•IFAS allowed low floc SRT operation for reliable EBPR while keeping high N removal.•P (98%) and N (91%) removal in domestic wastewater was achieved w/o org. C dosage.•Anammox biofilms contributed to ...44.5% of TIN removal.•HzsB expression further confirmed anammox activities in the anoxic/aerobic phases.•DPAOs were enriched while NOB and GAO growth was under control.
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
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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.
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•Biological nitrogen removal and recovery approaches are reviewed.•Anammox saves energy in side-stream treatment and is progressing toward mainstream.•Phototrophic nitrogen ...assimilation/recovery is attractive for tertiary treatment.•Microbial electrochemical cells are good for energy efficient recovery.•The ideal system utilizes several approaches and plays to the strength of each.
This review summarizes strategies for biological nitrogen removal (BNR) and recovery from wastewater. The most commonly used BNR technology nitrification/denitrification is also the most energy intensive, even though there are lower energy options, including nitritation/denitritation and more efficient partial nitritation/Anammox; the latter is well demonstrated for side-stream treatment and progressing toward mainstream applications. Nitrogen recovery can be done through cell assimilation with phototrophs, but bottlenecks with solids separation and space requirements limit applications to tertiary treatment. Whereas, microbial electrochemical cells are energy efficient at recovering nitrogen from side streams, but not capable of achieving low effluent levels. The combined strengths of these emerging approaches will improve wastewater nitrogen removal by reducing energy consumption, minimizing effluent nitrogen, and maximizing nitrogen recovery.
In this study, we analysed the nitrifying microbial community (ammonium-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB)) within three different aerobic granular sludge treatment systems ...as well as within one flocculent sludge system. Granular samples were taken from one pilot plant run on municipal wastewater as well as from two lab-scale reactors. Fluorescent in situ hybridization (FISH) and quantitative PCR (qPCR) showed that
Nitrobacter
was the dominant NOB in acetate-fed aerobic granules. In the conventional system, both
Nitrospira
and
Nitrobacter
were present in similar amounts. Remarkably, the NOB/AOB ratio in aerobic granular sludge was elevated but not in the conventional treatment plant suggesting that the growth of
Nitrobacter
within aerobic granular sludge, in particular, was partly uncoupled from the lithotrophic nitrite supply from AOB. This was supported by activity measurements which showed an approximately threefold higher nitrite oxidizing capacity than ammonium oxidizing capacity. Based on these findings, two hypotheses were considered: either
Nitrobacter
grew mixotrophically by acetate-dependent dissimilatory nitrate reduction (
ping-pong effect
) or a nitrite oxidation/nitrate reduction loop (
nitrite loop
) occurred in which denitrifiers reduced nitrate to nitrite supplying additional nitrite for the NOB apart from the AOB.
In aerobic granular sludge (AGS), the growth of nitrite oxidizing bacteria (NOB) can be uncoupled from the nitrite supply of ammonia oxidizing bacteria (AOB). Besides, unlike for conventional ...activated sludge, Nitrobacter was found to be the dominant NOB and not Nitrospira. To explain these experimental observations, two possible pathways have been put forward in literature. The first one involves the availability of additional nitrite from partial denitrification (nitrite-loop) and the second one consists of mixotrophic growth of Nitrobacter in the presence of acetate (ping-pong). In this contribution, mathematical models were set up to assess the possibility of these pathways to explain the reported observations. Simulation results revealed that both pathways influenced the nitrifier distribution in the granules. The nitrite-loop pathway led to an elevated NOB/AOB ratio, while mixotrophic growth of Nitrobacter guaranteed their predominance among the NOB population. Besides, mixotrophic growth of Nitrobacter could lead to NO emission from AGS. An increasing temperature and/or a decreasing oxygen concentration led to an elevated NOB/AOB ratio and increased NO emissions.
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.
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•Integration of simultaneous mainstream anaerobic digestion and PN-anammox•Creation of synthetic biofilm by coating of AOA and anammox onto a membrane•Encapsulation of anaerobic digester sludge in hydrogel beads•Successfully suppressing ordinary heterotrophs to minimize membrane fouling•Simultaneously high removal of carbon and nitrogen at 25, 16 and 10 °C
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; k
) of two freshwater AOA enrichments, an AOA soil isolate (N. viennensis), and a freshwater AnAOB enrichment. The AOA enrichments had similar kinetics (μ
≈ 0.36 d
, k
≈ 0.78 µM, and k
≈ 2.9 µM), whereas N. viennensis had similar k
values but lower μ
(0.23 d
). In agreement with the current paradigm, these AOA strains showed a higher affinity for ammonia (lower k
; 0.34-1.27 µM) than published AOB measurements (>20 µM). The slower growing AnAOB (μ
≈ 0.16 d
) had much higher k
values (k
≈ 132 µM, k
≈ 48 µM) and were inhibited by oxygen at low levels (half-oxygen inhibition; k
≈ 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.
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•Dissimilatory nitrate reduction to ammonium (DNRA) by anammox bacteria.•Limitation of COD and presence of ammonium favoured anammox bacteria to pursue DNRA.•DNRA by anammox bacteria ...could improve nitrogen removal in the main line.
Anammox bacteria can perform dissimilatory nitrate reduction to ammonium (DNRA) with nitrite as intermediate coupled to the oxidation of volatile fatty acids (VFA). Batch tests with enriched anammox and a co-culture of anammox and heterotrophic bacteria showed the capacity of Candidatus ‘Brocadia fulgida’ to perform the DNRA coupled to the anammox reaction (DNRA-anammox) at a high rate although the culture was not previously adapted to VFA. From thermodynamic calculations it could be stated that low COD/N influent ratios favour the DNRA-anammox transformation over heterotrophic conversions since more free energy is gained. A process scheme is proposed for an innovative nitrogen removal system in which the nitrate produced by nitrite oxidizing bacteria and/or anammox bacteria is converted during DNRA-anammox pathway, resulting in a sustainable nitrogen removal from municipal wastewater while circumventing the troublesome out-selection of nitrite oxidizing bacteria encountered in mainstream applications.
•Application of anammox-comammox to mainstream treatment of nitrogen from real municipal wastewater.•Hydrogel encapsulated community hold promise to enhance wastewater treatment.•Complete nitrogen ...removal at a temperature range of 25-10 °C.•Infrared heating allows the reactor to maitain good performance at 5 °C.
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.
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•Long term operation of hybrid granule/floc reactor achieved high nutrient removal.•Higher PAO activity in the floc/small granules (FSG) than large granules (LG).•PAO growth in LG was dependent on ...FSG.•Granule size reduction as small granules are less diffusion limited for carbon.•High NOB activity in LG and nitrite shunt (NOB suppression) in FSG.
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.
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