Microbes possess extraordinarily diverse and sophisticated physiologies, communication strategies, and mechanisms of evolution. Scientists and engineers are only beginning to understand and exploit the metabolic potential of these organisms and their communities. The broad objective of my research program is to improve our capacity to predict and model microbial behavior, while searching for novel biologically mediated transformations that can be harnessed for engineering applications.
My students and I study the microbial ecology of both natural and engineered systems. We use molecular tools to investigate microbial community structure and function in lakes and activated sludge. More recently, we have been using high-frequency environmental sensor networks to measure important variables that we know influence bacterial communities. Sensor data provided through the Global Lake Ecological Observatory Network (http://www.gleon.org) guides our adaptive sampling efforts and provides rich contextual data for our studies of lake bacterial community ecology. We are particularly interested in phosphorus as a nutrient driving eutrophication, and the role that bacteria play in phosphorus cycling. We are also engaged in metagenomic and post-genomic approaches to dissecting the metabolism of bacteria specialized in the sequestration of phosphorus in activated sludge. This information will ultimately lead to the construction of more predictive mechanistic and ecosystem-scale models to describe such processes as wastewater treatment and freshwater nutrient cycling.
Microbiology 425: Environmental Microbiology
Biocore 587: Biological Interactions
Trainer, Biotechnology Graduate Training Program
Women in Science and Engineering Residential College, past Co-Director
Chair, Graduate Admissions for Environmental Engineering Graduate Program
Associate Chair, Civil and Environmental Engineering Department
Member, UW-Madison Teaching Academy
Facilitator, I-LEAP Teaching Improvement Program
Faculty Co-Director, UW-Madison Delta Program
Member, WISCIENCE Executive Committee
Senior Editor, mSphere (ASM journal)
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.
" 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.
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.
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.
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.
Photosynthetic bacteria from the class Chlorobia (formerly phylum Chlorobi ) sustain carbon fixation in anoxic water columns. They harvest light at extremely low intensities and use various inorganic electron donors to fix carbon dioxide into biomass. Until now, most information on the functional ecology and local adaptations of Chlorobia members came from isolates and merely 26 sequenced genomes that may not adequately represent natural populations. To address these limitations, we analyzed global metagenomes to profile planktonic Chlorobia cells from the oxyclines of 42 freshwater bodies, spanning subarctic to tropical regions and encompassing all four seasons. We assembled and compiled over 500 genomes, including metagenome-assembled genomes (MAGs), single-amplified genomes (SAGs), and reference genomes from cultures, clustering them into 71 metagenomic operational taxonomic units (mOTUs or "species"). Of the 71 mOTUs, 57 were classified within the genus Chlorobium , and these mOTUs represented up to ∼60% of the microbial communities in the sampled anoxic waters. Several Chlorobium -associated mOTUs were globally distributed, whereas others were endemic to individual lakes. Although most clades encoded the ability to oxidize hydrogen, many lacked genes for the oxidation of specific sulfur and iron substrates. Surprisingly, one globally distributed Scandinavian clade encoded the ability to oxidize hydrogen, sulfur, and iron, suggesting that metabolic versatility facilitated such widespread colonization. Overall, these findings provide new insight into the biogeography of the Chlorobia and the metabolic traits that facilitate niche specialization within lake ecosystems. IMPORTANCE The reconstruction of genomes from metagenomes has helped explore the ecology and evolution of environmental microbiota. We applied this approach to 274 metagenomes collected from diverse freshwater habitats that spanned oxic and anoxic zones, sampling seasons, and latitudes. We demonstrate widespread and abundant distributions of planktonic Chlorobia -associated bacteria in hypolimnetic waters of stratified freshwater ecosystems and show they vary in their capacities to use different electron donors. Having photoautotrophic potential, these Chlorobia members could serve as carbon sources that support metalimnetic and hypolimnetic food webs.
Enhanced biological phosphorus removal (EBPR) is an economically and environmentally significant wastewater treatment process for removing excess phosphorus by harnessing the metabolic physiologies of enriched microbial communities. We present a genome-resolved metagenomic data set consisting of 86 metagenome-assembled genome sequences from a photosynthetically operated lab-scale bioreactor simulating EBPR.
Lake Tanganyika (LT) is the largest tropical freshwater lake, and the largest body of anoxic freshwater on Earth's surface. LT's mixed oxygenated surface waters float atop a permanently anoxic layer and host rich animal biodiversity. However, little is known about microorganisms inhabiting LT's 1470 meter deep water column and their contributions to nutrient cycling, which affect ecosystem-level function and productivity. Here, we applied genome-resolved metagenomics and environmental analyses to link specific taxa to key biogeochemical processes across a vertical depth gradient in LT. We reconstructed 523 unique metagenome-assembled genomes (MAGs) from 34 bacterial and archaeal phyla, including many rarely observed in freshwater lakes. We identified sharp contrasts in community composition and metabolic potential with an abundance of typical freshwater taxa in oxygenated mixed upper layers, and Archaea and uncultured Candidate Phyla in deep anoxic waters. Genomic capacity for nitrogen and sulfur cycling was abundant in MAGs recovered from anoxic waters, highlighting microbial contributions to the productive surface layers via recycling of upwelled nutrients, and greenhouse gases such as nitrous oxide. Overall, our study provides a blueprint for incorporation of aquatic microbial genomics in the representation of tropical freshwater lakes, especially in the context of ongoing climate change, which is predicted to bring increased stratification and anoxia to freshwater lakes.
Temperate phages are viruses of bacteria that can establish two types of infection: a lysogenic infection in which the virus replicates with the host cell without producing virions, and a lytic infection where the host cell is eventually destroyed, and new virions are released. While both lytic and lysogenic infections are routinely observed in the environment, the ecological and evolutionary processes regulating these viral dynamics are still not well understood, especially for uncultivated virus-host pairs. Here, we characterized the long-term dynamics of uncultivated viruses infecting green sulfur bacteria (GSB) in a model freshwater lake (Trout Bog Lake, TBL). As no GSB virus has been formally described yet, we first used two complementary approaches to identify new GSB viruses from TBL; one in vitro based on flow cytometry cell sorting, the other in silico based on CRISPR spacer sequences. We then took advantage of existing TBL metagenomes covering the 2005-2018 period to examine the interactions between GSB and their viruses across years and seasons. From our data, GSB populations in TBL were constantly associated with at least 2-8 viruses each, including both lytic and temperate phages. The dominant GSB population in particular was consistently associated with two prophages with a nearly 100% infection rate for >10 years. We illustrate with a theoretical model that such an interaction can be stable given a low, but persistent, level of prophage induction in low-diversity host populations. Overall, our data suggest that lytic and lysogenic viruses can readily co-infect the same host population, and that host strain-level diversity might be an important factor controlling virus-host dynamics including lytic/lysogeny switch.
Mercury (Hg) methylation is a microbially mediated process that converts inorganic Hg into bioaccumulative, neurotoxic methylmercury (MeHg). The metabolic activity of methylating organisms is highly dependent on biogeochemical conditions, which subsequently influences MeHg production. However, our understanding of the ecophysiology of methylators in natural ecosystems is still limited. Here, we identified potential locations of MeHg production in the anoxic, sulfidic hypolimnion of a freshwater lake. At these sites, we used shotgun metagenomics to characterize microorganisms with the Hg-methylation gene hgcA . Putative methylators were dominated by hgcA sequences divergent from those in well-studied, confirmed methylators. Using genome-resolved metagenomics, we identified organisms with hgcA (hgcA+) within the Bacteroidetes and the recently described Kiritimatiellaeota phyla. We identified hgcA+ genomes derived from sulfate-reducing bacteria, but these accounted for only 22% of hgcA+ genome coverage. The most abundant hgcA+ genomes were from fermenters, accounting for over half of the hgcA gene coverage. Many of these organisms also mediate hydrolysis of polysaccharides, likely from cyanobacterial blooms. This work highlights the distribution of the Hg-methylation genes across microbial metabolic guilds and indicate that primary degradation of polysaccharides and fermentation may play an important but unrecognized role in MeHg production in the anoxic hypolimnion of freshwater lakes.
Anaerobic ammonium-oxidizing (anammox) bacteria mediate a key step in the biogeochemical nitrogen cycle and have been applied worldwide for the energy-efficient removal of nitrogen from wastewater. However, outside their core energy metabolism, little is known about the metabolic networks driving anammox bacterial anabolism and use of different carbon and energy substrates beyond genome-based predictions. Here, we experimentally resolved the central carbon metabolism of the anammox bacterium Candidatus 'Kuenenia stuttgartiensis' using time-series C and H isotope tracing, metabolomics, and isotopically nonstationary metabolic flux analysis. Our findings confirm predicted metabolic pathways used for CO fixation, central metabolism, and amino acid biosynthesis in K. stuttgartiensis, and reveal several instances where genomic predictions are not supported by in vivo metabolic fluxes. This includes the use of the oxidative branch of an incomplete tricarboxylic acid cycle for alpha-ketoglutarate biosynthesis, despite the genome not having an annotated citrate synthase. We also demonstrate that K. stuttgartiensis is able to directly assimilate extracellular formate via the Wood-Ljungdahl pathway instead of oxidizing it completely to CO followed by reassimilation. In contrast, our data suggest that K. stuttgartiensis is not capable of using acetate as a carbon or energy source in situ and that acetate oxidation occurred via the metabolic activity of a low-abundance microorganism in the bioreactor's side population. Together, these findings provide a foundation for understanding the carbon metabolism of anammox bacteria at a systems-level and will inform future studies aimed at elucidating factors governing their function and niche differentiation in natural and engineered ecosystems.
There is growing evidence that phages with unusually large genomes are common across various microbiomes, but little is known about their genetic inventories or potential ecosystem impacts. In the present study, we reconstructed large phage genomes from freshwater lakes known to contain bacteria that oxidize methane. Of manually curated genomes, 22 (18 are complete), ranging from 159 kilobase (kb) to 527 kb in length, were found to encode the pmoC gene, an enzymatically critical subunit of the particulate methane monooxygenase, the predominant methane oxidation catalyst in nature. The phage-associated PmoC sequences show high similarity to (>90%), and affiliate phylogenetically with, those of coexisting bacterial methanotrophs, including members of Methyloparacoccus, Methylocystis and Methylobacter spp. In addition, pmoC-phage abundance patterns correlate with those of the coexisting bacterial methanotrophs, supporting host-phage relationships. Future work is needed to determine whether phage-associated PmoC has similar functions to additional copies of PmoC encoded in bacterial genomes, thus contributing to growth on methane. Transcriptomics data from Lake Rotsee (Switzerland) showed that some phage-associated pmoC genes were highly expressed in situ and, of interest, that the most rapidly growing methanotroph was infected by three pmoC-phages. Thus, augmentation of bacterial methane oxidation by pmoC-phages during infection could modulate the efflux of this potent greenhouse gas into the environment.
Methylmercury is a potent bioaccumulating neurotoxin that is produced by specific microorganisms that methylate inorganic mercury. Methylmercury production in diverse anaerobic bacteria and archaea was recently linked to the hgcAB genes. However, the full phylogenetic and metabolic diversity of mercury-methylating microorganisms has not been fully unraveled due to the limited number of cultured experimentally verified methylators and the limitations of primer-based molecular methods. Here, we describe the phylogenetic diversity and metabolic flexibility of putative mercury-methylating microorganisms by hgcAB identification in publicly available isolate genomes and metagenome-assembled genomes (MAGs) as well as novel freshwater MAGs. We demonstrate that putative mercury methylators are much more phylogenetically diverse than previously known and that hgcAB distribution among genomes is most likely due to several independent horizontal gene transfer events. The microorganisms we identified possess diverse metabolic capabilities spanning carbon fixation, sulfate reduction, nitrogen fixation, and metal resistance pathways. We identified 111 putative mercury methylators in a set of previously published permafrost metatranscriptomes and demonstrated that different methylating taxa may contribute to hgcA expression at different depths. Overall, we provide a framework for illuminating the microbial basis of mercury methylation using genome-resolved metagenomics and metatranscriptomics to identify putative methylators based upon hgcAB presence and describe their putative functions in the environment. IMPORTANCE Accurately assessing the production of bioaccumulative neurotoxic methylmercury by characterizing the phylogenetic diversity, metabolic functions, and activity of methylators in the environment is crucial for understanding constraints on the mercury cycle. Much of our understanding of methylmercury production is based on cultured anaerobic microorganisms within the Deltaproteobacteria , Firmicutes , and Euryarchaeota. Advances in next-generation sequencing technologies have enabled large-scale cultivation-independent surveys of diverse and poorly characterized microorganisms from numerous ecosystems. We used genome-resolved metagenomics and metatranscriptomics to highlight the vast phylogenetic and metabolic diversity of putative mercury methylators and their depth-discrete activities in thawing permafrost. This work underscores the importance of using genome-resolved metagenomics to survey specific putative methylating populations of a given mercury-impacted ecosystem.
Aquatic N-fixation is generally associated with the growth and mass development of Cyanobacteria in nitrogen-deprived photic zones. However, sequenced genomes and environmental surveys suggest active aquatic N-fixation also by many non-cyanobacterial groups. Here, we revealed the seasonal variation and genomic diversity of potential N-fixers in a humic bog lake using metagenomic data and nif gene clusters analysis. Groups with diazotrophic operons were functionally divergent and included Cholorobi , Geobacter , Desulfobacterales , Methylococcales , and Acidobacteria . In addition to nifH (a gene that encodes the dinitrogenase reductase component of the molybdenum nitrogenase), we also identified sequences corresponding to vanadium and iron-only nitrogenase genes. Within the Chlorobi population, the nitrogenase ( nifH ) cluster was included in a well-structured retrotransposon. Furthermore, the presence of light-harvesting photosynthesis genes implies that anoxygenic photosynthesis may fuel nitrogen fixation under the prevailing low-irradiance conditions. The presence of rnf genes (related to the expression of H/Na-translocating ferredoxin: NAD+ oxidoreductase) in Methylococcales and Desulfobacterales suggests that other energy-generating processes may drive the costly N-fixation in the absence of photosynthesis. The highly reducing environment of the anoxic bottom layer of Trout Bog Lake may thus also provide a suitable niche for active N-fixers and primary producers. While future studies on the activity of these potential N-fixers are needed to clarify their role in freshwater nitrogen cycling, the metagenomic data presented here enabled an initial characterization of previously overlooked diazotrophs in freshwater biomes.
Our current knowledge about nucleocytoplasmic large DNA viruses (NCLDVs) is largely derived from viral isolates that are co-cultivated with protists and algae. Here we reconstructed 2,074 NCLDV genomes from sampling sites across the globe by building on the rapidly increasing amount of publicly available metagenome data. This led to an 11-fold increase in phylogenetic diversity and a parallel 10-fold expansion in functional diversity. Analysis of 58,023 major capsid proteins from large and giant viruses using metagenomic data revealed the global distribution patterns and cosmopolitan nature of these viruses. The discovered viral genomes encoded a wide range of proteins with putative roles in photosynthesis and diverse substrate transport processes, indicating that host reprogramming is probably a common strategy in the NCLDVs. Furthermore, inferences of horizontal gene transfer connected viral lineages to diverse eukaryotic hosts. We anticipate that the global diversity of NCLDVs that we describe here will establish giant viruses-which are associated with most major eukaryotic lineages-as important players in ecosystems across Earth's biomes.
Virophages are small viruses with double-stranded DNA genomes that replicate along with giant viruses and co-infect eukaryotic cells. Due to the paucity of virophage reference genomes, a collective understanding of the global virophage diversity, distribution, and evolution is lacking.
Fonsibacter (LD12 subclade) is among the most abundant bacterioplankton in freshwater ecosystems. These bacteria belong to the order Pelagibacterales (SAR11) and are related to Pelagibacter (marine SAR11), which dominates many marine habitats. Although a few Pelagibacter phage (Pelagiphage) have been described, no phage that infect Fonsibacter have been reported. In this study, we describe two groups of Podoviridae phage that infect Fonsibacter A complete Fonsibacter genome containing a prophage was reconstructed from metagenomic data. A circularized and complete genome related to the prophage, referred to as uv-Fonsiphage-EPL (lysogenic strategy), shows high similarity to marine Pelagiphage HTVC025P. Additionally, we reconstructed three complete genomes and one draft genome of phage related to marine Pelagiphage HTVC010P and predicted a lytic strategy. The similarity in codon usage and cooccurrence patterns of HTVC010P-related phage and Fonsibacter suggested that these phage infect Fonsibacter Similar phage were detected in Lake Mendota, Wisconsin, where Fonsibacter is also present. A search of related phage revealed the worldwide distribution of some genotypes in freshwater ecosystems, suggesting their substantial role in shaping indigenous microbial assemblages and influence on biogeochemical cycling. However, the uv-Fonsiphage-EPL and one group of HTVC010P-related phage have a more limited distribution in freshwater ecosystems. Overall, the findings provide insights into the genomic features of phage that infect Fonsibacter and expand understanding of the ecology and evolution of these important bacteria. IMPORTANCE Fonsibacter represents a significant microbial group of freshwater ecosystems. Although the genomic and metabolic features of these bacteria have been well studied, no phage infecting them has been reported. In this study, we reconstructed complete genomes of Fonsibacter and infecting phage and revealed their close relatedness to the phage infecting marine SAR11 members. Also, we illustrated that phage that infect Fonsibacter are widely distributed in freshwater habitats. In summary, the results contribute new insights into the ecology and evolution of Fonsibacter and phage.
Despite broad scientific interest in harnessing the power of Earth's microbiomes, knowledge gaps hinder their efficient use for addressing urgent societal and environmental challenges. We argue that structuring research and technology developments around a design-build-test-learn (DBTL) cycle will advance microbiome engineering and spur new discoveries of the basic scientific principles governing microbiome function. In this Review, we present key elements of an iterative DBTL cycle for microbiome engineering, focusing on generalizable approaches, including top-down and bottom-up design processes, synthetic and self-assembled construction methods, and emerging tools to analyse microbiome function. These approaches can be used to harness microbiomes for broad applications related to medicine, agriculture, energy and the environment. We also discuss key challenges and opportunities of each approach and synthesize them into best practice guidelines for engineering microbiomes. We anticipate that adoption of a DBTL framework will rapidly advance microbiome-based biotechnologies aimed at improving human and animal health, agriculture and enabling the bioeconomy.
The ability of " Candidatus Accumulibacter phosphatis" to grow and remove phosphorus from wastewater under cycling anaerobic and aerobic conditions has also been investigated as a metabolism that could lead to simultaneous removal of nitrogen and phosphorus by a single organism. However, although phosphorus removal under cyclic anaerobic and anoxic conditions has been demonstrated, clarifying the role of " Ca . Accumulibacter phosphatis" in this process has been challenging, since (i) experimental research describes contradictory findings, (ii) none of the published " Ca . Accumulibacter phosphatis" genomes show the existence of a complete respiratory pathway for denitrification, and (iii) some genomes lacking a complete respiratory pathway have genes for assimilatory nitrate reduction. In this study, we used an integrated omics analysis to elucidate the physiology of a " Ca . Accumulibacter phosphatis" strain enriched in a reactor operated under cyclic anaerobic and microaerobic conditions. The reactor's performance suggested the ability of the enriched " Ca . Accumulibacter phosphatis" strain (clade IC) to simultaneously use oxygen and nitrate as electron acceptors under microaerobic conditions. A draft genome of this organism was assembled from metagenomic reads (" Ca . Accumulibacter phosphatis" UW-LDO-IC) and used as a reference to examine transcript abundance throughout one reactor cycle. The genome of UW-LDO-IC revealed the presence of a full pathway for respiratory denitrification. The observed transcript abundance patterns showed evidence of coregulation of the denitrifying genes along with a cbb cytochrome, which has been characterized as having high affinity for oxygen. Furthermore, we identified an FNR-like binding motif upstream of the coregulated genes, suggesting transcription-level regulation of both denitrifying and respiratory pathways in UW-LDO-IC. Taking the results together, the omics analysis provides strong evidence that " Ca . Accumulibacter phosphatis" UW-LDO-IC uses oxygen and nitrate simultaneously as electron acceptors under microaerobic conditions. IMPORTANCE " Candidatus Accumulibacter phosphatis" is widely found in full-scale wastewater treatment plants, where it has been identified as the key organism for biological removal of phosphorus. Since aeration can account for 50% of the energy use during wastewater treatment, microaerobic conditions for wastewater treatment have emerged as a cost-effective alternative to conventional biological nutrient removal processes. Our report provides strong genomics-based evidence not only that " Ca . Accumulibacter phosphatis" is the main organism contributing to phosphorus removal under microaerobic conditions but also that this organism simultaneously respires nitrate and oxygen in this environment, consequently removing nitrogen and phosphorus from the wastewater. Such activity could be harnessed in innovative designs for cost-effective and energy-efficient optimization of wastewater treatment systems.
Humic lakes and ponds receive large amounts of terrestrial carbon and are important components of the global carbon cycle, yet how their redox cycling influences the carbon budget is not fully understood. Here we compared metagenomes obtained from a humic bog and a clear-water eutrophic lake and found a much larger number of genes that might be involved in extracellular electron transfer (EET) for iron redox reactions and humic substance (HS) reduction in the bog than in the clear-water lake, consistent with the much higher iron and HS levels in the bog. These genes were particularly rich in the bog's anoxic hypolimnion and were found in diverse bacterial lineages, some of which are relatives of known iron oxidizers or iron-HS reducers. We hypothesize that HS may be a previously overlooked electron acceptor and that EET-enabled redox cycling may be important in pelagic respiration and greenhouse gas budget in humic-rich freshwater lakes.
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A central pursuit of microbial ecology is to accurately model changes in microbial community composition in response to environmental factors. This goal requires a thorough understanding of the drivers of variability in microbial populations. However, most microbial ecology studies focus on the effects of environmental factors on mean population abundances, rather than on population variability. Here, we imposed several experimental disturbances upon periphyton communities and analyzed the variability of populations within disturbed communities compared with those in undisturbed communities. We analyzed both the bacterial and the diatom communities in the periphyton under nine different disturbance regimes, including regimes that contained multiple disturbances. We found several similarities in the responses of the two communities to disturbance; all significant treatment effects showed that populations became less variable as the result of environmental disturbances. Furthermore, multiple disturbances to these communities were often interactive, meaning that the effects of two disturbances could not have been predicted from studying single disturbances in isolation. These results suggest that environmental factors had repeatable effects on populations within microbial communities, thereby creating communities that were more similar as a result of disturbances. These experiments add to the predictive framework of microbial ecology by quantifying variability in microbial populations and by demonstrating that disturbances can place consistent constraints on the abundance of microbial populations. Although models will never be fully predictive due to stochastic forces, these results indicate that environmental stressors may increase the ability of models to capture microbial community dynamics because of their consistent effects on microbial populations. IMPORTANCE There are many reasons why microbial community composition is difficult to model. For example, the high diversity and high rate of change of these communities make it challenging to identify causes of community turnover. Furthermore, the processes that shape community composition can be either deterministic, which cause communities to converge upon similar compositions, or stochastic, which increase variability in community composition. However, modeling microbial community composition is possible only if microbes show repeatable responses to extrinsic forcing. In this study, we hypothesized that environmental stress acts as a deterministic force that shapes microbial community composition. Other studies have investigated if disturbances can alter microbial community composition, but relatively few studies ask about the repeatability of the effects of disturbances. Mechanistic models implicitly assume that communities show consistent responses to stressors; here, we define and quantify microbial variability to test this assumption. Author Video: An author video summary of this article is available.
Lab- and pilot-scale simultaneous nitrification, denitrification and phosphorus removal-sequencing batch reactors were operated under cyclic anaerobic and micro-aerobic conditions. The use of oxygen, nitrite, and nitrate as electron acceptors by Candidatus Accumulibacter phosphatis during the micro-aerobic stage was investigated. A complete clade-level characterization of Accumulibacter in both reactors was performed using newly designed qPCR primers targeting the polyphosphate kinase gene (ppk1). In the lab-scale reactor, limited-oxygen conditions led to an alternated dominance of Clade IID and IC over the other clades. Results from batch tests when Clade IC was dominant (i.e., >92% of Accumulibacter) showed that this clade was capable of using oxygen, nitrite and nitrate as electron acceptors for P uptake. A more heterogeneous distribution of clades was found in the pilot-scale system (Clades IIA, IIB, IIC, IID, IA, and IC), and in this reactor, oxygen, nitrite and nitrate were also used as electron acceptors coupled to phosphorus uptake. However, nitrite was not an efficient electron acceptor in either reactor, and nitrate allowed only partial P removal. The results from the Clade IC dominated reactor indicated that either organisms in this clade can simultaneously use multiple electron acceptors under micro-aerobic conditions, or that the use of multiple electron acceptors by Clade IC is due to significant microdiversity within the Accumulibacter clades defined using the ppk1 gene.
The evolution of complex traits is hypothesized to occur incrementally. Identifying the transitions that lead to extant complex traits may provide a better understanding of the genetic nature of the observed phenotype. A keystone functional group in wastewater treatment processes are polyphosphate accumulating organisms (PAOs), however the evolution of the PAO phenotype has yet to be explicitly investigated and the specific metabolic traits that discriminate non-PAO from PAO are currently unknown. Here we perform the first comprehensive investigation on the evolution of the PAO phenotype using the model uncultured organism Candidatus Accumulibacter phosphatis (Accumulibacter) through ancestral genome reconstruction, identification of horizontal gene transfer, and a kinetic/stoichiometric characterization of Accumulibacter Clade IIA. The analysis of Accumulibacter's last common ancestor identified 135 laterally derived genes, including genes involved in glycogen, polyhydroxyalkanoate, pyruvate and NADH/NADPH metabolisms, as well as inorganic ion transport and regulatory mechanisms. In contrast, pathways such as the TCA cycle and polyphosphate metabolism displayed minimal horizontal gene transfer. We show that the transition from non-PAO to PAO coincided with horizontal gene transfer within Accumulibacter's core metabolism; likely alleviating key kinetic and stoichiometric bottlenecks, such as anaerobically linking glycogen degradation to polyhydroxyalkanoate synthesis. These results demonstrate the utility of investigating the derived genome of a lineage to identify key transitions leading to an extant complex phenotype.
Most free-living planktonic cells are streamlined and in spite of their limitations in functional flexibility, their vast populations have radiated into a wide range of aquatic habitats. Here we compared the metabolic potential of subgroups in the Alphaproteobacteria lineage SAR11 adapted to marine and freshwater habitats. Our results suggest that the successful leap from marine to freshwaters in SAR11 was accompanied by a loss of several carbon degradation pathways and a rewiring of the central metabolism. Examples for these are C1 and methylated compounds degradation pathways, the Entner-Doudouroff pathway, the glyoxylate shunt and anapleuretic carbon fixation being absent from the freshwater genomes. Evolutionary reconstructions further suggest that the metabolic modules making up these important freshwater metabolic traits were already present in the gene pool of ancestral marine SAR11 populations. The loss of the glyoxylate shunt had already occurred in the common ancestor of the freshwater subgroup and its closest marine relatives, suggesting that the adaptation to freshwater was a gradual process. Furthermore, our results indicate rapid evolution of TRAP transporters in the freshwater clade involved in the uptake of low molecular weight carboxylic acids. We propose that such gradual tuning of metabolic pathways and transporters toward locally available organic substrates is linked to the formation of subgroups within the SAR11 clade and that this process was critical for the freshwater clade to find and fix an adaptive phenotype.
Multiple models describe the formation and evolution of distinct microbial phylogenetic groups. These evolutionary models make different predictions regarding how adaptive alleles spread through populations and how genetic diversity is maintained. Processes predicted by competing evolutionary models, for example, genome-wide selective sweeps vs gene-specific sweeps, could be captured in natural populations using time-series metagenomics if the approach were applied over a sufficiently long time frame. Direct observations of either process would help resolve how distinct microbial groups evolve. Here, from a 9-year metagenomic study of a freshwater lake (2005-2013), we explore changes in single-nucleotide polymorphism (SNP) frequencies and patterns of gene gain and loss in 30 bacterial populations. SNP analyses revealed substantial genetic heterogeneity within these populations, although the degree of heterogeneity varied by >1000-fold among populations. SNP allele frequencies also changed dramatically over time within some populations. Interestingly, nearly all SNP variants were slowly purged over several years from one population of green sulfur bacteria, while at the same time multiple genes either swept through or were lost from this population. These patterns were consistent with a genome-wide selective sweep in progress, a process predicted by the 'ecotype model' of speciation but not previously observed in nature. In contrast, other populations contained large, SNP-free genomic regions that appear to have swept independently through the populations prior to the study without purging diversity elsewhere in the genome. Evidence for both genome-wide and gene-specific sweeps suggests that different models of bacterial speciation may apply to different populations coexisting in the same environment.
The National Center for Biotechnology Information [http://www.ncbi.nlm.nih.gov/guide/taxonomy/] database enlists more than 15,500 bacterial species. But this also includes a plethora of uncultured bacterial representations. Owing to their metabolism, they directly influence biogeochemical cycles, which underscores the the important status of bacteria on our planet. To study the function of a gene from an uncultured bacterium, we have undertaken a de novo gene synthesis approach. Actinobacteria of the acI-B subcluster are important but yet uncultured members of the bacterioplankton in temperate lakes of the northern hemisphere such as oligotrophic Lake Stechlin (NE Germany). This lake is relatively poor in phosphate (P) and harbors on average ~1.3 x 10 6 bacterial cells/ml, whereby Actinobacteria of the ac-I lineage can contribute to almost half of the entire bacterial community depending on seasonal variability. Single cell genome analysis of Actinobacterium SCGC AB141-P03, a member of the acI-B tribe in Lake Stechlin has revealed several phosphate-metabolizing genes. The genome of acI-B Actinobacteria indicates potential to degrade polyphosphate compound. To test for this genetic potential, we targeted the exoP-annotated gene potentially encoding polyphosphatase and synthesized it artificially to examine its biochemical role. Heterologous overexpression of the gene in Escherichia coli and protein purification revealed phosphatase activity. Comparative genome analysis suggested that homologs of this gene should be also present in other Actinobacteria of the acI lineages. This strategic retention of specialized genes in their genome provides a metabolic advantage over other members of the aquatic food web in a P-limited ecosystem. [Int Microbiol 2016; 19(1):39-47].
Previous studies on enhanced biological phosphorus removal (EBPR) have focused on reconstructing genomic blueprints for the model polyphosphate-accumulating organism Candidatus Accumulibacter phosphatis. Here, a time series metatranscriptome generated from enrichment cultures of Accumulibacter was used to gain insight into anerobic/aerobic metabolism and regulatory mechanisms within an EBPR cycle. Co-expressed gene clusters were identified displaying ecologically relevant trends consistent with batch cycle phases. Transcripts displaying increased abundance during anerobic acetate contact were functionally enriched in energy production and conversion, including upregulation of both cytoplasmic and membrane-bound hydrogenases demonstrating the importance of transcriptional regulation to manage energy and electron flux during anerobic acetate contact. We hypothesized and demonstrated hydrogen production after anerobic acetate contact, a previously unknown strategy for Accumulibacter to maintain redox balance. Genes involved in anerobic glycine utilization were identified and phosphorus release after anerobic glycine contact demonstrated, suggesting that Accumulibacter routes diverse carbon sources to acetyl-CoA formation via previously unrecognized pathways. A comparative genomics analysis of sequences upstream of co-expressed genes identified two statistically significant putative regulatory motifs. One palindromic motif was identified upstream of genes involved in PHA synthesis and acetate activation and is hypothesized to be a phaR binding site, hence representing a hypothetical PHA modulon. A second motif was identified ~35 base pairs (bp) upstream of a large and diverse array of genes and hence may represent a sigma factor binding site. This analysis provides a basis and framework for further investigations into Accumulibacter metabolism and the reconstruction of regulatory networks in uncultured organisms.
Microorganisms are usually studied either in highly complex natural communities or in isolation as monoclonal model populations that we manage to grow in the laboratory. Here, we uncover the biology of some of the most common and yet-uncultured bacteria in freshwater environments using a mixed culture from Lake Grosse Fuchskuhle. From a single shotgun metagenome of a freshwater mixed culture of low complexity, we recovered four high-quality metagenome-assembled genomes (MAGs) for metabolic reconstruction. This analysis revealed the metabolic interconnectedness and niche partitioning of these naturally dominant bacteria. In particular, vitamin- and amino acid biosynthetic pathways were distributed unequally with a member of Crenarchaeota most likely being the sole producer of vitamin B12 in the mixed culture. Using coverage-based partitioning of the genes recovered from a single MAG intrapopulation metabolic complementarity was revealed pointing to 'social' interactions for the common good of populations dominating freshwater plankton. As such, our MAGs highlight the power of mixed cultures to extract naturally occurring 'interactomes' and to overcome our inability to isolate and grow the microbes dominating in nature.
The environmental drivers contributing to cyanobacterial dominance in aquatic systems have been extensively studied. However, understanding of toxic vs. non-toxic cyanobacterial population dynamics and the mechanisms regulating cyanotoxin production remain elusive, both physiologically and ecologically. One reason is the disconnect between laboratory and field-based studies. Here, we combined 3 years of temporal data, including microcystin (MC) concentrations, 16 years of long-term ecological research, and 10 years of molecular data to investigate the potential factors leading to the selection of toxic Microcystis and MC production. Our analysis revealed that nitrogen (N) speciation and inorganic carbon (C) availability might be important drivers of Microcystis population dynamics and that an imbalance in cellular C: N ratios may trigger MC production. More specifically, precipitous declines in ammonium concentrations lead to a transitional period of N stress, even in the presence of high nitrate concentrations, that we call the "toxic phase." Following the toxic phase, temperature and cyanobacterial abundance remained elevated but MC concentrations drastically declined. Increases in ammonium due to lake turnover may have led to down regulation of MC synthesis or a shift in the community from toxic to non-toxic species. While total phosphorus (P) to total N ratios were relatively low over the time-series, MC concentrations were highest when total N to total P ratios were also highest. Similarly, high C: N ratios were also strongly correlated to the toxic phase. We propose a metabolic model that corroborates molecular studies and reflects our ecological observations that C and N metabolism may regulate MC production physiologically and ecologically. In particular, we hypothesize that an imbalance between 2-oxoglutarate and ammonium in the cell regulates MC synthesis in the environment.
Cyanobacterial harmful algal blooms (cyanoHABs) are a primary source of water quality degradation in eutrophic lakes. The occurrence of cyanoHABs is ubiquitous and expected to increase with current climate and land use change scenarios. However, it is currently unknown what environmental parameters are important for indicating the presence of cyanoHAB toxins making them difficult to predict or even monitor on time-scales relevant to protecting public health. Using qPCR, we aimed to quantify genes within the microcystin operon (mcy) to determine which cyanobacterial taxa, and what percentage of the total cyanobacterial community, were responsible for microcystin production in four eutrophic lakes. We targeted Microcystis-16S, mcyA, and Microcystis, Planktothrix, and Anabaena-specific mcyE genes. We also measured microcystins and several biological, chemical, and physical parameters--such as temperature, lake stability, nutrients, pigments and cyanobacterial community composition (CCC)--to search for possible correlations to gene copy abundance and MC production. All four lakes contained Microcystis-mcyE genes and high percentages of toxic Microcystis, suggesting Microcystis was the dominant microcystin producer. However, all genes were highly variable temporally, and in few cases, correlated with increased temperature and nutrients as the summer progressed. Interestingly, toxin gene abundances (and biomass indicators) were anti-correlated with microcystin in all lakes except the largest lake, Lake Mendota. Similarly, gene abundance and microcystins differentially correlated to CCC in all lakes. Thus, we conclude that the presence of microcystin genes are not a useful tool for eliciting an ecological role for toxins in the environment, nor are microcystin genes (e.g. DNA) a good indicator of toxins in the environment.
Members of the acI lineage of Actinobacteria are the most abundant microorganisms in most freshwater lakes; however, our understanding of the keys to their success and their role in carbon and nutrient cycling in freshwater systems has been hampered by the lack of pure cultures and genomes. We obtained draft genome assemblies from 11 single cells representing three acI tribes (acI-A1, acI-A7, acI-B1) from four temperate lakes in the United States and Europe. Comparative analysis of acI SAGs and other available freshwater bacterial genomes showed that acI has more gene content directed toward carbohydrate acquisition as compared to Polynucleobacter and LD12 Alphaproteobacteria, which seem to specialize more on carboxylic acids. The acI genomes contain actinorhodopsin as well as some genes involved in anaplerotic carbon fixation indicating the capacity to supplement their known heterotrophic lifestyle. Genome-level differences between the acI-A and acI-B clades suggest specialization at the clade level for carbon substrate acquisition. Overall, the acI genomes appear to be highly streamlined versions of Actinobacteria that include some genes allowing it to take advantage of sunlight and N-rich organic compounds such as polyamines, di- and oligopeptides, branched-chain amino acids and cyanophycin. This work significantly expands the known metabolic potential of the cosmopolitan freshwater acI lineage and its ecological and genetic traits.
For eutrophic lakes, patterns of phosphorus (P) measured by standard methods are well documented but provide little information about the components comprising standard operational definitions. Dissolved P (DP) and particulate P (PP) represents important but rarely characterized nutrient pools. Samples from Lake Mendota, Wisconsin, USA were characterized using 31-phosphorus nuclear magnetic resonance spectroscopy ((31)P NMR) during the open water season of 2011 in this unmatched temporal study of aquatic P dynamics. A suite of organic and inorganic P forms was detected in both dissolved and particulate fractions: orthophosphate, orthophosphate monoesters, orthophosphate diesters, pyrophosphate, polyphosphate, and phosphonates. Through time, phytoplankton biomass, temperature, dissolved oxygen, and water clarity were correlated with changes in the relative proportion of P fractions. Particulate P can be used as a proxy for phytoplankton-bound P, and in this study, a high proportion of polyphosphate within particulate samples suggested P should not be a limiting factor for the dominant primary producers, cyanobacteria. Hypolimnetic particulate P samples were more variable in composition than surface samples, potentially due to varying production and transport of sinking particles. Surface dissolved samples contained less P than particulate samples, and were typically dominated by orthophosphate, but also contained monoester, diester, polyphosphate, pyrophosphate, and phosphonate. Hydrologic inflows to the lake contained more orthophosphate and orthophosphate monoesters than in-lake samples, indicating transformation of P from inflowing waters. This time series explores trends of a highly regulated nutrient in the context of other water quality metrics (chlorophyll, mixing regime, and clarity), and gives insight on the variability of the structure and occurrence of P-containing compounds in light of the phosphorus-limited paradigm.
Little is known about the diversity and structuring of freshwater microbial communities beyond the patterns revealed by tracing their distribution in the landscape with common taxonomic markers such as the ribosomal RNA. To address this gap in knowledge, metagenomes from temperate lakes were compared to selected marine metagenomes. Taxonomic analyses of rRNA genes in these freshwater metagenomes confirm the previously reported dominance of a limited subset of uncultured lineages of freshwater bacteria, whereas Archaea were rare. Diversification into marine and freshwater microbial lineages was also reflected in phylogenies of functional genes, and there were also significant differences in functional beta-diversity. The pathways and functions that accounted for these differences are involved in osmoregulation, active transport, carbohydrate and amino acid metabolism. Moreover, predicted genes orthologous to active transporters and recalcitrant organic matter degradation were more common in microbial genomes from oligotrophic versus eutrophic lakes. This comparative metagenomic analysis allowed us to formulate a general hypothesis that oceanic- compared with freshwater-dwelling microorganisms, invest more in metabolism of amino acids and that strategies of carbohydrate metabolism differ significantly between marine and freshwater microbial communities.
Actinobacteria of the acI lineage are often the numerically dominant bacterial phylum in surface freshwaters, where they can account for > 50% of total bacteria. Despite their abundance, there are no described isolates. In an effort to obtain enrichment of these ubiquitous freshwater Actinobacteria, diluted freshwater samples from Lake Grosse Fuchskuhle, Germany, were incubated in 96-well culture plates. With this method, a successful enrichment containing high abundances of a member of the lineage acI was established. Phylogenetic classification showed that the acI Actinobacteria of the enrichment belonged to the acI-B2 tribe, which seems to prefer acidic lakes. This enrichment grows to low cell densities and thus the oligotrophic nature of acI-B2 was confirmed.
Members of the genus Candidatus Accumulibacter are important in many wastewater treatment systems performing enhanced biological phosphorus removal (EBPR). The Accumulibacter lineage can be subdivided phylogenetically into multiple clades, and previous work showed that these clades are ecologically distinct. The complete genome of Candidatus Accumulibacter phosphatis strain UW-1, a member of Clade IIA, was previously sequenced. Here, we report a draft genome sequence of Candidatus Accumulibacter spp. strain UW-2, a member of Clade IA, assembled following shotgun metagenomic sequencing of laboratory-scale bioreactor sludge. We estimate the genome to be 80-90% complete. Although the two clades share 16S rRNA sequence identity of >98.0%, we observed a remarkable lack of synteny between the two genomes. We identified 2317 genes shared between the two genomes, with an average nucleotide identity (ANI) of 78.3%, and accounting for 49% of genes in the UW-1 genome. Unlike UW-1, the UW-2 genome seemed to lack genes for nitrogen fixation and carbon fixation. Despite these differences, metabolic genes essential for denitrification and EBPR, including carbon storage polymer and polyphosphate metabolism, were conserved in both genomes. The ANI from genes associated with EBPR was statistically higher than that from genes not associated with EBPR, indicating a high selective pressure in EBPR systems. Further, we identified genomic islands of foreign origins including a near-complete lysogenic phage in the Clade IA genome. Interestingly, Clade IA appeared to be more phage susceptible based on it containing only a single Clustered Regularly Interspaced Short Palindromic Repeats locus as compared with the two found in Clade IIA. Overall, the comparative analysis provided a genetic basis to understand physiological differences and ecological niches of Accumulibacter populations, and highlights the importance of diversity in maintaining system functional resilience.
Activated sludge is one of the most abundant and effective wastewater treatment process used to treat wastewater, and has been used in developed countries for nearly a century. In all that time, several hundreds of studies have explored the bacterial communities responsible for treatment, but most studies were based on a handful of samples and did not consider temporal dynamics. In this study, we used the DNA fingerprinting technique called automated ribosomal intergenic spacer region analysis (ARISA) to study bacterial community dynamics over a two-year period in two different treatment trains. We also used quantitative PCR to measure the variation of five phylogenetically-defined clades within the Accumulibacter lineage, which is a model polyphosphate accumulating organism. The total bacterial community exhibited seasonal patterns of change reminiscent of those observed in lakes and oceans. Surprisingly, all five Accumulibacter clades were present throughout the study, and the total Accumulibacter community was relatively stable. However, the abundance of each clade did fluctuate through time. Clade IIA dynamics correlated positively with temperature (ρ = 0.65, p < 0.05) while Clade IA dynamics correlated negatively with temperature (ρ = -0.35, p < 0.05). This relationship with temperature hints at the mechanisms that may be driving the seasonal patterns in overall bacterial community dynamics and provides further evidence for ecological differentiation among clades within the Accumulibacter lineage. This work provides a valuable baseline for activated sludge bacterial community variation.
The objectives of this study were to characterize development of bacterial and archaeal populations during biodegradation of municipal solid waste (MSW) and to link specific methanogens to methane generation. Experiments were conducted in three 0.61-m-diameter by 0.90-m-tall laboratory reactors to simulate MSW bioreactor landfills. Pyrosequencing of 16S rRNA genes was used to characterize microbial communities in both leachate and solid waste. Microbial assemblages in effluent leachate were similar between reactors during peak methane generation. Specific groups within the Bacteroidetes and Thermatogae phyla were present in all samples and were particularly abundant during peak methane generation. Microbial communities were not similar in leachate and solid fractions assayed at the end of reactor operation; solid waste contained a more abundant bacterial community of cellulose-degrading organisms (e.g., Firmicutes). Specific methanogen populations were assessed using quantitative polymerase chain reaction. Methanomicrobiales, Methanosarcinaceae, and Methanobacteriales were the predominant methanogens in all reactors, with Methanomicrobiales consistently the most abundant. Methanogen growth phases coincided with accelerated methane production, and cumulative methane yield increased with increasing total methanogen abundance. The difference in methanogen populations and corresponding methane yield is attributed to different initial cellulose and hemicellulose contents of the MSW. Higher initial cellulose and hemicellulose contents supported growth of larger methanogen populations that resulted in higher methane yield.
Spatial and temporal variability in cyanobacterial community composition (CCC) within and between eutrophic lakes is not well-described using culture independent molecular methods. We analyzed CCC across twelve locations in four eutrophic lakes and within-lake locations in the Yahara Watershed, WI, on a weekly basis, for 5 months. Taxa were discriminated by length of MspI-digested cpcB/A intergenic spacer gene sequences and identified by comparison to a PCR-based clone library. CCC across all stations was spatially segregated by depth of sampling locations (ANOSIM R = 0.23, p < 0.001). Accordingly, CCC was correlated with thermal stratification, nitrate and soluble reactive phosphorus (SRP, R = 0.2-0.3). Spatial variability in CCC and temporal trends in taxa abundances were rarely correlative between sampling locations in the same lake indicating significant within lake spatiotemporal heterogeneity. Across all stations, a total of 37 bloom events were observed based on distinct increases in phycocyanin. Out of 97 taxa, a single Microcystis, and two different Aphanizomenon taxa were the dominant cyanobacteria detected during bloom events. The Microcystis and Aphanizomenon taxa rarely bloomed together and were significantly anti-correlated with each other at 9 of 12 stations with Pearson R values of -0.6 to -0.9 (p < 0.001). Of all environmental variables measured, nutrients, especially nitrate were significantly greater during periods of Aphanizomenon dominance while the nitrate+nitrite:SRP ratio was lower. This study shows significant spatial variability in CCC within and between lakes structured by depth of the sampling location. Furthermore, our study reveals specific genotypes involved in bloom formation. More in-depth characterization of these genotypes should lead to a better understanding of factors promoting bloom events in these lakes and more reliable bloom prediction models.
Phosphorus is a key element controlling the productivity of freshwater ecosystems, and microbes drive most of its relevant biogeochemistry. Eutrophic lakes are generally dominated by cyanobacteria that compete fiercely with algae and heterotrophs for the element. In wastewater treatment, engineers select for specialized bacteria capable of sequestering phosphorus from the water, to protect surface waters from further loading. The intracellular storage molecule polyphosphate plays an important role in both systems, allowing key taxa to control phosphorus availability. The importance of dissolved organic phosphorus in eutrophic lakes and mineralization mechanisms is still underappreciated and understudied. The need for functional redundancy through biological diversity in wastewater treatment plants is also clear. In both systems, a holistic ecosystems biology approach is needed to understand the molecular mechanisms controlling phosphorus metabolism and the ecological interactions and factors controlling ecosystem-level process rates.
With an unprecedented decade-long time series from a temperate eutrophic lake, we analyzed bacterial and environmental co-occurrence networks to gain insight into seasonal dynamics at the community level. We found that (1) bacterial co-occurrence networks were non-random, (2) season explained the network complexity and (3) co-occurrence network complexity was negatively correlated with the underlying community diversity across different seasons. Network complexity was not related to the variance of associated environmental factors. Temperature and productivity may drive changes in diversity across seasons in temperate aquatic systems, much as they control diversity across latitude. While the implications of bacterioplankton network structure on ecosystem function are still largely unknown, network analysis, in conjunction with traditional multivariate techniques, continues to increase our understanding of bacterioplankton temporal dynamics.
Toxic cyanobacterial blooms threaten freshwaters worldwide but have proven difficult to predict because the mechanisms of bloom formation and toxin production are unknown, especially on weekly time scales. Water quality management continues to focus on aggregated metrics, such as chlorophyll and total nutrients, which may not be sufficient to explain complex community changes and functions such as toxin production. For example, nitrogen (N) speciation and cycling play an important role, on daily time scales, in shaping cyanobacterial communities because declining N has been shown to select for N fixers. In addition, subsequent N pulses from N(2) fixation may stimulate and sustain toxic cyanobacterial growth. Herein, we describe how rapid early summer declines in N followed by bursts of N fixation have shaped cyanobacterial communities in a eutrophic lake (Lake Mendota, Wisconsin, USA), possibly driving toxic Microcystis blooms throughout the growing season. On weekly time scales in 2010 and 2011, we monitored the cyanobacterial community in a eutrophic lake using the phycocyanin intergenic spacer (PC-IGS) region to determine population dynamics. In parallel, we measured microcystin concentrations, N(2) fixation rates, and potential environmental drivers that contribute to structuring the community. In both years, cyanobacterial community change was strongly correlated with dissolved inorganic nitrogen (DIN) concentrations, and Aphanizomenon and Microcystis alternated dominance throughout the pre-toxic, toxic, and post-toxic phases of the lake. Microcystin concentrations increased a few days after the first significant N(2) fixation rates were observed. Then, following large early summer N(2) fixation events, Microcystis increased and became most abundant. Maximum microcystin concentrations coincided with Microcystis dominance. In both years, DIN concentrations dropped again in late summer, and N(2) fixation rates and Aphanizomenon abundance increased before the lake mixed in the fall. Estimated N inputs from N(2) fixation were large enough to supplement, or even support, the toxic Microcystis blooms.
A great challenge facing microbial ecology is how to define ecologically relevant taxonomic units. To address this challenge, we investigated how changing the definition of operational taxonomic units (OTUs) influences the perception of ecological patterns in microbial communities as they respond to a dramatic environmental change. We used pyrosequenced tags of the bacterial V2 16S rRNA region, as well as clone libraries constructed from the cytochrome oxidase C gene ccoN, to provide additional taxonomic resolution for the common freshwater genus Polynucleobacter. At the most highly resolved taxonomic scale, we show that distinct genotypes associated with the abundant Polynucleobacter lineages exhibit divergent spatial patterns and dramatic changes over time, while the also abundant Actinobacteria OTUs are highly coherent. This clearly demonstrates that different bacterial lineages demand different taxonomic definitions to capture ecological patterns. Based on the temporal distribution of highly resolved taxa in the hypolimnion, we demonstrate that change in the population structure of a single genotype can provide additional insight into the mechanisms of community-level responses. These results highlight the importance and feasibility of examining ecological change in microbial communities across taxonomic scales while also providing valuable insight into the ecological characteristics of ecologically coherent groups in this system.
Actinobacteria within the acI lineage are often numerically dominating in freshwater ecosystems, where they can account for >50% of total bacteria in the surface water. However, they remain uncultured to date. We thus set out to use single-cell genomics to gain insights into their genetic make-up, with the aim of learning about their physiology and ecological niche. A representative from the highly abundant acI-B1 group was selected for shotgun genomic sequencing. We obtained a draft genomic sequence in 75 larger contigs (sum=1.16 Mb), with an unusually low genomic G+C mol% (∼42%). Actinobacteria core gene analysis suggests an almost complete genome recovery. We found that the acI-B1 cell had a small genome, with a rather low percentage of genes having no predicted functions (∼15%) as compared with other cultured and genome-sequenced microbial species. Our metabolic reconstruction hints at a facultative aerobe microorganism with many transporters and enzymes for pentoses utilization (for example, xylose). We also found an actinorhodopsin gene that may contribute to energy conservation under unfavorable conditions. This project reveals the metabolic potential of a member of the global abundant freshwater Actinobacteria.
Disturbances act as powerful structuring forces on ecosystems. To ask whether environmental microbial communities have capacity to recover after a large disturbance event, we conducted a whole-ecosystem manipulation, during which we imposed an intense disturbance on freshwater microbial communities by artificially mixing a temperate lake during peak summer thermal stratification. We employed environmental sensors and water chemistry analyses to evaluate the physical and chemical responses of the lake, and bar-coded 16S ribosomal RNA gene pyrosequencing and automated ribosomal intergenic spacer analysis (ARISA) to assess the bacterial community responses. The artificial mixing increased mean lake temperature from 14 to 20 °C for seven weeks after mixing ended, and exposed the microorganisms to very different environmental conditions, including increased hypolimnion oxygen and increased epilimnion carbon dioxide concentrations. Though overall ecosystem conditions remained altered (with hypolimnion temperatures elevated from 6 to 20 °C), bacterial communities returned to their pre-manipulation state as some environmental conditions, such as oxygen concentration, recovered. Recovery to pre-disturbance community composition and diversity was observed within 7 (epilimnion) and 11 (hypolimnion) days after mixing. Our results suggest that some microbial communities have capacity to recover after a major disturbance.
Understanding characteristic variation in aquatic bacterial community composition (BCC) across space and time can inform us about processes driving community assembly and the ability of bacterial communities to respond to perturbations. In this study, we synthesize BCC data from north temperate lakes to evaluate our current understanding of how BCC varies across multiple scales in time and space. A hierarchy of average similarity emerged with the highest similarity found among samples collected within the same lake, especially within the same basin, followed by similarity among samples collected through time within the same lake, and finally similarity among samples collected from different lakes. Using decay of similarity across time and space, we identified equivalent temporal (1 day) and spatial (10 m) scales of BCC variation. Finally, we identify an intriguing pattern of contrasting patterns of intra- and inter-annual BCC variation in two lakes. We argue our synthesis of spatio-temporal variation of aquatic BCC informs expectations for the response of aquatic bacterial communities to perturbation and environmental change. However, further long-term temporal observations will be needed to develop a general understanding of inter-annual BCC variation and our ability to use aquatic BCC as a sensitive metric of environmental change.
Coastal lagoons, both hypersaline and freshwater, are common, but still understudied ecosystems. We describe, for the first time, using high throughput sequencing, the extant microbiota of two large and representative Mediterranean coastal lagoons, the hypersaline Mar Menor, and the freshwater Albufera de Valencia, both located on the south eastern coast of Spain. We show there are considerable differences in the microbiota of both lagoons, in comparison to other marine and freshwater habitats. Importantly, a novel uncultured sulfur oxidizing Alphaproteobacteria was found to dominate bacterioplankton in the hypersaline Mar Menor. Also, in the latter prokaryotic cyanobacteria were almost exclusively comprised by Synechococcus and no Prochlorococcus was found. Remarkably, the microbial community in the freshwaters of the hypertrophic Albufera was completely in contrast to known freshwater systems, in that there was a near absence of well known and cosmopolitan groups of ultramicrobacteria namely Low GC Actinobacteria and the LD12 lineage of Alphaproteobacteria.
Free-living Actinobacteria are universally recognized as high-GC organisms. Freshwater Actinobacteria have been identified as abundant and prevalent members of freshwater microbial communities, but the two most common lineages (acI and acIV) have remained impossible to culture to date. We have analysed metagenomic data from lakes and estuaries, and show that members of acI and acIV are indeed abundant. We then show that the majority of actinobacterial reads from metagenomic datasets (both lakes and estuaries) are consistently low GC. Analysis of assembled scaffolds from these datasets also confirms that actinobacterial scaffolds are primarily low GC, although high-GC scaffolds were also observed, indicating both types of Actinobacteria coinhabit. Phylogenetic analysis of 16S rRNA gene sequences, both from PCR-based clone libraries and metagenomic reads, and the discovery of a low-GC scaffold containing a partial 16S rRNA gene, points to the abundance of the well-known acI and acIV lineages of freshwater in these habitats, both of which appear to be low GC.
We describe the microbiota of two hypersaline saltern ponds, one of intermediate salinity (19%) and a NaCl saturated crystallizer pond (37%) using pyrosequencing. The analyses of these metagenomes (nearly 784 Mb) reaffirmed the vast dominance of Haloquadratum walsbyi but also revealed novel, abundant and previously unsuspected microbial groups. We describe for the first time, a group of low GC Actinobacteria, related to freshwater Actinobacteria, abundant in low and intermediate salinities. Metagenomic assembly revealed three new abundant microbes: a low-GC euryarchaeon with the lowest GC content described for any euryarchaeon, a high-GC euryarchaeon and a gammaproteobacterium related to Alkalilimnicola and Nitrococcus. Multiple displacement amplification and sequencing of the genome from a single archaeal cell of the new low GC euryarchaeon suggest a photoheterotrophic and polysaccharide-degrading lifestyle and its relatedness to the recently described lineage of Nanohaloarchaea. These discoveries reveal the combined power of an unbiased metagenomic and single cell genomic approach.
Recent studies indicate genetic diversity of cyanobacteria in eutrophic lakes is not represented well by culture collections or morphology. Yet, few studies have investigated genetic richness and evenness of cyanobacteria using culture-independent methods. We compared the genetic structure of cyanobacteria supported by four neighboring eutrophic lakes during the ice-free season. The partial phycobilincpcB/A genes plus intergenic spacer (PC-IGS) was used as a genetic marker.Sequences were phylogeneticallygrouped by maximum likelihood into genotypes representing sub-genera of the major taxa. Genotypes fell into genera commonly observed by microscopy in these lakes including Microcystis, Aphanizomenon, Chroococcus, Anabaena, and Cylindrospermopsis. Only three genotypes were shared among all four lakes, despite significant water flowage between lakes.A Parsimony P-test indicated lakes were significantly (p=0.01) clustered on the maximum likelihood tree. Pairwise differences using Unifrac distance were moderately or not significant. Analysis of molecular variance (AMOVA) indicated genetic variation among all genotypes (φ=0.06, p<0.001) and 94% of variability occurred within lakes rather than between lakes (6%), explaining the lack of pairwise differences between lakes. Lorenze curves of genotype abundance in each lake showed genetic structure was only moderately uneven (Gini coefficients of 0.37-0.5) indicating lakes did not support dominant genotypes. Overall, results from this study suggest diversity of cyanobacteria is shaped by heterogeneity within lakes (temporally or spatially) and relatively even population structures.
For lake microbes, water column mixing acts as a disturbance because it homogenizes thermal and chemical gradients known to define the distributions of microbial taxa. Our first objective was to isolate hypothesized drivers of lake bacterial response to water column mixing. To accomplish this, we designed an enclosure experiment with three treatments to independently test key biogeochemical changes induced by mixing: oxygen addition to the hypolimnion, nutrient addition to the epilimnion, and full water column mixing. We used molecular fingerprinting to observe bacterial community dynamics in the treatment and control enclosures, and in ambient lake water. We found that oxygen and nutrient amendments simulated the physical-chemical water column environment following mixing and resulted in similar bacterial communities to the mixing treatment, affirming that these were important drivers of community change. These results demonstrate that specific environmental changes can replicate broad disturbance effects on microbial communities. Our second objective was to characterize bacterial community stability by quantifying community resistance, recovery and resilience to an episodic disturbance. The communities in the nutrient and oxygen amendments changed quickly (had low resistance), but generally matched the control composition by the 10th day after treatment, exhibiting resilience. These results imply that aquatic bacterial assemblages are generally stable in the face of disturbance.
'Candidatus Accumulibacter' is a biotechnologically important bacterial group that can accumulate large amounts of intracellular polyphosphate, contributing to biological phosphorus removal in wastewater treatment. Since its first molecular identification more than a decade ago, this bacterial group has drawn significant research attention due to its high abundance in many biological phosphorus removal systems. In the past 6 years, our understanding of Accumulibacter microbiology and ecophysiology has advanced rapidly, largely owing to genomic information obtained through shotgun metagenomic sequencing efforts. In this review, we focus on the metabolism, physiology, fine-scale population structure and ecological distribution of Accumulibacter, aiming to integrate the information learned so far and to present a more complete picture of the microbiology of this important bacterial group.
River water is a small percentage of the total freshwater on Earth but represents an essential resource for mankind. Microbes in rivers perform essential ecosystem roles including the mineralization of significant quantities of organic matter originating from terrestrial habitats. The Amazon river in particular is famous for its size and importance in the mobilization of both water and carbon out of its enormous basin. Here we present the first metagenomic study on the microbiota of this river. It presents many features in common with the other freshwater metagenome available (Lake Gatun in Panama) and much less similarity with marine samples. Among the microbial taxa found, the cosmopolitan freshwater acI lineage of the actinobacteria was clearly dominant. Group I Crenarchaea and the freshwater sister group of the marine SAR11 clade, LD12, were found alongside more exclusive and well known freshwater taxa such as Polynucleobacter. A metabolism-centric analysis revealed a disproportionate representation of pathways involved in heterotrophic carbon processing, as compared to those found in marine samples. In particular, these river microbes appear to be specialized in taking up and mineralizing allochthonous carbon derived from plant material.
The predominant strategy for using algae to produce biofuels relies on the overproduction of lipids in microalgae with subsequent conversion to biodiesel (methyl-esters) or green diesel (alkanes). Conditions that both optimize algal growth and lipid accumulation rarely overlap, and differences in growth rates can lead to wild species outcompeting the desired lipid-rich strains. Here, we demonstrate an alternative strategy in which cellulose contained in the cell walls of multicellular algae is used as a feedstock for cultivating biofuel-producing microorganisms. Cellulose was extracted from an environmental sample of Cladophora glomerata-dominated periphyton that was collected from Lake Mendota, WI, USA. The resulting cellulose cake was hydrolyzed by commercial enzymes to release fermentable glucose. The hydrolysis mixture was used to formulate an undefined medium that was able to support the growth, without supplementation, of a free fatty acid (FFA)-overproducing strain of Escherichia coli (Lennen et. al 2010). To maximize free fatty acid production from glucose, an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible vector was constructed to express the Umbellularia californica acyl-acyl carrier protein (ACP) thioesterase. Thioesterase expression was optimized by inducing cultures with 50 μM IPTG. Cell density and FFA titers from cultures grown on algae-based media reached 50% of those (∼90 μg/mL FFA) cultures grown on rich Luria-Bertani broth supplemented with 0.2% glucose. In comparison, cultures grown in two media based on AFEX-pretreated corn stover generated tenfold less FFA than cultures grown in algae-based media. This study demonstrates that macroalgal cellulose is a potential carbon source for the production of biofuels or other microbially synthesized compounds.
The relationship between phage and their microbial hosts is difficult to elucidate in complex natural ecosystems. Engineered systems performing enhanced biological phosphorus removal (EBPR), offer stable, lower complexity communities for studying phage-host interactions. Here, metagenomic data from an EBPR reactor dominated by Candidatus Accumulibacter phosphatis (CAP), led to the recovery of three complete and six partial phage genomes. Heat-stable nucleoid structuring (H-NS) protein, a global transcriptional repressor in bacteria, was identified in one of the complete phage genomes (EPV1), and was most similar to a homolog in CAP. We infer that EPV1 is a CAP-specific phage and has the potential to repress up to 6% of host genes based on the presence of putative H-NS binding sites in the CAP genome. These genes include CRISPR associated proteins and a Type III restriction-modification system, which are key host defense mechanisms against phage infection. Further, EPV1 was the only member of the phage community found in an EBPR microbial metagenome collected seven months prior. We propose that EPV1 laterally acquired H-NS from CAP providing it with a means to reduce bacterial defenses, a selective advantage over other phage in the EBPR system. Phage encoded H-NS could constitute a previously unrecognized weapon in the phage-host arms race.
We examined the effects of nutrient amendments on epilimnetic freshwater bacteria during three distinct periods in the eutrophic Lake Mendota's seasonal cycle (spring overturn, summer stratification and autumn overturn). Microcosm treatments enriched solely with phosphorus containing compounds did not result in a large bacterial community composition (BCC) change or community activity response (assessed via alkaline phosphatase activity, APA) relative to the controls during any season. Treatments enriched with carbon- and nitrogen-containing compounds resulted in a dramatic BCC change and a large APA increase in the autumn and spring seasons, but only treatments receiving carbon, nitrogen and phosphorus (CNP) exhibited similar responses in the summer season. Despite the fact that the amendments created similar CNP concentration conditions across seasons, the BCC following amendment greatly varied among seasons. 16S rRNA gene sequence analysis indicated that many common freshwater bacterial lineages from the Alpha- and Betaproteobacteria class and Bacteroidetes phylum were favoured following nutrient (CNP) addition, but individual taxa were generally not favoured across all seasons. Targeted quantitative PCR analysis revealed that the abundance of the Actinobacteria acIB1 cluster decreased in all microcosms during all three seasons, while the Flavobacterium aquatile (spring) and ME-B0 (summer) clusters of Bacteroidetes increased following CNP addition. These results suggest a particular bacterial group is not universally favoured by increased nutrient loads to a lake; therefore, efforts to predict which bacteria are involved in nutrient cycling during these periods must take into account the seasonality of freshwater bacterial communities.
Freshwater bacteria are at the hub of biogeochemical cycles and control water quality in lakes. Despite this, little is known about the identity and ecology of functionally significant lake bacteria. Molecular studies have identified many abundant lake bacteria, but there is a large variation in the taxonomic or phylogenetic breadths among the methods used for this exploration. Because of this, an inconsistent and overlapping naming structure has developed for freshwater bacteria, creating a significant obstacle to identifying coherent ecological traits among these groups. A discourse that unites the field is sorely needed. Here we present a new freshwater lake phylogeny constructed from all published 16S rRNA gene sequences from lake epilimnia and propose a unifying vocabulary to discuss freshwater taxa. With this new vocabulary in place, we review the current information on the ecology, ecophysiology, and distribution of lake bacteria and highlight newly identified phylotypes. In the second part of our review, we conduct meta-analyses on the compiled data, identifying distribution patterns for bacterial phylotypes among biomes and across environmental gradients in lakes. We conclude by emphasizing the role that this review can play in providing a coherent framework for future studies.
Enhanced biological phosphorus removal (EBPR) activated sludge communities enriched in 'Candidatus Accumulibacter' relatives are widely used in wastewater treatment, but much remains to be learned about molecular-level controls on the EBPR process. The expression of genes found in the carbon and polyphosphate metabolic pathways in Accumulibacter was investigated using reverse transcription quantitative PCR. During a normal anaerobic/aerobic EBPR cycle, gene expression exhibited a dynamic change in response to external acetate, oxygen, phosphate concentrations and probably internal chemical pools. Anaerobic acetate addition induced expression of genes associated with the methylmalonyl-CoA pathway enabling the split mode of the tricarboxylic acid (TCA) cycle. Components of the full TCA cycle were induced after the switch to aerobic conditions. The induction of a key gene in the glyoxylate shunt pathway was observed under both anaerobic and aerobic conditions, with a higher induction by aeration. Polyphosphate kinase 1 from Accumulibacter was expressed, but did not appear to be regulated by phosphate limitation. To understand how Accumulibacter responds to disturbed electron donor and acceptor conditions, we perturbed the process by adding acetate aerobically. When high concentrations of oxygen were present simultaneously with acetate, phosphate-release was almost completely inhibited, and polyphosphate kinase 1 transcript abundance decreased. Genes associated with the methylmalonyl-CoA pathway were repressed and genes associated with the aerobic TCA cycle exhibited higher expression under this perturbation, suggesting that more acetyl-CoA was metabolized through the TCA cycle. These findings suggest that several genes involved in EBPR are tightly regulated at the transcriptional level.
"Candidatus Accumulibacter" and total bacterial community dynamics were studied in two lab-scale enhanced biological phosphorus removal (EBPR) reactors by using a community fingerprint technique, automated ribosomal intergenic spacer analysis (ARISA). We first evaluated the quantitative capability of ARISA compared to quantitative real-time PCR (qPCR). ARISA and qPCR provided comparable relative quantification of the two dominant "Ca. Accumulibacter" clades (IA and IIA) detected in our reactors. The quantification of total "Ca. Accumulibacter" 16S rRNA genes relative to that from the total bacterial community was highly correlated, with ARISA systematically underestimating "Ca. Accumulibacter" abundance, probably due to the different normalization techniques applied. During 6 months of normal (undisturbed) operation, the distribution of the two clades within the total "Ca. Accumulibacter" population was quite stable in one reactor while comparatively dynamic in the other reactor. However, the variance in the clade distribution did not appear to affect reactor performance. Instead, good EBPR activity was positively associated with the abundance of total "Ca. Accumulibacter." Therefore, we concluded that the different clades in the system provided functional redundancy. We disturbed the reactor operation by adding nitrate together with acetate feeding in the anaerobic phase to reach initial reactor concentrations of 10 mg/liter NO(3)-N for 35 days. The reactor performance deteriorated with a concomitant decrease in the total "Ca. Accumulibacter" population, suggesting that a population shift was the cause of performance upset after a long exposure to nitrate in the anaerobic phase.
Here we report the first metatranscriptomic analysis of gene expression and regulation of 'Candidatus Accumulibacter'-enriched lab-scale sludge during enhanced biological phosphorus removal (EBPR). Medium density oligonucleotide microarrays were generated with probes targeting most predicted genes hypothesized to be important for the EBPR phenotype. RNA samples were collected at the early stage of anaerobic and aerobic phases (15 min after acetate addition and switching to aeration respectively). We detected the expression of a number of genes involved in the carbon and phosphate metabolisms, as proposed by EBPR models (e.g. polyhydroxyalkanoate synthesis, a split TCA cycle through methylmalonyl-CoA pathway, and polyphosphate formation), as well as novel genes discovered through metagenomic analysis. The comparison between the early stage anaerobic and aerobic gene expression profiles showed that expression levels of most genes were not significantly different between the two stages. The majority of upregulated genes in the aerobic sample are predicted to encode functions such as transcription, translation and protein translocation, reflecting the rapid growth phase of Accumulibacter shortly after being switched to aerobic conditions. Components of the TCA cycle and machinery involved in ATP synthesis were also upregulated during the early aerobic phase. These findings support the predictions of EBPR metabolic models that the oxidation of intracellularly stored carbon polymers through the TCA cycle provides ATP for cell growth when oxygen becomes available. Nitrous oxide reductase was among the very few Accumulibacter genes upregulated in the anaerobic sample, suggesting that its expression is likely induced by the deprivation of oxygen.
The contribution of human activities to environmental reservoirs of antibiotic resistance is poorly understood. The purpose of this study was to determine if oxytetracycline (OTC) use in aquaculture facilities increased the detection frequency (i.e., prevalence) of tetracycline resistance (tet(R)) genes relative to facilities with no recent OTC treatment. We used polymerase chain reaction to screen water and sediment from four noncommercial fish farms in northwestern Wisconsin for the presence of ten tet(R) determinants: tet(A), tet(B), tet(D), tet(E), tet(G), tet(M), tet(O), tet(Q), tet(S), and tet(W). Water from farms with recent OTC use had significantly higher tet(R) detection frequencies than did water from farms without recent OTC use, with prevalence in raceways and rearing ponds of farms with recent OTC use exceeding by more than twofold that of farms not using OTC. Effluent from all farms, regardless of treatment regime, had higher tet(R) detection frequencies than their corresponding influent for all genes, but the specific combinations of tet(R) genes detected in a sample were not different from their corresponding influent. Although OTC use was associated with the increased occurrence and diversity of tet(R) genes in water samples, it was not found to relate to tet(R) gene occurrence in sediment samples. Sediment samples from facilities with no recent OTC use had significantly higher frequencies of tet(R) gene detection than did samples from facilities with recent OTC use. All of the tet(R) genes were detected in both the medicated and nonmedicated feed samples analyzed in this study. These findings suggest that both OTC treatment in aquaculture facilities and the farms themselves may be sources of tet(R) gene introduction to the environment. To our knowledge, this is the first study to use genotypic and cultivation-independent methods to examine tet(R) gene occurrence associated with OTC use in aquaculture.
Yuan Yang Lake (YYL), Taiwan, experiences both winter and typhoon-initiated mixing, and each type of mixing event is characterized by contrasting environmental conditions. Previous work suggested that after typhoon mixing, bacterial communities in YYL reset to a pioneer composition and then follow a predictable trajectory of change until the next typhoon. Our goal was to continue this investigation by observing bacterial community change after a range of mixing intensities, including seasonal winter mixing. We fingerprinted aquatic bacterial communities in the epilimnion and hypolimnion using automated ribosomal intergenic spacer analysis and then assessed community response using multivariate statistics. We found a significant linear relationship between water column stability and the epilimnion to hypolimnion divergences. In comparison to the summer, we found the winter community had a distinct composition and less variation. We divided the bacterial community into population subsets according to abundance (rare, common, or dominant) and occurrence (transient or persistent) and further explored the contribution of these subsets to the overall community patterns. We found that transient taxa did not drive bacterial community patterns following weak typhoon mixing events, but contributed substantially to patterns observed following strong events. Common taxa generally did not follow the community trajectory after weak or strong events. Our results suggest intensity, frequency, and seasonality jointly contribute to aquatic bacterial response to mixing disturbance.
Lake mixing disrupts chemical and physical gradients that structure bacterial communities. A transplant experiment was designed to investigate the influence of post-mixing environmental conditions and biotic interactions on bacterial community composition. The experimental design was 3x2 factorial, where water was incubated from three different sources (epilimnion, hypolimnion, and mixed epilimnion and hypolimnion) at two different locations in the water column (epilimnion or hypolimnion). Three replicate mesocosms of each treatment were removed every day for 5 days for bacterial community profiling, assessed by automated ribosomal intergenic spacer analysis. There were significant treatment effects observed, and temperature was the strongest measured driver of community change (r=-0.66). Epilimnion-incubated communities changed more than hypolimnion-incubated. Across all treatments, we classified generalist, layer-preferential and layer-specialist populations based on occurrence patterns. Most classified populations were generalists that occurred in both strata, suggesting that communities were robust to mixing. In a network analysis of the mixed-inocula treatments, there was correlative evidence of inter-population biotic interactions, where many of these interactions involved generalists. These results reveal differential responses of bacterial populations to lake mixing and highlight the role of generalist taxa in structuring an emergent community-level response.
The capability of "Candidatus Accumulibacter" to use nitrate as an electron acceptor for phosphorus uptake was investigated using two activated sludge communities. The two communities were enriched in Accumulibacter clade IA and clade IIA, respectively. By performing a series of batch experiments, we found that clade IA was able to couple nitrate reduction with phosphorus uptake, but clade IIA could not. These results agree with a previously proposed hypothesis that different populations of Accumulibacter have different nitrate reduction capabilities, and they will help to understand the ecological roles that these two clades provide.
Water entering lakes from the surrounding watershed often delivers large amounts of terrestrial-derived dissolved organic carbon (DOC) that can contribute to aquatic bacterial production. However, research suggests that phytoplankton-derived DOC is more labile than its terrestrial counterpart, owing to microbial processing of terrestrial-derived DOC along its flow path to surface waters. The ratio of water colour (absorbance at 440 nm) to chlorophyll a has been suggested as a simple measure of the relative contribution of terrestrial and aquatic primary production to aquatic secondary production. To explore the correlation between primary DOC source and the occurrence of bacterial taxonomic groups, we conducted a survey of bacterial 16S rRNA gene composition in 15 lakes positioned along a water colour : chlorophyll a gradient. Our goal was to identify bacterial taxa occurrence patterns along the colour : chlorophyll a gradient that may indicate a competitive advantage for bacterial taxa using terrestrial or aquatic carbon. We observed a large number of bacterial taxa occurrence patterns suggestive of carbon substrate niche partitioning, especially when relatively highly resolved taxonomic groups were considered. Our survey supports the hypothesis that bacterial taxa partition along a carbon substrate source gradient and highlights carbon source-bacterial interactions that should be explored further.
Automated sensing technologies, 'ASTs,' are tools that can monitor environmental or microbial-related variables at increasingly high temporal resolution. Microbial ecologists are poised to use AST data to couple microbial structure, function and associated environmental observations on temporal scales pertinent to microbial processes. In the context of aquatic microbiology, we discuss three applications of ASTs: windows on the microbial world, adaptive sampling and adaptive management. We challenge microbial ecologists to push AST potential in helping to reveal relationships between microbial structure and function.
Long distance atmospheric transport of bacterial cells is often implied as a driver of the apparent cosmopolitan distribution of bacterial taxa. Surprisingly, efforts to measure immigration in bacterial communities are rare. An 8-week time series of within-lake bacterial community composition and atmospheric deposition rates and composition were used to estimate the influence of immigration on bacterial community dynamics in two north temperate lakes. Characterization of bacterial community dynamics using automated ribosomal intergenic spacer analysis suggested moderate overlap in composition between the lakes and atmospherically deposited cells. However, taxa that appeared to be delivered by atmospheric deposition had a relatively minor influence on lake bacterial community dynamics. The weak influence of immigrating bacterial taxa suggests that a species-sorting concept best describes aquatic bacterial metacommunity dynamics.
Members of the uncultured bacterial genus Candidatus Accumulibacter are capable of intracellular accumulation of inorganic phosphate in activated sludge wastewater treatment plants (WWTPs) performing enhanced biological phosphorus removal, but were also recently shown to inhabit freshwater and estuarine sediments. Additionally, metagenomic sequencing of two bioreactor cultures enriched in Candidatus Accumulibacter, but housed on separate continents, revealed the potential for global dispersal of particular Candidatus Accumulibacter strains, which we hypothesize is facilitated by the ability of Candidatus Accumulibacter to persist in environmental habitats. In the current study, we used sequencing of a phylogenetic marker, the ppk1 gene, to characterize Candidatus Accumulibacter populations in diverse environments, at varying distances from WWTPs. We discovered several new lineages of Candidatus Accumulibacter which had not previously been detected in WWTPs, and also uncovered new diversity and structure within previously detected lineages. Habitat characteristics were found to be a key determinant of Candidatus Accumulibacter lineage distribution while, as predicted, geographic distance played little role in limiting dispersal on a regional scale. However, on a local scale, enrichment of particular Candidatus Accumulibacter lineages in WWTP appeared to impact local environmental populations. These results provide evidence of ecological differences among Candidatus Accumulibacter lineages.
Transmissible spongiform encephalopathies (TSEs, prion diseases) are a class of fatal neurodegenerative diseases affecting a variety of mammalian species including humans. A misfolded form of the prion protein (PrP(TSE)) is the major, if not sole, component of the infectious agent. Prions are highly resistant to degradation and to many disinfection procedures suggesting that, if prions enter wastewater treatment systems through sewers and/or septic systems (e.g., from slaughterhouses, necropsy laboratories, rural meat processors, private game dressing) or through leachate from landfills that have received TSE-contaminated material, prions could survive conventional wastewater treatment. Here, we report the results of experiments examining the partitioning and persistence of PrPTSE during simulated wastewater treatment processes including activated and mesophilic anaerobic sludge digestion. Incubation with activated sludge did not result in significant PrPTSE degradation. PrPTSE and prion infectivity partitioned strongly to activated sludge solids and are expected to enter biosolids treatment processes. A large fraction of PrPTSE survived simulated mesophilic anaerobic sludge digestion. The small reduction in recoverable PrPTSE after 20-d anaerobic sludge digestion appeared attributable to a combination of declining extractability with time and microbial degradation. Our results suggest that if prions were to enter municipal wastewater treatment systems, most would partition to activated sludge solids, survive mesophilic anaerobic digestion, and be present in treated biosolids.
Biogeographic patterns in microbial communities are an exciting but controversial topic in microbial ecology. Advances in theory pertaining to assembly of microbial communities have made strong assumptions about dispersal of bacteria without exploration. For this reason, we investigated rates of atmospheric bacterial deposition and compared the taxonomic composition of bacteria in rain with that of common freshwater bacterial communities. Our findings suggest that it is not appropriate to take for granted that atmospheric deposition of bacteria is a significant vector of immigration to freshwater ecosystems.
Multiple forces structure natural microbial communities, but the relative roles and interactions of these drivers are poorly understood. Gradients of physical and chemical parameters can be especially influential. In traditional ecological theory, variability in environmental conditions across space and time represents habitat heterogeneity, which may shape communities. Here we used aquatic microbial communities as a model to investigate the relationship between habitat heterogeneity and community composition and dynamics. We defined spatial habitat heterogeneity as vertical temperature and dissolved oxygen (DO) gradients in the water column, and temporal habitat heterogeneity as variation throughout the open-water season in these environmental parameters. Seasonal lake mixing events contribute to temporal habitat heterogeneity by destroying and re-creating these gradients. Because of this, we selected three lakes along a range of annual mixing frequency (polymictic, dimictic, meromictic) for our study. We found that bacterial community composition (BCC) was distinct between the epilimnion and hypolimnion within stratified lakes, and also more variable within the epilimnia through time. We found stark differences in patterns of epilimnion and hypolimnion dynamics over time and across lakes, suggesting that specific drivers have distinct relative importance for each community.
To understand factors that impact solar-powered electricity generation by Rhodobacter sphaeroides in a single-chamber microbial fuel cell (MFC). The MFC used submerged platinum-coated carbon paper anodes and cathodes of the same material, in contact with atmospheric oxygen. Power was measured by monitoring voltage drop across an external resistance. Biohydrogen production and in situ hydrogen oxidation were identified as the main mechanisms for electron transfer to the MFC circuit. The nitrogen source affected MFC performance, with glutamate and nitrate-enhancing power production over ammonium. Power generation depended on the nature of the nitrogen source and on the availability of light. With light, the maximum point power density was 790 mW m(-2) (2.9 W m(-3)). In the dark, power output was less than 0.5 mW m(-2) (0.008 W m(-3)). Also, sustainable electrochemical activity was possible in cultures that did not receive a nitrogen source. We show conditions at which solar energy can serve as an alternative energy source for MFC operation. Power densities obtained with these one-chamber solar-driven MFC were comparable with densities reported in nonphotosynthetic MFC and sustainable for longer times than with previous work on two-chamber systems using photosynthetic bacteria.
Using a combination of bacterial and phage-targeted metagenomics, we analyzed two geographically remote sludge bioreactors enriched in a single bacterial species Candidatus Accumulibacter phosphatis (CAP). We inferred unrestricted global movement of this species and identified aquatic ecosystems as the primary environmental reservoirs facilitating dispersal. Highly related and geographically remote CAP strains differed principally in genomic regions encoding phage defense mechanisms. We found that CAP populations were high density, clonal, and nonrecombining, providing natural targets for "kill-the-winner" phage predation. Community expression analysis demonstrated that phages were consistently active in the bioreactor community. Genomic signatures linking CAP to past phage exposures were observed mostly between local phage and host. We conclude that CAP strains disperse globally but must adapt to phage predation pressure locally.
This study investigated the role of Accumulibacter-related bacterial populations and factors influencing their distribution in enhanced biological phosphorus removal (EBPR) systems in the USA. For this purpose, five full-scale wastewater treatment facilities performing EBPR were surveyed. The facilities had different configurations but were all treating primarily domestic wastewater. Two facilities had history of poor EBPR performance. Batch-scale acetate uptake and inorganic phosphate (P(i)) release and uptake experiments were conducted to evaluate the EBPR activity of each sludge. Typical P(i) and acetate profiles were observed, and EBPR activity was found to be positively correlated to polyphosphate (polyP)-accumulating organism (PAO) abundance, as determined by staining intracellular polyP. The abundance of Accumulibacter-related organisms was investigated using fluorescent in situ hybridization. Accumulibacter-related organisms were present in all full-scale EBPR facilities, at levels ranging from 9 to 24% of total cells. More than 80% of Accumulibacter-related organisms were estimated to have high polyP content, confirming their involvement in EBPR in these five facilities. However, Accumulibacter-related PAOs were only a fraction (40-69%) of the total PAO population. The variation of Accumulibacter-related PAO abundance among these EBPR systems suggests that multiple interacting factors such as wastewater characteristics and operational conditions are structuring PAO communities.
The acI lineage of freshwater Actinobacteria is a cosmopolitan and often numerically dominant member of lake bacterial communities. We conducted a survey of acI 16S rRNA genes and 16S-23S rRNA internal transcribed spacer regions from 18 Wisconsin lakes and used standard nonphylogenetic and phylogenetic statistical approaches to investigate the factors that determine acI community composition at the local scale (within lakes) and at the regional scale (across lakes). Phylogenetic reconstruction of 434 acI 16S rRNA genes revealed a well-defined and highly resolved phylogeny. Eleven previously unrecognized monophyletic clades, each with > or =97.9% within-clade 16S rRNA gene sequence identity, were identified. Clade community similarity positively correlated with lake environmental similarity but not with geographic distance, implying that the lakes represent a single biotic region containing environmental filters for communities that have similar compositions. Phylogenetically disparate clades within the acI lineage were most abundant at the regional scale, and local communities were comprised of more closely related clades. Lake pH was a strong predictor of the community composition, but only when lakes with a pH below 6 were included in the data set. In the remaining lakes (pH above 6) biogeographic patterns in the landscape were instead a predictor of the observed acI community structure. The nonrandom distribution of the newly defined acI clades suggests potential ecophysiological differences between the clades, with acI clades AI, BII, and BIII preferring acidic lakes and acI clades AII, AVI, and BI preferring more alkaline lakes.
The performance of enhanced biological phosphorus removal (EBPR) wastewater treatment processes depends on the presence of bacteria that accumulate large quantities of polyphosphate. One such group of bacteria has been identified and named Candidatus Accumulibacter phosphatis. Accumulibacter-like bacteria are abundant in many EBPR plants, but not much is known about their community or population ecology. In this study, we used the polyphosphate kinase gene (ppk1) as a high-resolution genetic marker to study population structure in activated sludge. Ppk1 genes were amplified from samples collected from full-scale wastewater treatment plants of different configurations. Clone libraries were constructed using primers targeting highly conserved regions of ppk1, to retrieve these genes from activated sludge plants that did, and did not, perform EBPR. Comparative sequence analysis revealed that ppk1 fragments were retrieved from organisms affiliated with the Accumulibacter cluster from EBPR plants but not from a plant that did not perform EBPR. A new set of more specific primers was designed and validated to amplify a 1,100 bp ppk1 fragment from Accumulibacter-like bacteria. Our results suggest that the Accumulibacter cluster has finer-scale architecture than previously revealed by 16S ribosomal RNA-based analyses.
We investigated the fine-scale population structure of the "Candidatus Accumulibacter" lineage in enhanced biological phosphorus removal (EBPR) systems using the polyphosphate kinase 1 gene (ppk1) as a genetic marker. We retrieved fragments of "Candidatus Accumulibacter" 16S rRNA and ppk1 genes from one laboratory-scale and several full-scale EBPR systems. Phylogenies reconstructed using 16S rRNA genes and ppk1 were largely congruent, with ppk1 granting higher phylogenetic resolution and clearer tree topology and thus serving as a better genetic marker than 16S rRNA for revealing population structure within the "Candidatus Accumulibacter" lineage. Sequences from at least five clades of "Candidatus Accumulibacter" were recovered by ppk1-targeted PCR, and subsequently, specific primer sets were designed to target the ppk1 gene for each clade. Quantitative real-time PCR (qPCR) assays using "Candidatus Accumulibacter"-specific 16S rRNA and "Candidatus Accumulibacter" clade-specific ppk1 primers were developed and conducted on three laboratory-scale and nine full-scale EBPR samples and two full-scale non-EBPR samples to determine the abundance of the total "Candidatus Accumulibacter" lineage and the relative distributions and abundances of the five "Candidatus Accumulibacter" clades. The qPCR-based estimation of the total "Candidatus Accumulibacter" fraction as a proportion of the bacterial community as measured using 16S rRNA genes was not significantly different from the estimation measured using ppk1, demonstrating the power of ppk1 as a genetic marker for detection of all currently defined "Candidatus Accumulibacter" clades. The relative distributions of "Candidatus Accumulibacter" clades varied among different EBPR systems and also temporally within a system. Our results suggest that the "Candidatus Accumulibacter" lineage is more diverse than previously realized and that different clades within the lineage are ecologically distinct.
New high-throughput culture-independent molecular tools are allowing the scientific community to characterize and understand the microbial communities underpinning environmental biotechnology processes in unprecedented ways. By creatively leveraging these new data sources, microbial ecology has the potential to transition from a purely descriptive to a predictive framework, in which ecological principles are integrated and exploited to engineer systems that are biologically optimized for the desired goal. But to achieve this goal, ecology, engineering and microbiology curricula need to be changed from the very root to better promote interdisciplinarity.
Population dynamics are influenced by drivers acting from outside and from within an ecosystem. Extrinsic forces operating over broad spatial scales can impart synchronous behavior to separate populations, while internal, system-specific drivers often lead to idiosyncratic behavior. Here, we demonstrate synchrony in community-level dynamics among phytoplankton and bacteria in six north temperate humic lakes. The influence of regional meteorological factors explained much of the temporal variability in the phytoplankton community, and resulted in synchronous patterns of community change among lakes. Bacterial dynamics, in contrast, were driven by system-specific interactions with phytoplankton. Despite the importance of intrinsic factors for determining bacterial community composition and dynamics, we demonstrated that biological interactions transmitted the signal of the regional extrinsic drivers to the bacterial communities, ultimately resulting in synchronous community phenologies for bacterioplankton communities as well. This demonstrates how linkages between the components of a complex biological system can work to simplify the dynamics of the system and implies that it may be possible to predict the behavior of microbial communities responsible for important biogeochemical services in the landscape.
The development and proliferation of antibiotic resistance in pathogenic, commensal, and environmental microorganisms is a major public health concern. The extent to which human activities contribute to the maintenance of environmental reservoirs of antibiotic resistance is poorly understood. In the current study, wastewater treatment plants (WWTPs) were investigated as possible sources of tetracycline resistance via qualitative PCR and quantitative PCR (qPCR). Various WWTPs and two freshwater lakes were surveyed for the presence of an array of 10 tetracycline resistance determinants (tet(R)): tet(A)-(E), tet(G), tet(M), tet(O), tet(Q), tet(S). All WWTP samples contained more different types of tet(R) genes, as compared to the lake water samples. Gene copy numbers of tet(G) and tet(Q) in these samples were quantified via qPCR and normalized to both the volume of original sample and to the amount of DNA extracted per sample (a proxy for bacterial abundance). Concentrations of tet(Q) were found to be highest in wastewater influent while tet(G) concentrations were highest in activated sludge. Investigation of the effects of UV disinfection on wastewater effluent showed no reduction in the number of detectable tet(R) gene types.
Two primer sets for automated ribosomal intergenic spacer analysis (ARISA) were used to assess the bacterial community composition (BCC) in Lake Mendota, Wisconsin, over 3 years. Correspondence analysis revealed differences in community profiles generated by different primer sets, but overall ecological patterns were conserved in each case. ARISA is a powerful tool for evaluating BCC change through space and time, regardless of the specific primer set used.
Enhanced biological phosphorus removal (EBPR) is one of the best-studied microbially mediated industrial processes because of its ecological and economic relevance. Despite this, it is not well understood at the metabolic level. Here we present a metagenomic analysis of two lab-scale EBPR sludges dominated by the uncultured bacterium, "Candidatus Accumulibacter phosphatis." The analysis sheds light on several controversies in EBPR metabolic models and provides hypotheses explaining the dominance of A. phosphatis in this habitat, its lifestyle outside EBPR and probable cultivation requirements. Comparison of the same species from different EBPR sludges highlights recent evolutionary dynamics in the A. phosphatis genome that could be linked to mechanisms for environmental adaptation. In spite of an apparent lack of phylogenetic overlap in the flanking communities of the two sludges studied, common functional themes were found, at least one of them complementary to the inferred metabolism of the dominant organism. The present study provides a much needed blueprint for a systems-level understanding of EBPR and illustrates that metagenomics enables detailed, often novel, insights into even well-studied biological systems.
A lab-scale sequencing batch reactor (SBR) and six full-scale wastewater treatment plants (WWTPs) performing enhanced biological phosphorus removal (EBPR) were surveyed. The abundance of Accumulibacter-related organisms in the full-scale plants was investigated using fluorescent in situ hybridization. Accumulibacter-related organisms were present in all of the full-scale EBPR plants, at levels ranging from 9% to 24% of total cells. The high percentage of Accumulibacter-related organisms seemed to be associated with configurations which minimize the nitrate recycling to the anaerobic zone and low influent BOD:TP ratios. PCR-based clone libraries were constructed from the community 16S rRNA gene plus the internally transcribed spacer region amplified from the SBR and five of the full-scale WWTPs. Comparative sequence analysis was carried out using Accumulibacter-related clones, providing higher phylogenetic resolution and revealing finer-scale clustering of the sequences retrieved from the SBR and full-scale EBPR
A previous multiyear study observed correlations between bacterioplankton community composition (BCC) and abundance and the dynamics of phytoplankton populations and bacterivorous grazers in a humic lake. These observations generated hypotheses about the importance of trophic interactions (both top-down and bottom-up) for structuring bacterial communities in this lake, which were tested using two multifactorial food web manipulation experiments that separately manipulated the intensity of grazing and the composition of the phytoplankton community. Our results, combined with field observations, suggest that a hierarchy of drivers structures bacterial communities in this lake. While other studies have noted links between aggregate measures of phytoplankton and bacterioplankton communities, we demonstrate here correlations between succession of phytoplankton assemblages and BCC as assessed by automated ribosomal intergenic spacer analysis (ARISA). We used a novel approach linking community ARISA data to phylogenetic assignments from sequence analysis of 16S rRNA gene clone libraries to examine the responses of specific bacterial phylotypes to the experimental manipulations. The synchronous dynamics of these populations suggests that primary producers may mediate BCC and diversity through labile organic matter production, which evolves in quality and quantity during phytoplankton succession. Superimposed on this resource-mediated control of BCC are brief periods of intense bacterivory that impact bacterial abundance and composition.
In an effort to better understand the factors contributing to patterns in freshwater bacterioplankton community composition and diversity, we coupled automated ribosomal intergenic spacer analysis (ARISA) to analysis of 16S ribosomal RNA (rRNA) gene sequences to follow the persistence patterns of 46 individual phylotypes over 3 years in Crystal Bog Lake. Additionally, we sought to identify linkages between the observed phylotype variations and known chemical and biological drivers. Sequencing of 16S rRNA genes obtained from the water column indicated the presence of phylotypes associated with the Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, TM7 and Verrucomicrobia phyla, as well as phylotypes with unknown affiliation. Employment of the 16S rRNA gene/ARISA method revealed that specific phylotypes varied independently of the entire bacterial community dynamics. Actinobacteria, which were present on greater than 95% of sampling dates, did not share the large temporal variability of the other identified phyla. Examination of phylotype relative abundance patterns (inferred using ARISA fragment relative fluorescence) revealed a strong correlation between the dominant phytoplankton succession and the relative abundance patterns of the majority of individual phylotypes. Further analysis revealed covariation among unique phylotypes, which formed several distinct bacterial assemblages correlated with particular phytoplankton communities. These data indicate the existence of unique persistence patterns for different common freshwater phylotypes, which may be linked to the presence of dominant phytoplankton species.
Microbial population dynamics were investigated during start-up and during periods of overload conditions in anaerobic co-digesters treating municipal solid waste and sewage sludge. Changes in community structure were monitored using ribosomal RNA-based oligonucleotide probe hybridization to measure the abundance of syntrophic propionate-oxidizing bacteria (SPOB), saturated fatty acid-beta-oxidizing syntrophs (SFAS), and methanogens. These changes were linked to traditional performance parameters such as biogas production and volatile fatty acid (VFA) concentrations. Digesters with high levels of Archaea started up successfully. Methanosaeta concilii was the dominant aceticlastic methanogen in these systems. In contrast, digesters that experienced a difficult start-up period had lower levels of Archaea with proportionally more abundant Methanosarcina spp. Syntrophic propionate-oxidizing bacteria and saturated fatty acid-beta-oxidizing syntrophs were present at low levels in all digesters, and SPOB appeared to play a role in stabilizing propionate levels during start-up of one digester. Digesters with a history of poor performance tolerated a severe organic overload event better than digesters that had previously performed well. It is hypothesized that higher levels of SPOB and SFAS and their methanogenic partners in previously unstable digesters are responsible for this behavior.
A novel polyphosphate kinase (PPK) was retrieved from an uncultivated organism in activated sludge carrying out enhanced biological phosphorus removal (EBPR). Acetate-fed laboratory-scale sequencing batch reactors were used to maintain sludge with a high phosphorus content (approximately 11% of the biomass). PCR-based clone libraries of small subunit rRNA genes and fluorescent in situ hybridization (FISH) were used to verify that the sludge was enriched in Rhodocyclus-like beta-Proteobacteria known to be associated with sludges carrying out EBPR. These organisms comprised approximately 80% of total bacteria in the sludge, as assessed by FISH. Degenerate PCR primers were designed to retrieve fragments of putative ppk genes from a pure culture of Rhodocyclus tenuis and from organisms in the sludge. Four novel ppk homologs were found in the sludge, and two of these (types I and II) shared a high degree of amino acid similarity with R. tenuis PPK (86 and 87% similarity, respectively). Dot blot analysis of total RNA extracted from sludge demonstrated that the Type I ppk mRNA was present, indicating that this gene is expressed during EBPR. Inverse PCR was used to obtain the full Type I sequence from sludge DNA, and a full-length PPK was cloned, overexpressed, and purified to near homogeneity. The purified PPK has a specific activity comparable to that of other PPKs, has a requirement for Mg(2+), and does not appear to operate in reverse. PPK activity was found mainly in the particulate fraction of lysed sludge microorganisms.
The community structure and metabolic function of activated sludge carrying out enhanced biological phosphorus removal have been investigated. Laboratory-scale sequencing batch reactors were operated at several influent COD/P ratios to obtain sludges with a range of phosphorus contents. Molecular microbiological techniques based on small subunit ribosomal RNA were used to characterize the structure of these sludges. The dominant polyphosphate accumulating organism was a close relative of Rhodocyclus tenuis, a member of the beta subclass of the Proteobacteria. Fragments of genes coding for polyphosphate kinase (PPK), thought to be responsible for polyphosphate accumulation, were retrieved from one of the sludges. The relative abundance of PPK gene copies in genomic DNA extracted from sludges was determined to confirm that at least one of the PPK gene sequences was derived from the dominant polyphosphate accumulating organism.
Heavy metal and radionuclide contamination presents a significant environmental problem worldwide. Precipitation of heavy metals on membranes of cells that secrete phosphate has been shown to be an effective method of reducing the volume of these wastes, thus reducing the cost of disposal. A consortium of organisms, some of which secrete large quantities of phosphate, was enriched in a laboratory-scale sequencing batch reactor performing Enhanced Biological Phosphorus Removal, a treatment process widely used for removing phosphorus. Organisms collected after the aerobic phase of this process secreted phosphate and precipitated greater than 98% of the uranyl from a 1.5 mM uranyl nitrate solution when supplemented with an organic acid as a carbon source under anaerobic conditions. Transmission electron microscopy, energy dispersive x-ray spectroscopy, and fluorescence spectroscopy were used to identify the precipitate as membrane-associated uranyl phosphate, UO2HPO4.
Microbial population dynamics were evaluated in anaerobic codigesters treating municipal solid waste and sewage sludge. Ribosomal RNA based oligonucleotide probes were used to characterize changes in population abundance of syntrophic volatile fatty acid degrading bacteria and methanogens. Changes in community structure were linked to traditional performance parameters during the recovery of previously unstable codigesters induced by a reduction in mixing levels. Methanosarcina spp. were the most abundant aceticlastic methanogens in unstable codigesters with high acetate concentrations, while Methanosaeta concilii was dominant in stable systems with low levels of acetate. Growth of Syntrophobacter wolinii was enhanced during stabilization of a codigester with a well-developed population of Methanobacteriaceae, possibly because the presence of adequate numbers of these hydrogenotrophic methanogens encouraged the syntrophic oxidation of propionate. Mesophilic saturated fatty acid beta-oxidizing syntrophs were most abundant in previously unstable codigesters. One minimally mixed reactor became unstable after switching to continuously mixed conditions. After the switch, total archaeal abundance decreased sharply, though Methanobacteriaceae and Methanosarcina spp. levels increased as the fermentation became unbalanced. Based on the results presented here, mixing appears to inhibit the syntrophic oxidation of volatile fatty acids, possibly by disrupting the spatial juxtaposition of syntrophic bacteria and their methanogenic partners.
The feasibility of codigestion of the organic fraction of municipal solid waste, primary sludge, and waste activated sludge was evaluated in mesophilic (37 degrees C), laboratory-scale digesters. In a first experiment, different startup strategies were compared using four digesters, operated under continuously mixed conditions. After two weeks, the experiment was continued under minimally mixed conditions. Results demonstrated that reducing the level of mixing improved digester performance. Therefore, in a second experiment, six digesters were operated to compare performance under continuous mixing and reduced mixing levels at various loading rates and solids levels. The continuously mixed digesters exhibited unstable performance at the higher loading rates, while the minimally mixed digesters performed well for all loading rates evaluated. In a third experiment, it was demonstrated that an unstable, continuously mixed digester was quickly stabilized by reducing the mixing level. These experiments confirmed that continuous mixing was not necessary for good performance and was inhibitory at higher loading rates. In addition, reduction of mixing levels may be used as an operational tool to stabilize unstable digesters.
Abstract Nearly full-length, small subunit (SSU) rRNA was transcribed in vitro from clones of SSU rDNA genes. Comparing the use of in vitro-transcribed and native rRNA indicated that, when in vitro-transcribed rRNA was used as a standard for quantitative hybridizations with oligonucleotide probes, the population was consistently underestimated. The population abundance was expressed as a percentage of specific target SSU rRNA (determined with a specific oligonucleotide probe), relative to the total SSU rRNA (measured with a universal probe). Differences in hybridization signals could be related to specific probe target locations and rRNA denaturation conditions, suggesting that higher order structure is important in quantitative membrane hybridizations. Therefore, in vitro-transcribed rRNA cannot always be used for the absolute quantification of microbial populations, but can be employed as a standard to quantify shifts in population abundance over time, and to compare community structure in various environments.
An aggressive start-up strategy was used to initiate codigestion in two anaerobic, continuously mixed bench-top reactors at mesophilic (37 degrees C) and thermophilic (55 degrees C) conditions. The digesters were inoculated with mesophilic anaerobic sewage sludge and cattle manure and were fed a mixture of simulated municipal solid waste and biosolids in proportions that reflect U.S. production rates. The design organic loading rate was 3.1 kg volatile solids/m3/day and the retention time was 20 days. Ribosomal RNA-targeted oligonucleotide probes were used to determine the methanogenic community structure in the inocula and the digesters. Chemical analyses were performed to evaluate digester performance. The aggressive start-up strategy was successful for the thermophilic reactor, despite the use of a mesophilic inoculum. After a short start-up period (20 days), stable performance was observed with high gas production rates (1.52 m3/m3/day), high levels of methane in the biogas (59%), and substantial volatile solids (54%) and cellulose (58%) removals. In contrast, the mesophilic digester did not respond favorably to the start-up method. The concentrations of volatile fatty acids increased dramatically and pH control was difficult. After several weeks of operation, the mesophilic digester became more stable, but propionate levels remained very high. Methanogenic population dynamics correlated well with performance measures. Large fluctuations were observed in methanogenic population levels during the start-up period as volatile fatty acids accumulated and were subsequently consumed. Methanosaeta species were the most abundant methanogens in the inoculum, but their levels decreased rapidly as acetate built up. The increase in acetate levels was paralleled by an increase in Methanosarcina species abundance (up to 11.6 and 4.8% of total ribosomal RNA consisted of Methanosarcina species ribosomal RNA in mesophilic and thermophilic digesters, respectively). Methanobacteriaceae were the most abundant hydrogenotrophic methanogens in both digesters, but their levels were higher in the thermophilic digester.
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