Optimizing start-up strategies for the two-inflow nitritation/anammox process: Influence on biofilm microbial community composition.

Low-energy nitrogen removal from ammonium-rich wastewater is crucial in preserving the water environment. A one-stage nitritation/anammox process with two inflows treating ammonium-containing wastewater, supplied from inside and outside the wound filter, is expected to stably remove nitrogen. Laboratory-scale reactors were operated using different start-up strategies; the first involved adding nitritation inoculum after anammox biomass formation in the filter, which presented a relatively low nitrogen removal rate (0.171 kg N/m3 · d), at a nitrogen loading rate of 1.0 kg N/m3 · d. Conversely, the second involved the gradual cultivation of anammox and nitritation microorganisms, which increased the nitrogen removal rate (0.276 kg N/m3 · d). Furthermore, anammox (Candidatus Brocadia) and nitritation bacteria (Nitrosomonadaceae) coexisted in the biofilm formed on the filter surface. The abundance of nitritation bacteria (10.5%) in the reactor biofilm using the second start-up strategy was higher than that using the first (3.7%). Thus, the two-inflow nitritation/anammox process effectively induced habitat segregation using a suitable start-up strategy.

Anaerobic ammonium oxidation coupled with sulfate reduction links nitrogen with sulfur cycle.

Sulfate-dependent ammonium oxidation (Sulfammox) is a critical process linking nitrogen and sulfur cycles. However, the metabolic pathway of microbes driven Sulfammox is still in suspense. The study demonstrated that ammonium was not consumed with sulfate as the sole electron acceptor during long-term enrichment, probably due to inhibition from sulfide accumulation, while ammonium was removed at âˆ¼ 10 mg N/L/d with sulfate and nitrate as electron acceptors. Ammonium and sulfate were converted into nitrogen gas, sulfide, and elemental sulfur. Sulfammox was mainly performed by Candidatus Brocadia sapporoensis and Candidatus Brocadia fulgida, both of which encoded ammonium oxidation pathway and dissimilatory sulfate reduction pathway. Not sulfide-driven autotrophic denitrifiers but Candidatus Kuenenia stuttgartiensis converted nitrate to nitrite with sulfide. The results of this study reveal the specialized metabolism of Sulfammox bacteria (Candidatus Brocadia sapporoensis and Candidatus Brocadia fulgida) and provide insight into microbial relationships during the nitrogen and sulfur cycles.

Nitrogen removal strategy for real swine wastewater by combining partial nitrification-denitrification process with anammox.

Anammox process offers reduced operational cost and energy requirement compared to nitrification-denitrification methods due to lower biomass generation and no need for external carbon sources and aeration. High ammonia concetration and low biodegradable anaerobic digester of swaine wastewater provided an advantage for the growth of anammox microorangism. An anoxic/oxic (A/O) SBR and an anammox SBR were implemented parallelly to treat the same swine wastewater with partial nitrification/denitrification and partial nitrification/anammox process, respectively, and to compare their nitrogen removal efficiency. The nitrogen removal rates (NRRs) of the A/O SBR and anammox SBR were 0.054 and 0.26 kg-N/m3/day, respectively. The lower NRR of the A/O SBR could be attributed to insufficient biodegradable organic carbon sources in the denitrification process. The kinetic parameters obtained from the two SBRs were applied to estimate the time required for using the A/O process and partial nitrification/anammox process to treat the same amount of ammonia with the same reaction volume. Results showed that the A/O process required 3.3 times the reaction time of the partial nitrification/anammox process, suggesting that the partial nitrification/anammox process is a more efficient and economic nitrogen removal process for swine wastewater treatment. The next generation sequencing results revealed that Candidatus Brocadia, ranging from 10 to 23%, was the predominant anammox bacteria in the anammox SBR. More than 78.2 % of nitrite in the anammox SBR was removed through the anammox reaction.

Enhanced nitrogen removal from low COD/TIN mainstream wastewater in a continuous plug-flow reactor via partial nitrification, simultaneous anammox and endogenous denitrification (PN-SAED) process.

A continuous plug-flow reactor with anaerobic/front-aerobic/anoxic/post-aerobic zones, where partial nitrification occurred in the front-aerobic zone, followed by simultaneous anammox and endogenous denitrification in the anoxic zone (PN-SAED), was built up to treat municipal wastewater. Alternating anoxic/aerobic conditions and longer anoxic duration facilitated stable partial nitrification. The nitrite accumulation ratio (NAR) was maintained at 97.4 ± 1.2%, with temperatures between 13.3℃ to 19.8℃. Candidatus Brocadia were naturally enriched in-situ from the anoxic zone with relative abundances of 31.93% and 6.67% on the agitator blade and carriers, respectively. High removal efficiencies of total inorganic nitrogen (TIN) (95.1 ± 1.9%) and effluent TIN (2.6 ± 1.1 mg N/L) were acquired from low COD/TIN (3.4 ± 0.4) municipal wastewater with anammox contribution of 13.5%±5.8% to TIN removal. The PN-SAED process is a promising mainstream nitrogen removal method.

Effect of temperature on the compositions of ladderane lipids in globally surveyed anammox populations.

The adaptation of bacteria involved in anaerobic ammonium oxidation (anammox) to low temperatures will enable more efficient removal of nitrogen from sewage across seasons. At lower temperatures, bacteria typically tune the synthesis of their membrane lipids to promote membrane fluidity. However, such adaptation of anammox bacteria lipids, including unique ladderane phospholipids and especially shorter ladderanes with absent phosphatidyl headgroup, is yet to be described in detail. We investigated the membrane lipids composition (UPLC-HRMS/MS) and dominant anammox populations (16S rRNA gene amplicon sequencing, Fluorescence in situ hybridization) in 14 anammox enrichments cultivated at 10-37 °C. "Candidatus Brocadia" appeared to be the dominant organism in all but two laboratory enrichments of "Ca. Scalindua" and "Ca. Kuenenia". At lower temperatures, the membranes of all anammox populations were composed of shorter [5]-ladderane ester (reduced chain length demonstrated by decreased fraction of C20/(C18 + C20)). This confirmed the previous preliminary evidence on the prominent role of this ladderane fatty acid in low-temperature adaptation. "Ca. Scalindua" and "Ca. Kuenenia" had distinct profile of ladderane lipids compared to "Ca. Brocadia" biomasses with potential implications for adaptability to low temperatures. "Ca. Brocadia" membranes contained a much lower amount of C18 [5]-ladderane esters than reported in the literature for "Ca. Scalindua" at similar temperature and measured here, suggesting that this could be one of the reasons for the dominance of "Ca. Scalindua" in cold marine environments. Furthermore, we propose additional and yet unreported mechanisms for low-temperature adaptation of anammox bacteria, one of which involves ladderanes with absent phosphatidyl headgroup. In sum, we deepen the understanding of cold anammox physiology by providing for the first time a consistent comparison of anammox-based communities across multiple environments.

New Insight Into the Interspecies Shift of Anammox Bacteria Ca. "Brocadia" and Ca. "Jettenia" in Reactors Fed With Formate and Folate.

The sensitivity of anaerobic ammonium-oxidizing (anammox) bacteria to environmental fluctuations is a frequent cause of reactor malfunctions. It was hypothesized that the addition of formate and folate would have a stimulating effect on anammox bacteria, which in turn would lead to the stability of the anammox process under conditions of a sharp increase in ammonium load, i.e., it helps overcome a stress factor. The effect of formate and folate was investigated using a setup consisting of three parallel sequencing batch reactors equipped with a carrier. Two runs of the reactors were performed. The composition of the microbial community was studied by the 16S rRNA gene profiling and metagenomic analysis. Among anammox bacteria, Ca. "Brocadia" spp. dominated during the first run. A stimulatory effect of folate on the daily nitrogen removal rate (dN) was identified. The addition of formate led to progress in dissimilatory nitrate reduction and stimulated the growth of Ca. "Jettenia" spp. The spatial separation of two anammox species was observed in the formate reactor: Ca. "Brocadia" occupied the carrier and Ca. "Jettenia"-the walls of the reactors. Biomass storage at low temperature without feeding led to an interspecies shift in anammox bacteria in favor of Ca. "Jettenia." During the second run, a domination of Ca. "Jettenia" spp. was recorded along with a stimulating effect of formate, and there was no effect of folate on dN. A comparative genome analysis revealed the patterns suggesting different strategies used by Ca. "Brocadia" and Ca. "Jettenia" spp. to cope with environmental changes.

Application of xylitol on nitrogen removal from saline wastewater through "Candidatus Brocadia sinica"-dominated anammox process under low temperature.

Xylitol was first applied to enhance nitrogen removal from saline wastewater through "Candidatus Brocadia sinica"-dominated anammox process under low temperature. The reactor was maintained at 15°C, and the salinity of wastewater was 35 g/L. Ammonium removal rate (ARR) and nitrite removal rate (NRR) were stable at around 0.27 kg/(m3  d) without xylitol addition. As an osmotic pressure regulator and cryoprotective agent, optimal ARR and NRR were 0.51 kg/(m3  d) and 0.63 kg/(m3  d) at 0.3 mM xylitol. At the addition of 1 mM high-dosage xylitol, there existed dissimilatory reduction in nitrate to ammonium nitrogen and heterotrophic denitrification in the reactor. Remodified logistic model was suitable to simulate NH 4 + - N removal process with xylitol addition. As a result, xylitol dose should be controlled within 0.3 mM, which greatly promoted the nitrogen removal from saline wastewater under low temperature. PRACTITIONER POINTS: Xylitol could be used as osmotic pressure regulator and cryoprotective agent to enhance nitrogen removal. The optimal dose was achieved at 0.3 mM xylitol for "Candidatus Brocadia sinica" in low-temperature saline wastewater. High-dosage xylitol could interfere with nitrogen removal efficiency due to the presence of DNAR and HB. Remodified logistic model was suitable for the analysis and prediction of nitrogen removal process with xylitol addition.

Start-up of oxygen-limited autotrophic partial nitrification-anammox process for treatment of nitrite-free wastewater in a single-stage hybrid bioreactor.

This study presents effective ammonium removal from nitrite-free ammonium-rich synthetic wastewater through combined partial nitrification (PN) and anammox processes in a multi-zone hybrid airlift bioreactor (BioCAST). Removal efficiencies of ammonia-nitrogen and total nitrogen up to 85.6% and 81.2%, respectively, were achieved shortly after the start-up of bioreactor treating the nitrite-free ammonium-rich synthetic wastewater with ammonium concentrations of 10-350 mg/L. The hybrid (containing suspended and attached biomass) and multi-zone design of the bioreactor with different dissolved oxygen levels, along with the inoculation with anammox-containing sludge were the main factors in the successful start-up of the bioreactor. Nitrate accumulation problem due to the fast growth of nitrite-oxidizing bacteria in the bioreactor was controlled by two operating strategies including lowering the HRT from 4 days to 2 days and controlling the dissolved oxygen concentration in the aerobic zone of the bioreactor between 0.9 and 1.2 mg/L. Moreover, the 16S rRNA gene analysis confirmed that the partial nitrification of ammonia to nitrite occurred by Nitrosomonas sp. primarily in the suspended biomass in the aerobic zone, while the conversion of nitrite to N2 occurred by Candidatus Brocadia species in the anoxic zone. This study showed the effective removal of ammonium from a nitrite-free wastewater by providing a proper HRT, controlling the DO concentration between 0.9 and 1.2 mg/L in the aerobic zone, and preventing biomass loss using both suspended and attached microbial cultures in different zones of the bioreactor.

Nitrogen management in landfill leachate using single-stage anammox process-illustrating key nitrogen pathways under an ecogenomics framework.

Simultaneous functional gene expressions using mRNA, rate measurements, and biochemical analysis proved the consistent contribution of ammonia oxidizers, heterotrophic denitrifiers, and anammox bacteria in a single-stage attached growth partial nitritation/anammox system for nitrogen management in landfill leachate. Average removal efficiencies of ammonia-nitrogen, total inorganic nitrogen, and COD were 94%, 88%, and 26%, respectively, in the reactor. Off-gas N2O fluxes increased at relatively higher dissolved oxygen. Batch activity tests revealed the occurrence of significant anammox activity even in the presence of high concentrations of organic carbon in the influent. mRNA based functional expressions of nitrite reductase (nirK and nirS) and hydrazine synthase (hzsA) suggested simultaneous active heterotrophic denitrification and anammox, respectively. 16S rRNA amplicon sequencing revealed Proteobacteria (36-56%), Planctomycetes (10-31%), and Bacteroidetes (6-39%) as dominant phyla in the reactor. Candidatus brocadia was observed as the most abundant genus representing anammox community.

Comparative Genome-Centric Analysis of Freshwater and Marine ANAMMOX Cultures Suggests Functional Redundancy in Nitrogen Removal Processes.

There is a lack of understanding of the interaction between anammox bacteria and the flanking microbial communities in both freshwater (non-saline) and marine (saline) ecosystems. Here, we present a comparative genome-based exploration of two different anammox bioreactors, through the analysis of 23 metagenome-assembled genomes (MAGs), 12 from freshwater anammox reactor (FWR), and 11 from marine anammox reactor (MWR). To understand the contribution of individual members to community functions, we applied the index of replication (iRep) to determine bacteria that are actively replicating. Using genomic content and iRep information, we provided a potential ecological role for the dominant members of the community based on the reactor operating conditions. In the non-saline system, anammox (Candidatus Brocadia sinica) and auxotrophic neighboring bacteria belonging to the phyla Ignavibacteriae and Chloroflexi might interact to reduce nitrate to nitrite for direct use by anammox bacteria. Whereas, in the saline reactor, anammox bacterium (Ca. Scalindua erythraensis) and flanking community belonging to phyla Planctomycetes (different than anammox bacteria)-which persistently growing in the system-may catabolize detritus and extracellular material and recycle nitrate to nitrite for direct use by anammox bacteria. Despite different microbial communities, there was functional redundancy in both ecosystems. These results signify the potential application of marine anammox bacteria for treating saline N-rich wastewaters.

Metagenomic data of the microbial community of lab-scale nitritation-anammox sequencing-batch bioreactor performing nitrogen removal from synthetic wastewater.

The nitritation-anammox process, which involves partial aerobic oxidation of the ammonium to nitrite and following oxidation of ammonium by nitrite to molecular nitrogen, is an efficient and cost-effective approach for biological nitrogen removal from wastewater. To characterize the microbial communities involved in the nitrogen and carbon cycles in wastewater treatment bioreactors employing this process, we sequenced the metagenome of a sludge sample collected from the lab-scale nitritation-anammox sequencing-batch reactor. At the phylum level, Proteobacteria and Chloroflexi were the most numerous groups. Anammox bacteria belonged to the genus Candidatus Brocadia. The obtained data will help to investigate the taxonomical and functional diversity the microbial communities involved in nitritation-anammox process, and will be used for genome-based analysis of uncultured bacterial lineages. The raw sequencing data is available from the NCBI Sequence Read Archive (SRR9831403) database under the BioProject PRJN0A55627.

Autofluorescent characteristics of Candidatus Brocadia fulgida and the consequences for FISH and microscopic detection.

An enrichment culture of Candidatus Brocadia fulgida was identified by three independent methods: analysis of autofluorescence using different microscope filter blocks and a fluorescence spectrometer, fluorescence in situ hybridization (FISH) with anammox-specific probes and partial sequencing of the 16S rDNA, hydrazine synthase hzsA and hydrazine oxidoreductase hzo. The filter block BV-2A (400-440, 470 LP, Nikon) was suitable for preliminary detection of Ca. B. fulgida. An excitation-emission matrix revealed three pairs of excitation-emission maxima: 288-330 nm, 288-478 nm and 417-478 nm. Several autofluorescent cell clusters could not be stained with DAPI or by FISH, suggesting empty but intact cells (ghost cells) or inhibited permeability. Successful staining of autofluorescent cells with the FISH probes Ban162 and Bfu613, even at higher formamide concentrations, suggested insufficient specificity of Ban162. Under certain conditions, Ca. B. fulgida lost its autofluorescence, which reduced the reliability of autofluorescence for identification and detection. Non-fluorescent Ca. Brocadia cells could not be stained with Ban162, but with Bfu613 at higher formamide concentrations, suggesting a dependency between both parameters. The phylogenetic analysis showed only good taxonomical clustering of the 16S rDNA and hzsA. In conclusion, careful consideration of autofluorescent characteristics is recommended when analysing and presenting FISH observations of Ca. B. fulgida to avoid misinterpretations and misidentifications.

Microbial community response to influent shift and lowering temperature in a two-stage mainstream deammonification process.

The effects of influent shift from synthetic wastewater to anaerobically pretreated actual sewage coupling with lowering temperature on microbial community of a two-stage partial nitritation (PN)-anammox process were evaluated through high-throughput sequencing. Venn diagrams and Hill numbers showed the significantly increased bacterial diversity both in the PN and anammox reactor. However, taxonomic analysis indicated that outstanding enrichment of heterotrophic bacteria and reduction of autotrophic species mainly occurred in the PN reactor, while nearly all of the dominant bacteria in the anammox reactor only slightly decreased in abundance. Moreover, immigrant bacteria from the PN reactor to the following anammox reactor had no negative effect on the anammox function. These results implied the positive role of the first-stage PN in maintaining the stability of the following anammox community. Nitrosomonas europaea (17.9-52.9%) and one cluster (19.2-27.7%) within Candidatus Brocadia remained as the dominant functional species in the PN and anammox reactor, respectively.

Nitrogen removal through "Candidatus Brocadia sinica" treating high-salinity and low-temperature wastewater with glycine addition: Enhanced performance and kinetics.

Freshwater-derived anaerobic ammonia oxidation (anammox) bacteria ("Candidatus Brocadia sinica") were investigated to remove nitrogen from high-salinity and low-temperature wastewater with glycine addition. The reactor was operated at 15 ±â€¯0.5 °C with influent pH of 7.5 ±â€¯0.1. When glycine were 0.2, 0.4, and 0.6 mM, respectively, nitrite removal rate (NRR) increased by 27.7%, 47.3%, and 70.4% accordingly. Optimal ammonia removal rate (0.32 kg/(m3·d)) and NRR (0.45 kg/(m3·d)) were achieved at 0.8 mM glycine. Effect resulting from glycine on nitrite reductase was higher than hydrazine synthase. Moreover, ΔNO2--N/ΔNH4+-N increased with glycine addition while ΔNO3--N/ΔNH4+-N first increased and then decreased. The remodified Logistic model and modified Boltzmann model were appropriate to describe nitrogen removal with glycine addition. Kinetic parameter λ achieved through the remodified Logistic model revealed that "Candidatus Brocadia sinica" had a shorter lag phase than that of marine anammox bacteria.

Anaerobic Ammonium Oxidation in Acidic Red Soils.

Anaerobic ammonium oxidation (anammox) has been proven to be an important nitrogen removal process in terrestrial ecosystems, particularly paddy soils. However, the contribution of anammox in acidic red soils to nitrogen loss has not been well-documented to date. Here, we investigated the activity, abundance, and distribution of anammox bacteria in red soils collected from nine provinces of Southern China. High-throughput sequencing analysis showed that Candidatus Brocadia dominates the anammox bacterial community (93.03% of sequence reads). Quantification of the hydrazine synthase gene (hzsB) and anammox 16S rRNA gene indicated that the abundance of anammox bacteria ranged from 6.20 × 106 to 1.81 × 109 and 4.81 × 106 to 4.54 × 108 copies per gram of dry weight, respectively. Contributions to nitrogen removal by anammox were measured by a 15N isotope-pairing assay. Anammox rates in red soil ranged from 0.01 to 0.59 nmol N g-1 h-1, contributing 16.67-53.27% to N2 production in the studied area, and the total amount of removed nitrogen by anammox was estimated at 2.33 Tg N per year in the natural red soils of southern China. Pearson correlation analyses revealed that the distribution of anammox bacteria significantly correlated with the concentration of nitrate and pH, whereas the abundance and activity of anammox bacteria were significantly influenced by the nitrate and total nitrogen concentrations. Our findings demonstrate that Candidatus Brocadia dominates anammox bacterial communities in acidic red soils and plays an important role in nitrogen loss of the red soil in Southern China.

Discovery and metagenomic analysis of an anammox bacterial enrichment related to Candidatus "Brocadia caroliniensis" in a full-scale glycerol-fed nitritation-denitritation separate centrate treatment process.

A distinctive red biofilm was observed in a glycerol-fed digester liquid effluent treatment process coupling partial nitrification (nitritation) and partial denitrification (denitritation) processes. Based on initial phylogenetic screening using 16S rRNA clone libraries and quantitative polymerase chain reaction, the biofilm was enriched in novel anaerobic ammonium oxidizing bacteria (AMX/anammox) closely related to Candidatus "Brocadia caroliniensis". The metabolic functionality of the C. "Brocadia caroliniensis" enrichment was further explored using high-throughput sequencing and de novo metagenome assembly. The population anammox genome that was binned from the metagenome consisted of 209 contigs with a total of 3.73 Mbp consensus sequences having 43.3% GC content, and 27.4 average coverage depth. The assembled metagenome bin was comprised of 3582 open reading frames (ORFs). Based on 16S rRNA similarity the binned metagenome was closely related with Candidatus "Brocadia caroliniensis", Candidatus "Brocadia fulgida", planctomycete KSU-1, and Candidatus "Kuenenia stuttgartiensis" with 99%, 96%, 92% and 93% similarity, respectively. Essential genes in anammox metabolic functions including ammonium and nitrite transport, hydrazine synthesis, electron transfer for catabolism, and inorganic carbon fixation, among several other anabolic pathways, were also observed in the population genome of the C. "Brocadia caroliniensis" related enrichment. Our results demonstrate the wider profusion of anammox bacteria in engineered nitrogen removal systems than expected. The utility of metagenomics approaches to deciphering such novel functionality in these systems is also highlighted.

Effects of porous carrier size on biofilm development, microbial distribution and nitrogen removal in microaerobic bioreactors.

In this study, effects of porous carrier's size (polyurethane-based) on microbial characteristics were systematically investigated in addition to nitrogen removal performance in six microaerobic bioreactors. Among different sized carriers (50, 30, 20, 15,10, 5mm), 15mm carrier showed highest nitrogen removal (98%) due to optimal micro-environments created for aerobic nitrifiers in outer layer (0-7mm), nitrifiers and denitrifiers in middle layer (7-10mm) and anaerobic denitrifiers in inner layer (10-15mm). Candidatus brocadia, a dominant anammox bacteria, was solely concentrated close to centroid (0-70µm) and strongly co-aggregated with other bacterial communities in the middle layer of the carrier. Contrarily, carriers with a smaller (<15mm) or larger size (>15mm) either destroy the effective zone for anaerobic denitrifiers or damage the microaerobic environments due to poor mass transfer. This study is of particular use for optimal design of carriers in enhancing simultaneous nitrification-denitrification in microaerobic wastewater treatment processes.

Metagenomic insights into the effects of volatile fatty acids on microbial community structures and functional genes in organotrophic anammox process.

To explore the metabolic versatility of "Candidatus Brocadia sinica" in the presence of VFAs, the impacts of VFAs on anammox activity and nitrogen removal were investigated in this study. Results found that low VFAs concentrations has no affect on anammox activity and the removal efficiencies of NH4(+)-N and NO2(-)-N. However, "Ca. Brocadia sinica" showed higher adaptability to some VFAs stresses. Furthermore, Illumina MiSeq pyrosequencing results indicated that the microbial community structures varied significantly and the phyla Chloroflexi, Proteobacteria, Bacteroidetes and Chlorobi were dominated. Finally, qPCR was performed to validate the growth of anammox bacteria and gain the quantitative insights into the correlation between nitrogen transformation rates and the key functional genes in the organotrophic anammox system. Combined analysis clearly demonstrated that the coupling of the anammox, denitrification and DNRA were the noteworthy pathway for the simultaneous removal of nitrogen and organic carbon.

Activity and growth of anammox biomass on aerobically pre-treated municipal wastewater.

Direct treatment of municipal wastewater (MWW) based on anaerobic ammonium oxidizing (anammox) bacteria holds promise to turn the energy balance of wastewater treatment neutral or even positive. Currently, anammox processes are successfully implemented at full scale for the treatment of high-strength wastewaters, whereas the possibility of their mainstream application still needs to be confirmed. In this study, the growth of anammox organisms on aerobically pre-treated municipal wastewater (MWW(pre-treated)), amended with nitrite, was proven in three parallel reactors. The reactors were operated at total N concentrations in the range 5-20 mg(N)∙L(-1), as expected for MWW. Anammox activities up to 465 mg(N)∙L(-1)∙d(-1) were reached at 29 °C, with minimum doubling times of 18 d. Lowering the temperature to 12.5 °C resulted in a marked decrease in activity to 46 mg(N)∙L(-1)∙d(-1) (79 days doubling time), still in a reasonable range for autotrophic nitrogen removal from MWW. During the experiment, the biomass evolved from a suspended growth inoculum to a hybrid system with suspended flocs and wall-attached biofilm. At the same time, MWW(pre-treated) had a direct impact on process performance. Changing the influent from synthetic medium to MWW(pre-treated) resulted in a two-month delay in net anammox growth and a two to three-fold increase in the estimated doubling times of the anammox organisms. Interestingly, anammox remained the primary nitrogen consumption route, and high-throughput 16S rRNA gene-targeted amplicon sequencing analyses revealed that the shift in performance was not associated with a shift in dominant anammox bacteria ("Candidatus Brocadia fulgida"). Furthermore, only limited heterotrophic denitrification was observed in the presence of easily biodegradable organics (acetate, glucose). The observed delays in net anammox growth were thus ascribed to the acclimatization of the initial anammox population or/and the development of a side population beneficial for them. Additionally, by combining microautoradiography and fluorescence in situ hybridization it was confirmed that the anammox organisms involved in the process did not directly incorporate or store the amended acetate and glucose. In conclusion, these investigations strongly support the feasibility of MWW treatment via anammox.

Enriquecimento de bactérias anaeróbias oxidadoras de amônia - anammox / Enrichment of anaerobic ammonium oxidizing bacteria - anammox

Bactérias anaeróbias oxidadoras de amônia (bactérias Anammox, do inglês anaerobic ammonium oxidizing bacteria) foram enriquecidas em reator em batelada sequencial (RBS), a partir de lodo proveniente de um sistema convencional de lodos ativados tratando esgoto doméstico de Belo Horizonte (MG). Após três meses de cultivo, atividade Anammox foi detectada no sistema pelo consumo de quantidades estequiométricas de NO2- e NH4+. Análises de hibridação in situ fluorescente (FISH, do inglês fluorescent in situ hybridization) confirmaram a presença de bactérias Anammox, provavelmente Candidatus Brocadia anammoxidans, e revelaram que estas representavam 53 por cento do total de células (após 6 meses de cultivo). O desempenho do reator ao longo dos sete meses de operação demonstrou remoção quase que total de nitrito, baseada em concentração afluente de 61 a 95 mg N-NO2-/L. A eficiência máxima de remoção de amônia alcançada foi de 95 por cento, a partir de concentração afluente de 55 a 82 mg N-NH4+/L.

Anaerobic ammonium-oxidizing (Anammox) bacteria were enriched from sludge collected at a conventional activated sludge system treating domestic wastewater of Belo Horizonte(MG), Brazil, employing a sequencing batch reactor (SBR). After three months of cultivation, Anammox activity was detected in the system by the consumption of stoichiometric amounts of NO2- and NH4+. Fluorescent in situ hybridization (FISH) results revealed the presence of Anammox bacteria (probably Candidatus Brocadia anammoxidans) and showed that they accounted for 53 percent of the total bacterial population (after 6 months of cultivation). The reactor performance during the seven months of operation showed a near perfect removal of nitrite, based on the influent NO2--N concentration of 61-95 mg/L. The maximum ammonia removal efficiency was 95 percent from the influent N-NH4+ concentration of 55-82 mg/L.