Hydrodynamic regimes modulate nitrogen fixation and the mode of diazotrophy in Lake Tanganyika.

The factors that govern the geographical distribution of nitrogen fixation are fundamental to providing accurate nitrogen budgets in aquatic environments. Model-based insights have demonstrated that regional hydrodynamics strongly impact nitrogen fixation. However, the mechanisms establishing this physical-biological coupling have yet to be constrained in field surveys. Here, we examine the distribution of nitrogen fixation in Lake Tanganyika - a model system with well-defined hydrodynamic regimes. We report that nitrogen fixation is five times higher under stratified than under upwelling conditions. Under stratified conditions, the limited resupply of inorganic nitrogen to surface waters, combined with greater light penetration, promotes the activity of bloom-forming photoautotrophic diazotrophs. In contrast, upwelling conditions support predominantly heterotrophic diazotrophs, which are uniquely suited to chemotactic foraging in a more dynamic nutrient landscape. We suggest that these hydrodynamic regimes (stratification versus mixing) play an important role in governing both the rates and the mode of nitrogen fixation.

Elucidating the role of extracellular polymeric substances (EPS) in dewaterability of fecal sludge from onsite sanitation systems, and changes during anaerobic storage.

As the importance of fecal sludge management (FSM) is increasingly being realized, the need for adequately designed and functioning fecal sludge (FS) treatment plants is also increasing. Research to fill this gap is only emerging and dewatering is a key challenge for developing sustainable treatment solutions. This study evaluated the effect of extracellular polymeric substances (EPS) on dewaterability of FS, and how EPS and dewaterability change during anaerobic storage (as a proxy for time in onsite containment). EPS was extracted from FS and activated sludge using Na2CO3 and sonication and added to sludge samples to determine the effect on dewaterability. The results confirmed that an increase in EPS had a direct impact of decreasing FS dewaterability (as capillary suction time). In this context, we evaluated FS degradation during anaerobic storage, the effect of anaerobic storage time on EPS, EPS fractions and particle size distribution, and the effect of variations in these factors on FS dewaterability. Variations in EPS, EPS fraction and particle size distribution during anaerobic storage were less than expected and average VS reduction of 20% was recorded over 7 weeks. Although anaerobic digestion was verified (biogas production), the results indicate that kinetics of degradation of FS is different from wastewater sludges. Comparatively, EPS fractions in FS were 70 - 75% lower and with higher fractions of humic-like substances than wastewater sludges. Although EPS significantly affects FS dewaterability, anaerobic storage time is not a predictor of dewaterability.

Microbial Nitrogen Transformation Potential in Sediments of Two Contrasting Lakes Is Spatially Structured but Seasonally Stable.

The nitrogen (N) cycle is of global importance, as N is an essential element and a limiting nutrient in terrestrial and aquatic ecosystems. Excessive anthropogenic N fertilizer usage threatens sensitive downstream aquatic ecosystems. Although freshwater lake sediments remove N through various microbially mediated processes, few studies have investigated the microbial communities involved. In an integrated biogeochemical and microbiological study on a eutrophic and oligotrophic lake, we estimated N removal rates from pore water concentration gradients in sediments. Simultaneously, the abundance of different microbial N transformation genes was investigated using metagenomics on a seasonal and spatial scale. We observed that contrasting nutrient concentrations in sediments were associated with distinct microbial community compositions and significant differences in abundances of various N transformation genes. For both characteristics, we observed a more pronounced spatial than seasonal variability within each lake. The eutrophic Lake Baldegg showed a higher denitrification potential with higher nosZ gene (N2O reductase) abundances and higher nirS:nirK (nitrite reductase) ratios, indicating a greater capacity for complete denitrification. Correspondingly, this lake had a higher N removal efficiency. The oligotrophic Lake Sarnen, in contrast, had a higher potential for nitrification. Specifically, it harbored a high abundance of Nitrospira, including some with the potential for comammox. Our results demonstrate that knowledge of the genomic N transformation potential is important for interpreting N process rates and understanding how the lacustrine sedimentary N cycle responds to variations in trophic conditions. IMPORTANCE Anthropogenic nitrogen (N) inputs can lead to eutrophication in surface waters, especially in N-limited coastal ecosystems. Lakes effectively remove reactive N by transforming it to N2 through microbial denitrification or anammox. The rates and distributions of these microbial processes are affected by factors such as the amount and quality of settling organic material and nitrate concentrations. However, the microbial communities mediating these N transformation processes in freshwater lake sediments remain largely unknown. We provide the first seasonally and spatially resolved metagenomic analysis of the N cycle in sediments of two lakes with different trophic states. We show that lakes with different trophic states select for distinct communities of N-cycling microorganisms with contrasting functional potentials for N transformation.

Robustness of mainstream anammox activity at bench and pilot scale.

New technologies and processes, such as mainstream anammox, aim to reduce energy requirements of wastewater treatment and improve effluent quality. However, in municipal wastewater (MWW) anammox system are often unstable due to process control disturbance, influent variability, or unwanted nitrite oxidizing bacteria (NOB). This study examines the anammox system by focusing on anammox activity and its robustness in a mainstream environment. An 8 m3 pilot-scale sequencing batch reactor (SBR) receiving pretreated MWW (with external nitrite addition) was seeded with pre-colonized carriers. Within six months at 12-20 °C an anammox activity of 200 gN·m-3·d-1 was achieved. After the startup an anammox activity of 260 ± 83 gN·m-3·d-1 was maintained over 450 days. The robustness of the anammox activity was analyzed through three disturbance experiments. Anammox biofilm on carriers were exposed to dissolved oxygen (DO = 1.6 mg·L-1, intermittent aeration), organic loading rate (OLR, C/N increased from 2:1 to 5:1) and temperature disturbances (20 °C to 12 °C) in triplicate 12 L bench scale reactors. The anammox activity and microbial community was monitored during these disturbances. The DO and OLR disturbance experiments were replicated at pilot scale to investigate upscaling effects. Bench and pilot scale anammox activity were unaffected by the DO disturbance. Similarly, an increase in OLR did not deteriorate the bench and pilot scale anammox activity, if nitrate was available. When, at bench scale, the reactor temperature was reduced from 20 °C to 12 °C overnight, anammox activity decreased significantly, this was not the case for the slow seasonal temperature changes (12-25 °C) at pilot scale where no strong temperature dependency was detected in winter. Metagenomic analysis revealed a broad range of Brocadiaceae species with no single dominant anammox species. Anammox thrive under mainstream conditions and can withstand typical process disruptions.

Distinct growth stages controlled by the interplay of deterministic and stochastic processes in functional anammox biofilms.

Mainstream anaerobic ammonium oxidation (anammox) represents one of the most promising energy-efficient mechanisms of fixed nitrogen elimination from wastewaters. However, little is known about the exact processes and drivers of microbial community assembly within the complex microbial biofilms that support anammox in engineered ecosystems. Here, we followed anammox biofilm development on fresh carriers in an established 8m3 mainstream anammox reactor that is exposed to seasonal temperature changes (~25-12°C) and varying NH4+ concentrations (5-25 mg/L). We use fluorescence in situ hybridization and 16S rRNA gene sequencing to show that three distinct stages of biofilm development emerge naturally from microbial community composition and biofilm structure. Neutral modelling and network analysis are employed to elucidate the relative importance of stochastic versus deterministic processes and synergistic and antagonistic interactions in the biofilms during their development. We find that the different phases are characterized by a dynamic succession and an interplay of both stochastic and deterministic processes. The observed growth stages (Colonization, Succession and Maturation) appear to be the prerequisite for the anticipated growth of anammox bacteria and for reaching a biofilm community structure that supports the desired metabolic and functional capacities observed for biofilm carriers already present in the system (~100gNH4-N m3 d-1). We discuss the relevance of this improved understanding of anammox-community ecology and biofilm development in the context of its practical application in the start-up, configuration, and optimization of anammox biofilm reactors.

Nitrogen isotope effects can be used to diagnose N transformations in wastewater anammox systems.

Anaerobic ammonium oxidation (anammox) plays an important role in aquatic systems as a sink of bioavailable nitrogen (N), and in engineered processes by removing ammonium from wastewater. The isotope effects anammox imparts in the N isotope signatures (15N/14N) of ammonium, nitrite, and nitrate can be used to estimate its role in environmental settings, to describe physiological and ecological variations in the anammox process, and possibly to optimize anammox-based wastewater treatment. We measured the stable N-isotope composition of ammonium, nitrite, and nitrate in wastewater cultivations of anammox bacteria. We find that the N isotope enrichment factor 15ε for the reduction of nitrite to N2 is consistent across all experimental conditions (13.5‰ ± 3.7‰), suggesting it reflects the composition of the anammox bacteria community. Values of 15ε for the oxidation of nitrite to nitrate (inverse isotope effect, - 16 to - 43‰) and for the reduction of ammonium to N2 (normal isotope effect, 19-32‰) are more variable, and likely controlled by experimental conditions. We argue that the variations in the isotope effects can be tied to the metabolism and physiology of anammox bacteria, and that the broad range of isotope effects observed for anammox introduces complications for analyzing N-isotope mass balances in natural systems.

Linking seasonal N2O emissions and nitrification failures to microbial dynamics in a SBR wastewater treatment plant.

Nitrous oxide (N2O) is a strong greenhouse gas and causal for stratospheric ozone depletion. During biological nitrogen removal in wastewater treatment plants (WWTP), high N2O fluxes to the atmosphere can occur, typically exhibiting a seasonal emission pattern. Attempts to explain the peak emission phases in winter and spring using physico-chemical process data from WWTP were so far unsuccessful and new approaches are required. The complex and diverse microbial community of activated sludge used in biological treatment systems also exhibit substantial seasonal patterns. However, a potentially causal link between the seasonal patterns of microbial diversity and N2O emissions has not yet been investigated. Here we show that in a full-scale WWTP nitrification failure and N2O peak emissions, bad settleability of the activated sludge and a turbid effluent strongly correlate with a significant reduction in the microbial community diversity and shifts in community composition. During episodes of impaired performance, we observed a significant reduction in abundance for filamentous and nitrite oxidizing bacteria in all affected reactors. In some reactors that did not exhibit nitrification and settling failures, we observed a stable microbial community and no drastic loss of species. Standard engineering approaches to stabilize nitrification, such as increasing the aerobic sludge age and oxygen availability failed to improve the plant performance on this particular WWTP and replacing the activated sludge was the only measure applied by the operators to recover treatment performance in affected reactors. Our results demonstrate that disturbances of the sludge microbiome affect key structural and functional microbial groups, which lead to seasonal N2O emission patterns. To reduce N2O emissions from WWTP, it is therefore crucial to understand the drivers that lead to the microbial population dynamics in the activated sludge.

Temperature modulates stress response in mainstream anammox reactors.

Autotrophic nitrogen removal by anaerobic ammonium oxidizing (anammox) bacteria is an energy-efficient nitrogen removal process in wastewater treatment. However, full-scale deployment under mainstream conditions remains challenging for practitioners due to the high stress susceptibility of anammox bacteria towards fluctuations in dissolved oxygen (DO) and temperature. Here, we investigated the response of microbial biofilms with verified anammox activity to DO shocks under 20 °C and 14 °C. While pulse disturbances of 0.3 mg L-1 DO prompted only moderate declines in the NH4+ removal rates, 1.0 mg L-1 DO led to complete but reversible inhibition of the NH4+ removal activity in all reactors. Genome-centric metagenomics and metatranscriptomics were used to investigate the stress response on various biological levels. We show that temperature regime and strength of DO perturbations induced divergent responses from the process level down to the transcriptional profile of individual taxa. Community-wide gene expression differed significantly depending on the temperature regime in all reactors, and we found a noticeable impact of DO disturbances on genes involved in transcription, translation, replication and posttranslational modification at 20 °C but not 14 °C. Genome-centric analysis revealed that different anammox species and other key biofilm taxa differed in their transcriptional responses to distinct temperature regimes and DO disturbances.

Ecological strategies and metabolic trade-offs of complex environmental biofilms.

Microorganisms aggregated into matrix-enclosed biofilms dominate microbial life in most natural, engineered, and medical systems. Despite this, the ecological adaptations and metabolic trade-offs of the formation of complex biofilms are currently poorly understood. Here, exploring the dynamics of bacterial ribosomal RNA operon (rrn) copy numbers, we unravel the genomic underpinning of the formation and success of stream biofilms that contain hundreds of bacterial taxa. Experimenting with stream biofilms, we found that nascent biofilms in eutrophic systems had reduced lag phases and higher growth rates, and more taxa with higher rrn copy number than biofilms from oligotrophic systems. Based on these growth-related traits, our findings suggest that biofilm succession was dominated by slow-but-efficient bacteria likely with leaky functions, such as the production of extracellular polymeric substances at the cost of rapid growth. Expanding our experimental findings to biofilms from 140 streams, we found that rrn copy number distribution reflects functional trait allocation and ecological strategies of biofilms to be able to thrive in fluctuating environments. These findings suggest that alternative trade-offs dominating over rate-yield trade-offs contribute to the evolutionary success of stream biofilms.

Attached biofilms and suspended aggregates are distinct microbial lifestyles emanating from differing hydraulics.

Small-scale hydraulics affects microbial behaviour at the cell level1, trophic interactions in marine aggregates2 and the physical structure and function of stream biofilms3,4. However, it remains unclear how hydraulics, predictably changing from small streams to large rivers, impacts the structure and biodiversity of complex microbial communities in these ecosystems. Here, we present experimental evidence unveiling hydraulics as a hitherto poorly recognized control of microbial lifestyle differentiation in fluvial ecosystems. Exposing planktonic source communities from stream and floodplain ecosystems to different hydraulic environments revealed strong selective hydraulic pressures but only minor founder effects on the differentiation of attached biofilms and suspended aggregates and their biodiversity dynamics. Key taxa with a coherent phylogenetic underpinning drove this differentiation. Only a few resident and phylogenetically related taxa formed the backbone of biofilm communities, whereas numerous resident taxa characterized aggregate communities. Our findings unveil fundamental differences between biofilms and aggregates and build the basis for a mechanistic understanding of how hydraulics drives the distribution of microbial diversity along the fluvial continuum5-7.