Impact of aerobic granular sludge sizes and dissolved oxygen concentration on greenhouse gas N2O emission.

Aerobic granular sludge (AGS) at wastewater treatment plants (WWTPs) are known to produce nitrous oxide (N2O), a greenhouse gas which has a ∼300 times higher global warming potential than carbon dioxide. In this research, we studied N2O emissions from different sizes of AGS developed at a dissolved oxygen (DO) level of 2 mgO2/L while exposing them to disturbances at various DO concentrations ranging from 1 to 4 mgO2/L. Five different AGS size classes were studied: 212-600 µm, 600-1000 µm, 1000-1400 µm, 1400-2000 µm, and > 2000 µm. Metagenomic data showed N2O reductase genes (nosZ) were more abundant in the smaller AGS sizes which aligned with the observation of higher N2O reduction rates in small AGS under anaerobic conditions. However, when oxygen was present, the activity measurements of N2O emission showed an opposite trend compared to metagenomic data, smaller AGS (212 to 1000 µm) emitted significantly higher N2O (p < 0.05) than larger AGS (1000 µm to >2000 µm) at DO of 2, 3, and 4 mgO2/L. The N2O emission rate showed positive correlation with both oxygen levels and nitrification rate. This pattern indicates a connection between N2O emission and nitrification. In addition, the data suggested the penetration of oxygen into the anoxic zone of granules might have hindered nitrous oxide reduction, resulting in incomplete denitrification stopping at N2O and consequently contributing to an increase in N2O emissions. This work sets the stage to better understand the impacts of AGS size on N2O emissions in WWTPs under different disturbance of DO conditions, and thus ensure that wastewater treatment will comply with possible future regulations demanding lowering greenhouse gas emissions in an effort to combat climate change.

Integrating socio-economic vulnerability factors improves neighborhood-scale wastewater-based epidemiology for public health applications.

Wastewater Based Epidemiology (WBE) of COVID-19 is a low-cost, non-invasive, and inclusive early warning tool for disease spread. Previously studied WBE focused on sampling at wastewater treatment plant scale, limiting the level at which demographic and geographic variations in disease dynamics can be incorporated into the analysis of certain neighborhoods. This study demonstrates the integration of demographic mapping to improve the WBE of COVID-19 and associated post-COVID disease prediction (here kidney disease) at the neighborhood level using machine learning. WBE was conducted at six neighborhoods in Seattle during October 2020 - February 2022. Wastewater processing and RT-qPCR were performed to obtain SARS-CoV-2 RNA concentration. Census data, clinical data of COVID-19, as well as patient data of acute kidney injury (AKI) cases reported during the study period were collected and the distribution across the city was studied using Geographic Information System (GIS) mapping. Further, we analyzed the data set to better understand socioeconomic impacts on disease prevalence of COVID-19 and AKI per neighborhood. The heterogeneity of eleven demographic factors (such as education and age among others) was observed within neighborhoods across the city of Seattle. Dynamics of COVID-19 clinical cases and wastewater SARS-CoV-2 varied across neighborhood with different levels of demographics. Machine learning models trained with data from the earlier stages of the pandemic were able to predict both COVID-19 and AKI incidence in the later stages of the pandemic (Spearman correlation coefficient of 0·546 - 0·904), with the most predictive model trained on the combination of wastewater data and demographics. The integration of demographics strengthened machine learning models' capabilities to predict prevalence of COVID-19, and of AKI as a marker for post-COVID sequelae. Demographic-based WBE presents an effective tool to monitor and manage public health beyond COVID-19 at the neighborhood level.

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.

Mainstream nitrogen removal from low temperature and low ammonium strength municipal wastewater using hydrogel-encapsulated comammox and anammox.

Application of partial nitritation (PN)-anammox to mainstream wastewater treatment faces challenges in low water temperature and low ammonium strength. In this study, a continuous flow PN-anammox reactor with hydrogel-encapsulated comammox and anammox was designed and operated for nitrogen removal from mainstream wastewater with low temperature. Long-term operation with synthetic and real wastewater as the feed demonstrated nearly complete ammonium and total inorganic nitrogen (TIN) removal by the reactor at temperatures as low as 10 °C. A significantly decreased nitrogen removal performance and biomass activity was observed in the reactor at 4 °C before a selective heating strategy was employed. A novel heating technology using radiation to heat carbon black co-encapsulated in the hydrogel matrix with biomass was used to selectively heat biomass but not water in the treatment system. This selective heating technology enabled nearly complete ammonium removal and 89.4 ± 4.3 % TIN removal at influent temperature of 4 °C and reactor temperature 5 °C. Activity tests suggested selective heating brought the biomass activity at influent temperatures of 4 °C and reactor temperature 5 °C to a level comparable to that at 10 °C. Comammox and anammox were consistently present in the system and spatially organized in the hydrogel beads as revealed by qPCR and fluorescence in-situ hybridization (FISH). The abundance of comammox largely decreased by 3 orders of magnitude during the operation at 4 °C, and rapidly recovered after the application of selective heating. The anammox-comammox technology tested in this study essentially enabled mainstream shortcut nitrogen removal, and the selective heating ensured good performance of the technology at temperature as low as 5 °C.

Simultaneous anaerobic carbon and nitrogen removal from primary municipal wastewater with hydrogel encapsulated anaerobic digestion sludge and AOA-anammox coated hollow fiber membrane.

In this study, a one-stage continuous-flow membrane-hydrogel reactor integrating both partial nitritation-anammox (PN-anammox) and anaerobic digestion (AD) was designed and operated for simultaneous autotrophic nitrogen (N) and anaerobic carbon (C) removal from mainstream municipal wastewater. In the reactor, a synthetic biofilm consisting of anammox biomass and pure culture ammonia oxidizing archaea (AOA) were coated onto and maintained on a counter-diffusion hollow fiber membrane to autotrophically remove nitrogen. Anaerobic digestion sludge was encapsulated in hydrogel beads and placed in the reactor to anaerobically remove COD. During the pilot operation at three operating temperature (25, 16 and 10 °C), the membrane-hydrogel reactor demonstrated stable anaerobic COD removal (76.2 ± 15.5 %) and membrane fouling was successfully suppressed allowing a relatively stable PN-anammox process. The reactor demonstrated good nitrogen removal efficiency, with an overall removal efficiency of 95.8 ± 5.0 % for NH4+-N and 78.9 ± 13.2 % for total inorganic nitrogen (TIN) during the entire pilot operation. Reducing the temperature to 10 °C caused a temporary reduction in nitrogen removal performance and abundances of AOA and anammox. However, the reactor and microbes demonstrated the ability to adapt to the low temperature spontaneously with recovered nitrogen removal performance and microbial abundances. Methanogens in hydrogel beads and AOA and anammox on the membrane were observed in the reactor by qPCR and 16S sequencing across all operational temperatures.

Integration of EBPR with mainstream anammox process to treat real municipal wastewater: Process performance and microbiology.

The mainstream application of anaerobic ammonium oxidation (anammox) for sustainable N removal remains a challenge. Similarly, with recent additional stringent regulations for P discharges, it is imperative to integrate N with P removal. This research studied integrated fixed film activated sludge (IFAS) technology to simultaneously remove N and P in real municipal wastewater by combining biofilm anammox with flocculent activated sludge for enhanced biological P removal (EBPR). This technology was assessed in a sequencing batch reactor (SBR) operated as a conventional A2O (anaerobic-anoxic-oxic) process with a hydraulic retention time of 8.8 h. After a steady state operation was reached, robust reactor performance was obtained with average TIN and P removal efficiencies of 91.3 ± 4.1% and 98.4 ± 2.4%, respectively. The average TIN removal rate recorded over the last 100 d of reactor operation was 118 mg/L·d, which is a reasonable number for mainstream applications. The activity of denitrifying polyphosphate accumulating organisms (DPAOs) accounted for nearly 15.9% of P-uptake during the anoxic phase. DPAOs and canonical denitrifiers removed approximately 5.9 mg TIN/L in the anoxic phase. Batch activity assays, which showed that nearly 44.5% of TIN were removed by the biofilms during the aerobic phase. The functional gene expression data also confirmed anammox activities. The IFAS configuration of the SBR allowed operation at a low solid retention time (SRT) of 5-d without washing out biofilm ammonium-oxidizing and anammox bacteria. The low SRT, combined with low dissolved oxygen and intermittent aeration, provided a selective pressure to washout nitrite-oxidizing bacteria and glycogen-accumulating organisms, as relative abundances of.

Wastewater surveillance of SARS-CoV-2 at intra-city level demonstrated high resolution in tracking COVID-19 and calibration using chemical indicators.

Wastewater-based epidemiology has proven to be a supportive tool to better comprehend the dynamics of the COVID-19 pandemic. As the disease moves into endemic stage, the surveillance at wastewater sub-catchments such as pump station and manholes is providing a novel mechanism to examine the reemergence and to take measures that can prevent the spread. However, there is still a lack of understanding when it comes to wastewater-based epidemiology implementation at the smaller intra-city level for better granularity in data, and dilution effect of rain precipitation at pump stations. For this study, grab samples were collected from six areas of Seattle between March-October 2021. These sampling sites comprised five manholes and one pump station with population ranging from 2580 to 39,502 per manhole/pump station. The wastewater samples were analyzed for SARS-CoV-2 RNA concentrations, and we also obtained the daily COVID-19 cases (from individual clinical testing) for each corresponding sewershed, which ranged from 1 to 12 and the daily incidence varied between 3 and 64 per 100,000 of population. Rain precipitation lowered viral RNA levels and sensitivity of viral detection but wastewater total ammonia (NH4+-N) and phosphate (PO43--P) were shown as potential chemical indicators to calibrate/level out the dilution effect. These chemicals showed the potential in improving the wastewater surveillance capacity of COVID-19.

Case Study: Impact of Diurnal Variations and Stormwater Dilution on SARS-CoV-2 RNA Signal Intensity at Neighborhood Scale Wastewater Pumping Stations.

Wastewater based epidemiology (WBE) has emerged as a tool to track the spread of SARS-CoV-2. However, sampling at wastewater treatment plants (WWTPs) cannot identify transmission hotspots within a city. Here, we sought to understand the diurnal variations (24 h) in SARS-CoV-2 RNA titers at the neighborhood level, using pump stations that serve vulnerable communities (e.g., essential workers, more diverse communities). Hourly composite samples were collected from wastewater pump stations located in (i) a residential area and (ii) a shopping district. In the residential area, SARS-CoV-2 RNA concentration (N1, N2, and E assays) varied by up to 42-fold within a 24 h period. The highest viral load was observed between 5 and 7 am, when viral RNA was not diluted by stormwater. Normalizing peak concentrations during this time window with nutrient concentrations (N and P) enabled correcting for rainfall to connect sewage to clinical cases reported in the sewershed. Data from the shopping district pump station were inconsistent, probably due to the fluctuation of customers shopping at the mall. This work indicates pump stations serving the residential area offer a narrow time period of high signal intensity that could improve the sensitivity of WBE, and tracer compounds (N, P concentration) can be used to normalize SARS-CoV-2 signals during rainfall.

Partitioning of nutrient removal contribution between granules and flocs in a hybrid granular activated sludge system.

Sludge granulation in continuous-flow systems is an emerging technology to intensify existing activated sludge infrastructure for nutrient removal. In these systems, the nutrient removal contributions and partitioning of microbial functions between granules and flocs can offer insights into process implementations. To this end, a reactor system that simulates the continuous-flow environment using an equal amount of initial granule and floc biomass was investigated. The two operational strategies for maintaining granule growth in the continuous-flow system were (a) the higher solids retention time (SRT) for the granules versus flocs, as well as (b) selective feeding of carbon to the granules. The SRT of the large granule fractions (>425 µm, LG) and floc/small granule fractions (<425 µm, FSG) were controlled at 20 and 2.7-6.0 days, respectively. Long term operation of the hybrid granule/floc system achieved high PO43- and NH4+ removal efficiencies. Higher polyphosphate-accumulating organisms (PAO) activity was observed in the FSG than LG, while ammonia-oxidizing bacteria (AOB) activities were similar in the two biomass fractions. Nitrite shunt was observed in the FSG, possibly due to out-competition by the high NOB activity in LG. More importantly, washing out the FSG caused a reduction in LG's AOB and PAO activity, indicating a possible dependency of LG on FSG for maintaining its nutrient removal capacity. Our findings highlighted the partitioning and potential competition/cooperation of key microbial functional groups between LG and FSG, facilitating nutrient removal in a hybrid granular activated sludge system, as well as implications for practical application of the treatment platform.

Application of aerobic kenaf granules for biological nutrient removal in a full-scale continuous flow activated sludge system.

Aerobic granular sludge (AGS) is a biofilm technology that offers more treatment capacity in comparison to activated sludge. The integration of AGS into existing continuous-flow activated sludge systems is of great interest as process intensification can be achieved without the use of plastic-based biofilm carriers. Such integration should allow good separation of granules/flocs and ideally with minor retrofitting, making it an ongoing challenge. This study utilized an all-organic media carrier made of porous kenaf plant stalks with high surface areas to facilitate biofilm attachment and granule development. A 5-stage Bardenpho plant was upgraded with the addition of kenaf media and a rotary drum screen to retain the larger particles from the secondary clarifier underflow whereas flocs were selectively wasted. Startup took 5 months with a sludge volume index (SVI) reduction from >200 to 50 mL g-1. Most of the kenaf granules fell in the size range of 600-1400 µm and had a clear biofilm layer. The wet biomass density, SVI30, and SVI30/SVI5 of the kenaf granules were 1035 g L-1, 30.6 mL g-1, and 1.0, respectively, which met the standards of aerobic granules. Improved stability of biological phosphorus removal performance enabled a 25% reduction in sodium aluminate usage. Microbial activities of kenaf granules were compared with aerobic granules, showing comparable N and P removal rates and presence of ammonium-oxidizing bacteria and polyphosphate-accumulating organisms in the outer 50-60 µm layer of the granule. This work is the first viable example for integrating fully organic biofilm particles in existing continuous-flow systems.

Elucidating the Competition between Heterotrophic Denitrification and DNRA Using the Resource-Ratio Theory.

Heterotrophic denitrification and dissimilatory nitrate reduction to ammonium (DNRA) are two microbial processes competing for two shared resources, namely, nitrate and organic carbon (COD). Their competition has great implications for nitrogen loss, conservation, and greenhouse gas emissions. Nevertheless, a comprehensive and mechanistic understanding of the governing factors for this competition is still lacking. We applied the resource-ratio theory to study this competition and validated the theory with experimental data from continuous cultures reported in the literature. Based on this theory, we revealed that influent COD/N ratio alone was not sufficient to predict the competition outcome as the boundary values for different competition outcomes changed substantially with influent resource concentrations. The stoichiometry of the two processes was determinative for the boundaries, whereas the affinity for the shared resources (KS), maximum specific growth rate (µmax) of the two species, and the dilution rate had significant impacts as well but mainly at low influent resource concentrations (e.g., <100 µM nitrate). The presented approach allows for a more comprehensive understanding of the parameters controlling microbial competition. The computational comparison between continuous and batch cultures could explain seemingly conflicting experimental results as to the impact of the COD/N ratio. The results also include testable hypotheses and tools for understanding and managing the fate of nitrate in ecosystems, which could also be applied more widely to other species competing for two shared resources.

Alternative strategies of nutrient acquisition and energy conservation map to the biogeography of marine ammonia-oxidizing archaea.

Ammonia-oxidizing archaea (AOA) are among the most abundant and ubiquitous microorganisms in the ocean, exerting primary control on nitrification and nitrogen oxides emission. Although united by a common physiology of chemoautotrophic growth on ammonia, a corresponding high genomic and habitat variability suggests tremendous adaptive capacity. Here, we compared 44 diverse AOA genomes, 37 from species cultivated from samples collected across diverse geographic locations and seven assembled from metagenomic sequences from the mesopelagic to hadopelagic zones of the deep ocean. Comparative analysis identified seven major marine AOA genotypic groups having gene content correlated with their distinctive biogeographies. Phosphorus and ammonia availabilities as well as hydrostatic pressure were identified as selective forces driving marine AOA genotypic and gene content variability in different oceanic regions. Notably, AOA methylphosphonate biosynthetic genes span diverse oceanic provinces, reinforcing their importance for methane production in the ocean. Together, our combined comparative physiological, genomic, and metagenomic analyses provide a comprehensive view of the biogeography of globally abundant AOA and their adaptive radiation into a vast range of marine and terrestrial habitats.

Effective nitrogen removal from ammonium-depleted wastewater by partial nitritation and anammox immobilized in granular and thin layer gel carriers.

This study investigates the effect of physicochemical conditions on the partial nitritation and anammox treatment by immobilized ammonia oxidizers under ammonium-deplete conditions. The impact of oxygen and temperature was studied by measuring the activity of immobilized aerobic and anaerobic ammonia-oxidizing organisms (Ammonia-oxidizing bacteria (AOB) and archaea (AOA), and Anammox bacteria) embedded in polyvinyl alcohol - sodium alginate (PVA-SA) beads and in thin layer poly-ethylene glycol hydrogels. Beads and flat hydrogels were incubated in a fluidized bed reactor (FBR) and in two flow cells, respectively. Both systems were fed with synthetic wastewater (15 mg N-NH4+/L) at different temperatures (20 °C and/or 30 °C) and different dissolved oxygen (DO) concentrations (0.1, 0.3, 0.5 and/or 1 mg/L) over 152 and 207 days, respectively. The FBR system had a maximum removal rate of 1.7 g-N/m3/d at 0.1 mg O2/L, corresponding to 80% removal efficiency, while a high aerobic ammonia-oxidizing activity but a partial oxygen inhibition of Anammox bacteria were observed at higher DO concentrations. In both flow cells, nitrogen removal efficiency was highest (80%) at 30 °C and 1 mg O2/L while removal was less favorable at lower DO and lower temperature. Our results indicate a potential use of hydrogel beads for an energy efficient technology with reduced aeration demand for treating low ammonia wastewater, while layered hydrogels are a possible first step for biological treatments of wastewater using tangential flow. In addition, we provide blueprint drawings of the flow cells, which may be used to 3D-print the apparatus for other applications.

Correlating sludge constituents with digester foaming risk using sludge foam potential and rheology.

Foam potential and viscometer ramp tests (VRTs) were conducted for three municipal wastewater treatment plants to determine if these methods can relate to mechanisms of foaming to physical and biological constituents in sludge. At all plants, digester volatile solids (VS) concentration correlated (R2 > 0.41) with increases in plastic viscosity, a VRT parameter corresponding to foaming risk. Plastic viscosity also correlated with foam-causing bacteria Gordonia (R2 = 0.38). Foam potential test values increased with Microthrix parvicella (R2> 0.28). For one plant, suspected foam-causing bacteria Mycobacterium negatively correlated with parameters representing foam risk. Microscopic filament counting correlated (R2 = 0.97) with quantitative polymerase chain reaction (qPCR) for Gordonia, suggesting that the more accessible counting method can reliably quantify foam-causing bacteria. Foam potential tests and VRTs resulted in plant-specific correlations with foam-related constituents. Therefore, these tests may provide useful evidence when investigating causes of digester foam events.

Flocs in disguise? High granule abundance found in continuous-flow activated sludge treatment plants.

To date, high performance of full-scale aerobic granular sludge (AGS) technology has been demonstrated on a global scale. Its further integration with existing continuous flow activated sludge (CFAS) treatment plants is the next logical step. All granular sludge reactors operated in sequencing batch reactors (SBR) mode with anaerobic feeding conditions select for growth of phosphorus and glycogen accumulating organisms (PAO and GAO, respectively), which are known to enhance sludge settling characteristics. Therefore, we hypothesized that AGS are commonly present at full-scale CFAS processes with enhanced biological phosphorus removal (EBPR) and low sludge volume index (SVI). This hypothesis was confirmed at 13 EBPR plants, where granules were found present (at plants where SVI was lower than 100 ml/g) with a strong correlation between high granule abundance and low SVI. A wide range of granule abundance was found among the plants, ranging from 0.5% to as high as 80%. Evaluations of the EBPR plant process configurations showed that high granule abundances may be related to selector design features such as high anaerobic food to mass (F/M) ratios, unmixed in-line fermentation, and high influent soluble COD fraction. Granules were also observed at a non-EBPR plant with an aerobic selector receiving high F/M feeds. Quantitative PCR and 16S rRNA gene sequencing analyses revealed higher relative gene abundance of Accumulibacter PAO and Competibacter GAO in the granules over flocs, as well as a correlation between granule abundance and some possible EPS producers such as Flavobacterium and Competibacter. Our results indicated that process configurations that select for slow-growing or EPS-producing heterotrophs play an important role for granule formation in full-scale CFAS systems as previously shown in SBR configurations.

Reducing Cost and Environmental Impact of Wastewater Treatment with Denitrifying Methanotrophs, Anammox, and Mainstream Anaerobic Treatment.

In water resource recovery facilities, sidestream biological nitrogen removal via anaerobic ammonium oxidation (anammox) is more energy and cost efficient than conventional nitrification-denitrification. However, under mainstream conditions, nitrite oxidizing bacteria (NOB) out-select anammox bacteria for nitrite produced by ammonium oxidizing bacteria (AOB). Therefore, nitrite production is the bottleneck in mainstream anammox nitrogen removal. Nitrate-dependent denitrifying anaerobic methane oxidizing archaea (n-damo) oxidize methane and reduce nitrate to nitrite. The nitrite supply challenge in mainstream anammox implementation could be solved with a microbial community of AOB, NOB, n-damo, and anammox with methane from anaerobic sludge digestion or a mainstream anaerobic membrane bioreactor (AnMBR). The cost and environmental impact of traditional nitrification/dentrification relative to AOB/anammox and AOB/anammox/n-damo systems, with and without an AnMBR, were compared with a stoichiometric model. AnMBR implementation reduced costs and emission rates at moderate to high nutrient loading by lowering aeration and sludge handling demands while increasing methane available for cogeneration. AnMBR/AOB/anammox systems reduced cost and GHG emission by up to $0.303/d/m3 and 1.72 kg equiv. CO2/d/m3, respectively, while AnMBR/AOB/anammox/n-damo systems saw a similar reduction of at least $0.300/d/m3 and 1.65 kg equiv. CO2/d/m3 in addition to alleviating the necessity to stop nitrification at nitrate, allowing easier aeration control.

Kinetic implication of moving warm side-stream Anaerobic ammonium oxidizing bacteria to cold mainstream wastewater.

Decreased activity of Anaerobic ammonia oxidation (Anammox) at low temperatures is widely reported and one of the key challenges to applying Anammox to mainstream wastewater treatment. In this work, we systematically measured Anammox across a range of temperatures for biomass taken from a side-stream (25-30 °C) Anammox system. Using the Cardinal temperature model with inflection (CTMI) and parameters Tmin = 10 °C, Topt = 31 °C, Tmax = 40 °C, and µmax = 0.29 d-1, the data was accurately represented. Alternatively, the popularly used Arrhenius equation was not able to consistently capture the trend we observed. The CTMI was further used to compute the relative increase in retention time and/or biomass concentration to treat the same quantity of nitrogen at sub-optimal temperatures, requiring a two-fold increase at 21 °C and three-fold increase at 19 °C. We conclude that cold adapted cultures are necessary to evaluate the possibility of cold Anammox wastewater treatment.

Affinity informs environmental cooperation between ammonia-oxidizing archaea (AOA) and anaerobic ammonia-oxidizing (Anammox) bacteria.

Anaerobic ammonia-oxidizing (Anammox) bacteria (AnAOB) rely on nitrite supplied by ammonia-oxidizing bacteria (AOB) and archaea (AOA). Affinities for ammonia and oxygen play a crucial role in AOA/AOB competition and their association with AnAOB. In this work we measured the affinity constants for ammonia and oxygen (half-saturation; km) of two freshwater AOA enrichments, an AOA soil isolate (N. viennensis), and a freshwater AnAOB enrichment. The AOA enrichments had similar kinetics (µmax ≈ 0.36 d-1, km,NH4 ≈ 0.78 µM, and km,O2 ≈ 2.9 µM), whereas N. viennensis had similar km values but lower µmax (0.23 d-1). In agreement with the current paradigm, these AOA strains showed a higher affinity for ammonia (lower km,NH4; 0.34-1.27 µM) than published AOB measurements (>20 µM). The slower growing AnAOB (µmax ≈ 0.16 d-1) had much higher km values (km,NH4 ≈ 132 µM, km,NO2 ≈ 48 µM) and were inhibited by oxygen at low levels (half-oxygen inhibition; ki,O2 ≈ 0.092 µM). The higher affinity of AOA for ammonia relative to AnAOB, suggests AOA/AnAOB cooperation is only possible where AOA do not outcompete AnAOB for ammonia. Using a biofilm model, we show that environments of ammonia/oxygen counter diffusion, such as stratified lakes, favors this cooperation.

Perchlorate bioreduction linked to methane oxidation in a membrane biofilm reactor: Performance and microbial community structure.

Perchlorate bioreduction coupled to methane oxidation was successfully achieved without the addition of nitrate or nitrite in a membrane biofilm reactor (MBfR) inoculated with a mixture of freshwater sediments and anaerobic digester sludge as well as return activated sludge. The reactor was operated at different methane pressures (60, 40 and 20 Kpa) and influent perchlorate concentrations (1, 5 and 10 mg/L) to evaluate the biochemical process of perchlorate bioreduction coupled to methane oxidation. Perchlorate was completely reduced with a higher removal flux of 92.75 mg/m2·d using methane as the sole carbon source and electron donor, other than hydrogen or other limiting organics. Quantitative real-time PCR showed that bacteria prevailed over archaea and the abundances of mcrA, pMMO, pcrA, and nirS genes were correlated with the influent perchlorate flux. High-throughput sequencing of 16S rRNA genes demonstrated that the functional community consisted of methanotrophs, methylotrophs, perchlorate-reducing bacteria, as well as various denitrifiers.

Integration of methane removal in aerobic anammox-based granular sludge reactors.

Combined partial nitritation-anaerobic ammonium oxidation (anammox) processes have been widely applied for nitrogen removal from anaerobic digestion reject water. However, such streams also contain dissolved methane that can escape to the atmosphere, hence contributing to global warming. This study investigates the possibility of integrating methane removal in aerobic anammox-based granular sludge reactors, through modelling and simulation. Methane removal could be established through aerobic methane-oxidizing bacteria (MOB), denitrifying anaerobic methane-oxidizing bacteria (damoB, NO2- + CH4 → N2 + CO2), and/or archaea (damoA, NO3- + CH4 → NO2- + CO2). The simulation results demonstrated that the combined removal of nitrogen and methane was feasible at low dissolved oxygen conditions. Aerobic MOB were the main responsible microorganisms for removing methane. A sensitivity analysis of key kinetic parameters showed a shift in the methanotrophic populations depending on the most favourable parameters for each microbial group, while keeping high nitrogen and methane removal efficiencies. Possible methane stripping during aeration could be limited by increasing the depth within the reactor column at which aeration was supplied. Overall, the integration of methane removal in aerobic anammox-based granular sludge reactors seems to be a promising process option to reduce the carbon footprint from wastewater treatment.