exponential growth phase. The proteomic adaptations involved a clear increase in amino acid degradation capabilities and a loss of transcriptional as well as translational capacity. The final entrance to the stationary phase was accompanied by increased oxidative stress protection, although the stress and stationary sigma factor RpoS increased in abundance already during mid-exponential growth. The results show that it is important to consider significant sample variations when exponentially growing cultures are studied alone or compared across proteomic or transcriptomic literature. All MS data have been deposited in the ProteomeXchange with identifier PXD001219 (http://proteomecentral.proteomexchange.org/dataset/PXD001219).

Intracellular Accumulation of Glycine in Polyphosphate-Accumulating Organisms in Activated Sludge, a Novel Storage Mechanism under Dynamic Anaerobic-Aerobic Conditions.

Dynamic anaerobic-aerobic feast-famine conditions are applied to wastewater treatment plants to select polyphosphate-accumulating organisms to carry out enhanced biological phosphorus removal. Acetate is a well-known substrate to stimulate this process, and here we show that different amino acids also are suitable substrates, with glycine as the most promising. (13)C-labeled glycine and nuclear magnetic resonance (NMR) were applied to investigate uptake and potential storage products when activated sludge was fed with glycine under anaerobic conditions. Glycine was consumed by the biomass, and the majority was stored intracellularly as free glycine and fermentation products. Subsequently, in the aerobic phase without addition of external substrate, the stored glycine was consumed. The uptake of glycine and oxidation of intracellular metabolites took place along with a release and uptake of orthophosphate, respectively. Fluorescence in situ hybridization combined with microautoradiography using (3)H-labeled glycine revealed uncultured actinobacterial Tetrasphaera as a dominant glycine consumer. Experiments with Tetrasphaera elongata as representative of uncultured Tetrasphaera showed that under anaerobic conditions it was able to take up labeled glycine and accumulate this and other labeled metabolites to an intracellular concentration of approximately 4 mM. All components were consumed under subsequent aerobic conditions. Intracellular accumulation of amino acids seems to be a novel storage strategy for polyphosphate-accumulating bacteria under dynamic anaerobic-aerobic feast-famine conditions.