An integrated evaluation of bioenhanced in situ LNAPL dissolution.

Performance evaluation of in situ bioremediation processes in the field is difficult due to uncertainty created by matrix and contaminant heterogeneity, inaccessibility to direct observation, expense of sampling, and limitations of some measurements. The goal of this research was to develop a strategy for evaluating in situ bioremediation of light nonaqueous-phase liquid (LNAPL) contamination and demonstrating the occurrence of bioenhanced LNAPL dissolution by: (1) integrating a suite of analyses into a rational evaluation strategy; and (2) demonstrating the strategy's application in intermediate-scale flow-cell (ISFC) experiments simulating an aquifer contaminated with a pool of LNAPL (naphthalene dissolved in dodecane). Two ISFCs were operated to evaluate how the monitored parameters changed between a "no bioremediation" scenario and an "intrinsic in situ bioremediation" scenario. Key was incorporating different measures of microbial activity and contaminant degradation relevant to bioremediation: contaminant loss; consumption of electron acceptors; and changes in total alkalinity, pH, dissolved total inorganic carbon, carbon-stable isotopes, microorganisms, and intermediate metabolites. These measurements were integrated via mass-flux modeling and mass-balance analyses to document that in situ biodegradation of naphthalene was strongly accelerated in the "intrinsic in situ bioremediation" scenario versus "no bioremediation." Furthermore, the integrated strategy provided consistent evidence of bioenhancement of LNAPL dissolution through intrinsic bioremediation by a factor of approximately 2 due to the biodegradation of the naphthalene near the pool/water interface.

Contribution of Microorganisms with the Clade II Nitrous Oxide Reductase to Suppression of Surface Emissions of Nitrous Oxide.

The sources and sinks of nitrous oxide, as control emissions to the atmosphere, are generally poorly constrained for most environmental systems. Initial depth-resolved analysis of nitrous oxide flux from observation wells and the proximal surface within a nitrate contaminated aquifer system revealed high subsurface production but little escape from the surface. To better understand the environmental controls of production and emission at this site, we used a combination of isotopic, geochemical, and molecular analyses to show that chemodenitrification and bacterial denitrification are major sources of nitrous oxide in this subsurface, where low DO, low pH, and high nitrate are correlated with significant nitrous oxide production. Depth-resolved metagenomes showed that consumption of nitrous oxide near the surface was correlated with an enrichment of Clade II nitrous oxide reducers, consistent with a growing appreciation of their importance in controlling release of nitrous oxide to the atmosphere. Our work also provides evidence for the reduction of nitrous oxide at a pH of 4, well below the generally accepted limit of pH 5.

Genomic and environmental controls on Castellaniella biogeography in an anthropogenically disturbed subsurface.

Castellaniella species have been isolated from a variety of mixed-waste environments including the nitrate and multiple metal-contaminated subsurface at the Oak Ridge Reservation (ORR). Previous studies examining microbial community composition and nitrate removal at ORR during biostimulation efforts reported increased abundances of members of the Castellaniella genus concurrent with increased denitrification rates. Thus, we asked how genomic and abiotic factors control the Castellaniella biogeography at the site to understand how these factors may influence nitrate transformation in an anthropogenically impacted setting. We report the isolation and characterization of several Castellaniella strains from the ORR subsurface. Five of these isolates match at 100% identity (at the 16S rRNA gene V4 region) to two Castellaniella amplicon sequence variants (ASVs), ASV1 and ASV2, that have persisted in the ORR subsurface for at least 2 decades. However, ASV2 has consistently higher relative abundance in samples taken from the site and was also the dominant blooming denitrifier population during a prior biostimulation effort. We found that the ASV2 representative strain has greater resistance to mixed metal stress than the ASV1 representative strains. We attribute this resistance, in part, to the large number of unique heavy metal resistance genes identified on a genomic island in the ASV2 representative genome. Additionally, we suggest that the relatively lower fitness of ASV1 may be connected to the loss of the nitrous oxide reductase (nos) operon (and associated nitrous oxide reductase activity) due to the insertion at this genomic locus of a mobile genetic element carrying copper resistance genes. This study demonstrates the value of integrating genomic, environmental, and phenotypic data to characterize the biogeography of key microorganisms in contaminated sites.

Novel order-level lineage of ammonia-oxidizing archaea widespread in marine and terrestrial environments.

Ammonia-oxidizing archaea (AOA) are among the most ubiquitous and abundant archaea on Earth, widely distributed in marine, terrestrial, and geothermal ecosystems. However, the genomic diversity, biogeography, and evolutionary process of AOA populations in subsurface environments are vastly understudied compared to those in marine and soil systems. Here, we report a novel AOA order Candidatus (Ca.) Nitrosomirales which forms a sister lineage to the thermophilic Ca. Nitrosocaldales. Metagenomic and 16S rRNA gene-read mapping demonstrates the abundant presence of Nitrosomirales AOA in various groundwater environments and their widespread distribution across a range of geothermal, terrestrial, and marine habitats. Terrestrial Nitrosomirales AOA show the genetic capacity of using formate as a source of reductant and using nitrate as an alternative electron acceptor. Nitrosomirales AOA appear to have acquired key metabolic genes and operons from other mesophilic populations via horizontal gene transfer, including genes encoding urease, nitrite reductase, and V-type ATPase. The additional metabolic versatility conferred by acquired functions may have facilitated their radiation into a variety of subsurface, marine, and soil environments. We also provide evidence that each of the four AOA orders spans both marine and terrestrial habitats, which suggests a more complex evolutionary history for major AOA lineages than previously proposed. Together, these findings establish a robust phylogenomic framework of AOA and provide new insights into the ecology and adaptation of this globally abundant functional guild.

Environmental stress mediates groundwater microbial community assembly.

Community assembly describes how different ecological processes shape microbial community composition and structure. How environmental factors impact community assembly remains elusive. Here we sampled microbial communities and >200 biogeochemical variables in groundwater at the Oak Ridge Field Research Center, a former nuclear waste disposal site, and developed a theoretical framework to conceptualize the relationships between community assembly processes and environmental stresses. We found that stochastic assembly processes were critical (>60% on average) in shaping community structure, but their relative importance decreased as stress increased. Dispersal limitation and 'drift' related to random birth and death had negative correlations with stresses, whereas the selection processes leading to dissimilar communities increased with stresses, primarily related to pH, cobalt and molybdenum. Assembly mechanisms also varied greatly among different phylogenetic groups. Our findings highlight the importance of microbial dispersal limitation and environmental heterogeneity in ecosystem restoration and management.

Ammonia-oxidizing bacteria and archaea exhibit differential nitrogen source preferences.

Ammonia-oxidizing microorganisms (AOM) contribute to one of the largest nitrogen fluxes in the global nitrogen budget. Four distinct lineages of AOM: ammonia-oxidizing archaea (AOA), beta- and gamma-proteobacterial ammonia-oxidizing bacteria (ß-AOB and γ-AOB) and complete ammonia oxidizers (comammox), are thought to compete for ammonia as their primary nitrogen substrate. In addition, many AOM species can utilize urea as an alternative energy and nitrogen source through hydrolysis to ammonia. How the coordination of ammonia and urea metabolism in AOM influences their ecology remains poorly understood. Here we use stable isotope tracing, kinetics and transcriptomics experiments to show that representatives of the AOM lineages employ distinct regulatory strategies for ammonia or urea utilization, thereby minimizing direct substrate competition. The tested AOA and comammox species preferentially used ammonia over urea, while ß-AOB favoured urea utilization, repressed ammonia transport in the presence of urea and showed higher affinity for urea than for ammonia. Characterized γ-AOB co-utilized both substrates. These results reveal contrasting niche adaptation and coexistence patterns among the major AOM lineages.

Cross-Feedings, Competition, and Positive and Negative Synergies in a Four-Species Synthetic Community for Anaerobic Degradation of Cellulose to Methane.

Complex interactions exist among microorganisms in a community to carry out ecological processes and adapt to changing environments. Here, we constructed a quad-culture consisting of a cellulolytic bacterium (Ruminiclostridium cellulolyticum), a hydrogenotrophic methanogen (Methanospirillum hungatei), an acetoclastic methanogen (Methanosaeta concilii), and a sulfate-reducing bacterium (Desulfovibrio vulgaris). The four microorganisms in the quad-culture cooperated via cross-feeding to produce methane using cellulose as the only carbon source and electron donor. The community metabolism of the quad-culture was compared with those of the R. cellulolyticum-containing tri-cultures, bi-cultures, and mono-culture. Methane production was higher in the quad-culture than the sum of the increases in the tri-cultures, which was attributed to a positive synergy of four species. In contrast, cellulose degradation by the quad-culture was lower than the additive effects of the tri-cultures which represented a negative synergy. The community metabolism of the quad-culture was compared between a control condition and a treatment condition with sulfate addition using metaproteomics and metabolic profiling. Sulfate addition enhanced sulfate reduction and decreased methane and CO2 productions. The cross-feeding fluxes in the quad-culture in the two conditions were modeled using a community stoichiometric model. Sulfate addition strengthened metabolic handoffs from R. cellulolyticum to M. concilii and D. vulgaris and intensified substrate competition between M. hungatei and D. vulgaris. Overall, this study uncovered emergent properties of higher-order microbial interactions using a four-species synthetic community. IMPORTANCE A synthetic community was designed using four microbial species that together performed distinct key metabolic processes in the anaerobic degradation of cellulose to methane and CO2. The microorganisms exhibited expected interactions, such as cross-feeding of acetate from a cellulolytic bacterium to an acetoclastic methanogen and competition of H2 between a sulfate reducing bacterium and a hydrogenotrophic methanogen. This validated our rational design of the interactions between microorganisms based on their metabolic roles. More interestingly, we also found positive and negative synergies as emergent properties of high-order microbial interactions among three or more microorganisms in cocultures. These microbial interactions can be quantitatively measured by adding and removing specific members. A community stoichiometric model was constructed to represent the fluxes in the community metabolic network. This study paved the way toward a more predictive understanding of the impact of environmental perturbations on microbial interactions sustaining geochemically significant processes in natural systems.

Ecophysiological and genomic analyses of a representative isolate of highly abundant Bacillus cereus strains in contaminated subsurface sediments.

Bacillus cereus strain CPT56D-587-MTF (CPTF) was isolated from the highly contaminated Oak Ridge Reservation (ORR) subsurface. This site is contaminated with high levels of nitric acid and multiple heavy metals. Amplicon sequencing of the 16S rRNA genes (V4 region) in sediment from this area revealed an amplicon sequence variant (ASV) with 100% identity to the CPTF 16S rRNA sequence. Notably, this CPTF-matching ASV had the highest relative abundance in this community survey, with a median relative abundance of 3.77% and comprised 20%-40% of reads in some samples. Pangenomic analysis revealed that strain CPTF has expanded genomic content compared to other B. cereus species-largely due to plasmid acquisition and expansion of transposable elements. This suggests that these features are important for rapid adaptation to native environmental stressors. We connected genotype to phenotype in the context of the unique geochemistry of the site. These analyses revealed that certain genes (e.g. nitrate reductase, heavy metal efflux pumps) that allow this strain to successfully occupy the geochemically heterogenous microniches of its native site are characteristic of the B. cereus species while others such as acid tolerance are mobile genetic element associated and are generally unique to strain CPTF.

Nitrosomonas supralitoralis sp. nov., an ammonia-oxidizing bacterium from beach sand in a supralittoral zone.

A betaproteobacterial chemolithotrophic ammonia-oxidizing bacterium designated APG5T was isolated from supralittoral sand of the Edmonds City Beach, WA, USA. Growth was observed at 10-35 °C (optimum, 30 °C), pH 5-9 (optimum, pH 8) and ammonia concentrations as high as 100 mM (optimum, 1-30 mM NH4Cl). The strain grows optimally in a freshwater medium but tolerates up to 400 mM NaCl. It is most closely related to 'Nitrosomonas ureae' (96.7% 16S rRNA and 92.4% amoA sequence identity). The 3.75-Mbp of AGP5T draft genome contained a single rRNA operon and all necessary tRNA genes and has the lowest G+C content (43.5%) when compared to the previously reported genomes of reference strains in cluster 6 Nitrosomonas. Based on an average nucleotide identity of 82% with its closest relative ('N. ureae' Nm10T) and the suggested species boundary of 95-96%, a new species Nitrosomonas supralitoralis sp. nov. is proposed. The type strain of Nitrosomonas supralitoralis is APG5T (= NCIMB 14870T = ATCC TSD-116T).

Microbial maintenance energy quantified and modeled with microcalorimetry.

Refining the energetic costs of cellular maintenance is essential for predicting microbial growth and survival in the environment. Here, we evaluate a simple batch culture method to quantify energy partitioning between growth and maintenance using microcalorimetry and thermodynamic modeling. The constants derived from the batch culture system were comparable to those that have been reported from meta-analyses of data derived from chemostat studies. The model accurately predicted temperature-dependent biomass yield and the upper temperature limit of growth for Desulfovibrio alaskensis G20, suggesting the method may have broad application. An Arrhenius temperature dependence for the specific energy consumption rate, inferred from substrate consumption and heat evolution, was observed over the entire viable temperature range. By combining this relationship for specific energy consumption rates and observed specific growth rates, the model describes an increase in nongrowth associated maintenance at higher temperatures and the corresponding decrease in energy available for growth. This analytical and thermodynamic formulation suggests that simply monitoring heat evolution in batch culture could be a useful complement to the recognized limitations of estimating maintenance using extrapolation to zero growth in chemostats.

Metagenomic Insights Into Competition Between Denitrification and Dissimilatory Nitrate Reduction to Ammonia Within One-Stage and Two-Stage Partial-Nitritation Anammox Bioreactor Configurations.

Anaerobic ammonia oxidizing bacteria (Anammox) are implemented in high-efficiency wastewater treatment systems operated in two general configurations; one-stage systems combine aerobic ammonia oxidizing bacteria (AOB) and Anammox within a single aerated reactor, whereas two-stage configurations separate these processes into discrete tanks. Within both configurations heterotrophic populations that perform denitrification or dissimilatory nitrate reduction to ammonia (DNRA) compete for carbon and nitrate or nitrite and can impact reactor performance because DNRA retains nitrogen in the system. Therefore, it is important to understand how selective pressures imposed by one-stage and two-stage reactor configurations impact the microbial community structure and associated nitrogen transforming functions. We performed 16S rRNA gene and metagenomic sequencing on different biomass fractions (granules, flocs, and suspended biomass) sampled from two facilities treating sludge dewatering centrate: a one-stage treatment facility (Chambers Creek, Tacoma, WA) and a two-stage system (Rotterdam, Netherlands). Similar microbial populations were identified across the different samples, but relative abundances differed between reactor configurations and biomass sources. Analysis of metagenome assembled genomes (MAGs) indicated different lifestyles for abundant heterotrophic populations. Acidobacteria, Bacteroidetes, and Chloroflexi MAGs had varying capacity for DNRA and denitrification. Acidobacteria MAGs possessed high numbers of glycosyl hydrolases and glycosyl transferases indicating a role in biomass degradation. Ignavibacteria and Phycosphaerae MAGs contributed to the greater relative abundance of DNRA associated nrf genes in the two-stage granules and contained genomic features suggesting a preference for an anoxic or microoxic niche. In the one-stage granules a MAG assigned to Burkholderiales accounted for much of the abundant denitrification genes and had genomic features, including the potential for autotrophic denitrification using reduced sulfur, that indicate an ability to adapt its physiology to varying redox conditions. Overall, the competition for carbon substrates between denitrifying and DNRA performing heterotrophs may be impacted by configuration specific selective pressures. In one-stage systems oxygen availability in the bulk liquid and the oxygen gradient within granules would provide a greater niche space for heterotrophic populations capable of utilizing both oxygen and nitrate or nitrite as terminal electron acceptors, compared to two-stage systems where a homogeneous anoxic environment would favor heterotrophic populations primarily adapted to anaerobic metabolism.

Application of pyritic sludge with an anaerobic granule consortium for nitrate removal in low carbon systems.

Granules recovered from a highly reduced anaerobic digester were capable of active nitrogen removal in the absence of exogenous electron donors, averaging 0.25 mg mgNO3--N /gVSS/d over 546 days of operation. Electron mass balance indicated that about half the influent nitrate was converted to ammonia via DNRA and another half denitrified. This capacity was associated with an onion-like structure of multiple layers enriched in reduced iron and sulfur, and a complex microbial community shown by metagenomic sequencing to consist of multiple physiological groups and associated activities, including methanogenesis, denitrification, dissimilatory nitrate reduction to ammonia (DNRA), iron oxidation and reduction, and sulfur reduction and oxidation. Nitrate reduction was supported by both entrained organic material and reduced iron and sulfur species, corresponding to 2.13 mg COD/gVSS/d. Batch incubations showed that approximately 15% of denitrified nitrate was coupled to the oxidation of sulfur derived from both sulfate respiration and granular material enriched in iron-sulfide. Inhibition of sulfate reduction resulted in redirection of electron flow to methanogenesis and, in combination with other batch tests, showed that these granules supported a complex microbial community in which cryptic redox cycles linked carbon, sulfur, and iron oxidation with nitrate, sulfate, iron, and carbon dioxide reduction. This system shows promise for treatment of nitrate contaminated ground water without addition of an external organic carbon source as well as wastewater treatment in combination with (granular) sludge elimination leading in a net reduction of solid treatment costs.

The influence of alfalfa-switchgrass intercropping on microbial community structure and function.

The use of nitrogen fertilizer on bioenergy crops such as switchgrass results in increased costs, nitrogen leaching and emissions of N2 O, a potent greenhouse gas. Intercropping with nitrogen-fixing alfalfa has been proposed as an environmentally sustainable alternative, but the effects of synthetic fertilizer versus intercropping on soil microbial community functionality remain uncharacterized. We analysed 24 metagenomes from the upper soil layer of agricultural fields from Prosser, WA over two growing seasons and representing three agricultural practices: unfertilized switchgrass (control), fertilized switchgrass and switchgrass intercropped with alfalfa. The synthetic fertilization and intercropping did not result in major shifts of microbial community taxonomic and functional composition compared with the control plots, but a few significant changes were noted. Most notably, mycorrhizal fungi, ammonia-oxidizing archaea and bacteria increased in abundance with intercropping and fertilization. However, only betaproteobacterial ammonia-oxidizing bacteria abundance in fertilized plots significantly correlated to N2 O emission and companion qPCR data. Collectively, a short period of intercropping elicits minor but significant changes in the soil microbial community toward nitrogen preservation and that intercropping may be a viable alternative to synthetic fertilization.

An automated multiplexed turbidometric and data collection system for measuring growth kinetics of anaerobes dependent on gaseous substrates.

Standard methods of monitoring the growth kinetics of anaerobic microorganisms are generally impractical when there is a protracted or indeterminate period of active growth, and when high numbers of samples or replications are required. As part of our studies of the adaptive evolution of a simple anaerobic syntrophic mutualism, requiring the characterization of many isolates and alternative syntrophic pairings, we developed a multiplexed growth monitoring system using a combination of commercially available electronics and custom designed circuitry and materials. This system automatically monitors up to 64 sealed, and as needed pressurized, culture tubes and reports the growth data in real-time through integration with a customized relational database. The utility of this system was demonstrated by resolving minor differences in growth kinetics associated with the adaptive evolution of a simple microbial community comprised of a sulfate reducing bacterium, Desulfovibrio vulgaris, grown in syntrophic association with Methanococcus maripaludis, a hydrogenotrophic methanogen.

Nitrosopumilus zosterae sp. nov., an autotrophic ammonia-oxidizing archaeon of phylum Thaumarchaeota isolated from coastal eelgrass sediments of Japan.

A novel mesophilic and aerobic ammonia-oxidizing archaeon of the phylum Thaumarchaeota, strain NM25T, was isolated from coastal eelgrass zone sediment sampled in Shimoda (Japan). The cells were rod-shaped with an S-layer cell wall. The temperature range for growth was 20-37 °C, with an optimum at 30 °C. The pH range for growth was pH 6.1-7.7, with an optimum at pH 7.1. The salinity range for growth was 5-40 %, with an optimum range of 15-32 %. Cells obtained energy from ammonia oxidation and used bicarbonate as a carbon source. Utilization of urea was not observed for energy generation and growth. Strain NM25T required a hydrogen peroxide scavenger, such as α-ketoglutarate, pyruvate or catalase, for sustained growth on ammonia. Growth of strain NM25T was inhibited by addition of low concentrations of some organic compounds and organic mixtures, including complete inhibition by glycerol, peptone and yeast extract. Phylogenetic analysis of four concatenated housekeeping genes (16S rRNA, rpoB, rpsI and atpD) and concatenated AmoA, AmoB, AmoC amino acid sequences indicated that the isolate is similar to members of the genus Nitrosopumilus. The closest relative is Nitrosopumilus ureiphilus PS0T with sequence similarities of 99.5 % for the 16S rRNA gene and 97.2 % for the amoA gene. Genome relatedness between strain NM25T and N. ureiphilus PS0T was assessed by average nucleotide identity and digital DNA-DNA hybridization, giving results of 85.4 and 40.2 %, respectively. On the basis of phenotypic, genotypic and phylogenetic data, strain NM25T represents a novel species of the genus Nitrosopumilus, for which the name sp. nov, is proposed. The type strain is NM25T (=NBRC 111181T=ATCC TSD-147T).

Sustained nitrogen loss in a symbiotic association of Comammox Nitrospira and Anammox bacteria.

The discovery of anaerobic ammonia-oxidizing bacteria (Anammox) and, more recently, aerobic bacteria common in many natural and engineered systems that oxidize ammonia completely to nitrate (Comammox) have significantly altered our understanding of the global nitrogen cycle. A high affinity for ammonia (Km(app),NH3 ≈ 63nM) and oxygen place Comammox Nitrospira inopinata, the first described isolate, in the same trophic category as organisms such as some ammonia-oxidizing archaea. However, N. inopinata has a relatively low affinity for nitrite (Km,NO2 ≈ 449.2µM) suggesting it would be less competitive for nitrite than other nitrite-consuming aerobes and anaerobes. We examined the ecological relevance of the disparate substrate affinities by coupling it with the Anammox bacterium Candidatus Brocadia anammoxidans. Synthetic communities of the two were established in hydrogel granules in which Comammox grew in the aerobic outer layer to provide Anammox with nitrite in the inner anoxic core to form dinitrogen gas. This spatial organization was confirmed with FISH imaging, supporting a mutualistic or commensal relationship. The functional significance of interspecies spatial organization was informed by the hydrogel encapsulation format, broadening our limited understanding of the interplay between these two species. The resulting low nitrate formation and the competitiveness of Comammox over other aerobic ammonia- and nitrite-oxidizers sets this ecological cooperation apart and points to potential biotechnological applications. Since nitrate is an undesirable product of wastewater treatment effluents, the Comammox-Anammox symbiosis may be of economic and ecological importance to reduce nitrogen contamination of receiving waters.

An investigation into the optimal granular sludge size for simultaneous nitrogen and phosphate removal.

An aerobic granular sludge (AGS) pilot plant fed with a mixture of acetate amended centrate and secondary effluent was used to investigate the optimal granule size range for simultaneous nitrification and denitrification (SND) and ortho-phosphate removal. The anaerobic phase was mixed to understand how AGS will perform if integrated with a continuous flow activated sludge system that cannot feed the influent through the settled sludge bed. Five different granule size fractions were taken from the pilot (operated at DO setpoint of 2mgO2/L) and each size was subjected to activity tests in a well-controlled lab-scale AGS reactor at four dissolved oxygen (DO) concentrations of 1, 2, 3, and 4 mgO2/L. The size fractions were: 212 - 600 µm, 600 - 1000 µm, 1000 - 1400 µm, 1400 - 2000 µm, and >2000 µm. The smallest size range (212 - 600 µm) had the highest nitrification and phosphate removal rates at DO setpoints from 1 - 3 mgO2/L, which was attributed to the higher aerobic volume fraction in small granules and hence a higher abundance of phosphorus accumulating organisms (PAO) and ammonia oxidizing bacteria (AOB). In comparison, large granules (>1000 µm) had 1.4 - 4.7 times lower ammonia oxidation rates than the smallest size range, which aligned with their lower AOB abundance relative to granule biomass. The granules with the highest anoxic volume fraction had the highest abundance of nitrite reductase genes (nir gene) but did not show the highest specific nitrogen removal rate. Instead, smaller granules (212 - 600 and 600 - 1000 µm), which had a lower nir gene abundance, had the highest specific nitrogen removal rates (1.2 - 3.1 times higher than larger granules) across all DO values except at 4 mgO2/L. At a DO setpoint of 4 mgO2/L, nitrite production by ammonia oxidation (ammonia monooxygenase) exceeded nitrite reduction by nitrite reductase in granules smaller than 1000 µm, in addition, some denitrifying heterotrophs switched to oxygen utilization in deeper layers hence suppressing denitrification activity. At the DO range of 2 - 4 mg/L, granular size had a greater effect on nutrient removal than DO. Therefore, for AGS developed at an average DO setpoint of 2 mgO2/L, selecting for size fractions in the range of 212 - 1000 µm and avoiding DO values higher than 3 mgO2/L can achieve both a higher nitrogen removal capacity and energy savings. This study is the first to investigate the influence of different DO values on SND and biological phosphorus removal performance of different aerobic granular sludge sizes.

Pairing denitrifying phosphorus accumulating organisms with anaerobic ammonium oxidizing bacteria for simultaneous N and P removal.

Coupling of denitrifying polyphosphate accumulating organisms (DPAO) with anaerobic ammonium oxidizing (Anammox) bacteria in a single treatment scheme has so far been unsuccessful but could offer substantial energy savings, minimize sludge production, while achieving simultaneous carbon, nitrogen and phosphate removal. However, both organisms compete for nitrite and have vastly different growth rates and therefore the goal of this study was to uncouple their solid retention time (SRT) by growing them in different sludge fractions and to determine their biomass specific kinetic properties. Anammox bacteria were grown in a biofilm for longer SRTs and DPAO in flocs to allow shorter SRTs. Exposure of DPAO to anaerobic conditions was accomplished by recycling the flocs to a separate reactor by which simultaneous P, N, and C removal was accomplished. The diffusion limited biofilm lowered the biomass specific nitrite affinity for Anammox (KsAMX = 0.091 mM), which gave DPAO a competitive edge to consume nitrite (KsDPAO = 0.022 mM) in the suspended floc fraction. However, DPAO are more sensitive to nitrite (KiDPAO = 0.377 mM) than Anammox bacteria and (KiAMX > 1.786 mM), and therefore the DPAO would be better suited to grow in the protective biofilm, showing that both biomass growth types (flocs and granules) have advantages (and disadvantages) depending on the setting. This work is an important steppingstone to understanding resource competition amongst Anammox and DPAO and SRT management strategies to allow their pairing in combined reactor configurations.

Mechanism Across Scales: A Holistic Modeling Framework Integrating Laboratory and Field Studies for Microbial Ecology.

Over the last century, leaps in technology for imaging, sampling, detection, high-throughput sequencing, and -omics analyses have revolutionized microbial ecology to enable rapid acquisition of extensive datasets for microbial communities across the ever-increasing temporal and spatial scales. The present challenge is capitalizing on our enhanced abilities of observation and integrating diverse data types from different scales, resolutions, and disciplines to reach a causal and mechanistic understanding of how microbial communities transform and respond to perturbations in the environment. This type of causal and mechanistic understanding will make predictions of microbial community behavior more robust and actionable in addressing microbially mediated global problems. To discern drivers of microbial community assembly and function, we recognize the need for a conceptual, quantitative framework that connects measurements of genomic potential, the environment, and ecological and physical forces to rates of microbial growth at specific locations. We describe the Framework for Integrated, Conceptual, and Systematic Microbial Ecology (FICSME), an experimental design framework for conducting process-focused microbial ecology studies that incorporates biological, chemical, and physical drivers of a microbial system into a conceptual model. Through iterative cycles that advance our understanding of the coupling across scales and processes, we can reliably predict how perturbations to microbial systems impact ecosystem-scale processes or vice versa. We describe an approach and potential applications for using the FICSME to elucidate the mechanisms of globally important ecological and physical processes, toward attaining the goal of predicting the structure and function of microbial communities in chemically complex natural environments.

Sulfur Metabolites Play Key System-Level Roles in Modulating Denitrification.

Competition between nitrate-reducing bacteria (NRB) and sulfate-reducing bacteria (SRB) for resources in anoxic environments is generally thought to be governed largely by thermodynamics. It is now recognized that intermediates of nitrogen and sulfur cycling (e.g., hydrogen sulfide, nitrite, etc.) can also directly impact NRB and SRB activities in freshwater, wastewater, and sediment and therefore may play important roles in competitive interactions. Here, through comparative transcriptomic and metabolomic analyses, we have uncovered mechanisms of hydrogen sulfide- and cysteine-mediated inhibition of nitrate respiratory growth for the NRB Intrasporangium calvum C5. Specifically, the systems analysis predicted that cysteine and hydrogen sulfide inhibit growth of I. calvum C5 by disrupting distinct steps across multiple pathways, including branched-chain amino acid (BCAA) biosynthesis, utilization of specific carbon sources, and cofactor metabolism. We have validated these predictions by demonstrating that complementation with BCAAs and specific carbon sources relieves the growth inhibitory effects of cysteine and hydrogen sulfide. We discuss how these mechanistic insights give new context to the interplay and stratification of NRB and SRB in diverse environments.IMPORTANCE Nitrate-reducing bacteria (NRB) and sulfate-reducing bacteria (SRB) colonize diverse anoxic environments, including soil subsurface, groundwater, and wastewater. NRB and SRB compete for resources, and their interplay has major implications on the global cycling of nitrogen and sulfur species, with undesirable outcomes in some contexts. For instance, the removal of reactive nitrogen species by NRB is desirable for wastewater treatment, but in agricultural soils, NRB can drive the conversion of nitrates from fertilizers into nitrous oxide, a potent greenhouse gas. Similarly, the hydrogen sulfide produced by SRB can help sequester and immobilize toxic heavy metals but is undesirable in oil wells where competition between SRB and NRB has been exploited to suppress hydrogen sulfide production. By characterizing how reduced sulfur compounds inhibit growth and activity of NRB, we have gained systems-level and mechanistic insight into the interplay of these two important groups of organisms and drivers of their stratification in diverse environments.