Brocadia Anammoxidans Research Articles

Nitrate removal by anammox bacteria utilizing photoexcited electrons via inward extracellular electron transfer channel

Dissimilatory nitrate reduction to ammonium (DNRA) has been found to occur in some anammox bacteria species, and the DNRA metabolites (nitrite and ammonium) can further be removed to nitrogen from water. However, the activation of DNRA pathway of anammox bacteria is usually limited by the access to electron donors. Herein, we constructed a photosensitized hybrid system combining anammox bacteria (Candidatus Kuenenia stuttgartiensis and Candidatus Brocadia anammoxidans) with CdS nanoparticles semiconductor for energy-efficient NO3− removal. Such photosensitized anammox-CdS hybrid systems achieved NO3− removal with an average efficiency of 88% (the maximum of 91%) and a N2 selectivity of 72%, only with photoexcited electrons as donors. The DNRA-anammox metabolism of anammox bacteria was proved to responsible for NO3− removal via inward extracellular electron transfer channel. The greatly up-regulated genes encoding c-type cytochrome proteins (5 or 11 hemes) in the outer membrane, c-type cytochrome protein (4 hemes) and electron transport protein RnfA-E in the inner membrane, ferredoxin (2Fe-2S) in the cytoplasm and c-type cytochrome bc1 in anammoxosome membrane were supposed to play key roles in the inward extracellular electron transfer pathway. This work provides a novel insight into the design of the biotic-abiotic hybrid photosynthetic systems, and opens a new strategy for light-driven NO3− removal from the perspective of light energy input.

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

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

Adaptation of flocculent anammox culture to low temperature by cold shock: long-term response of the microbial population

ABSTRACT Partial nitritation-anammox (PN/A) process will substantially reduce the costs for the removal of nitrogen in the mainstream of municipal sewage. However, one of the mainstream PN/A challenges is to reduce the time necessary for the adaptation of anammox bacteria to lower temperatures in mild climates. In this study, we exposed anammox flocculent culture to cold shocks [35°C → 5°C (8 h) → 15°C] and evaluated long-term cold shock response. Over a post-shock period of 40 d at 15°C, the nitrogen removal rates in the shocked culture were significantly higher compared to control, with maximum rates up to 0.082 and 0.033 kg-N/kg-VSS/d or 0.164 and 0.076 kg-N/m3/d, for shocked culture and control, respectively. In the corresponding semi-batch cycles, the shocked culture was on average 136 ± 101% more active than the control, due to the negative effect of cold shock on side populations and more active anammox cells. Per FISH, Ca. Brocadia anammoxidans and Ca. Scalindua survived the shock and remained present throughout. Thus, both anammox microorganisms seem to respond favourably to cold shocks. In sum, we provide further evidence that cold shocks accelerate the adaptation of anammox to the mainstream of municipal WWTPs. Further, for the first time, we report the long-term adaptive response of anammox to cold shocks.

Bacterial communities of sponges from the wetland ecosystem of Little Rann of Kutch, India with particular reference to Planctomycetes.

Five sponge specimens belonging to the generaSpongillaandCiocalyptawere collected from Little Rann of Kutch (in Gujarat, India) and analysed for associated microbiomes. Critical analysis was done with respect to members of the phylum Planctomycetes, using two different strategies; 1. Culture-independent metagenomic approach and 2. culture-dependent anaerobic enrichment for anammox-planctomycetes. The 16S rRNA gene (V1-V3 region) amplicon metagenome analysis revealed significant divergence in bacterial diversity, including Planctomycetes among the sponges analysed. Community metagenomics revealed a total of 376 Operational Taxonomic Units (OTUs) belonging to 41 different phyla. OTUs belonging to Proteobacteria was the most abundant (38%) among the sponge analysed. The KEGG annotation predicted a total of 6909 KEGG orthologs (KOs); most of the KOs are associated with membrane transport, xenobiotic degradation, production of secondary metabolites, amino acid metabolism and carbohydrate metabolism. In the culture-dependent study, FISH analysis confirmed the association of anammox-planctomycetes with sponges. Partial 16S rRNA gene sequences of two planctomycetes (JC545, JC543) were cladding with those of unculturedPhycisphaeraeclass. The other three putative anammox bacteria (JC541, JC542, JC544) formed a different clade with "CandidatusBrocadia anammoxidans". These three putative bacteria believably represent new species/genus related to "CandidatusBrocadia".

Nanoarchitectured structure and population dynamics of anaerobic ammonium oxidizing (anammox) bacteria in a wastewater treatment plant

Studies on microbial community and population dynamics are essential for the successful development, monitoring and operation of biological wastewater treatment systems. Especially for novel or sustainable systems such as the anaerobic ammonium oxidizing (anammox) process that are not yet well explored. Here we collected granular microbial sludge samples and investigated a community of anammox bacteria over a period of four years, divided into eight stages in a full scale simultaneous partial nitrification, anammox and denitrification (SNAD) process for treating landfill leachate. Specific qPCR primers were designed to target and quantify the two most abundant anammox species, Candidatus Kuenenia stuttgartiensis (KS) and Candidatus Brocadia anammoxidans (BA). The two species were monitored and could explain the dynamic shift of the anammox community corresponding to the operating conditions. Using the newly designed KS-specific primer (KSqF3/KSqR3) and BA-specific primer (BAqF/BAqR), we estimated the amounts of KS and BA to be in the range of 6.2 × 106 to 5.9 × 108 and 1.1 × 105 to 4.1 × 107 copies μg−1 DNA, respectively. KS was found to be the dominant species in all anammox granules studied and played an important role in the formation of granules. The KS/BA ratio was positively correlated to the size of granules in the reactor and ammonia nitrogen removal efficiency of the treatment plant.

Effect of hydraulic retention time on nitrogen removal in domestic wastewater by partial nitritation and Anammox processes

The nitrogen treatment technology using the Anammox process is known to have advantages over conventional technology of nitrification - denitrification. For the purpose of evaluating the effect of hydraulic retention time to nitrogen removal in domestic wastewater by Anammox process, the authors conducted the study on partial nitritation and Anammox reactors, separately. Partial nitritation (PN) reactor used Felibendy plate material with Nitrosomonas bacteria while Anammox (AX) reactor used Felibendy cubes carrier material with strain Candidatus Brocadia anammoxidans. This study was implemented during 210 days. The nitrogen treatment efficiency of the system was evaluated with different hydraulic retention times (HRTs). The short HRT of 4.5 hours in the AX reactor affected to the total nitrogen treatment efficiency is low of 52.76 ± 1.29%. With the hydraulic retention times in PN + AX reactors of 9 and 6 hours, the effluent quality met the requirements of B-column according to QCVN 14:2008/BTNMT or QCVN 40:2011/BTNMT.
 Keywords:
 Nitrosomonas; Candidatus Brocadia anammoxidans; partial nitritation process; Anammox process; nitrogen treatment.

Adaptation and characterization of thermophilic anammox in bioreactors

Anammox, the oxidation of ammonium with nitrite, is a key microbial process in the nitrogen cycle. Under mesophilic conditions (below 40 °C), it is widely implemented to remove nitrogen from wastewaters lacking organic carbon. Despite evidence of the presence of anammox bacteria in high-temperature environments, reports on the cultivation of thermophilic anammox bacteria are limited to a short-term experiment of 2 weeks. This study showcases the adaptation of a mesophilic inoculum to thermophilic conditions, and its characterization. First, an attached growth technology was chosen to obtain the process. In an anoxic fixed-bed biofilm bioreactor (FBBR), a slow linear temperature increase from 38 to over 48 °C (0.05–0.07 °C d−1) was imposed to the community over 220 days, after which the reactor was operated at 48 °C for over 200 days. Maximum total nitrogen removal rates reached up to 0.62 g N L−1 d−1. Given this promising performance, a suspended growth system was tested. The obtained enrichment culture served as inoculum for membrane bioreactors (MBR) operated at 50 °C, reaching a maximum total nitrogen removal rate of 1.7 g N L−1 d−1 after 35 days. The biomass in the MBR had a maximum specific anammox activity of 1.1 ± 0.1 g NH4+-N g−1 VSS d−1, and the growth rate was estimated at 0.075–0.19 d−1. The thermophilic cultures displayed nitrogen stoichiometry ratios typical for mesophilic anammox: 0.93–1.42 g NO2--Nremoved g−1 NH4+-Nremoved and 0.16–0.35 g NO3--Nproduced g−1 NH4+-Nremoved. Amplicon and Sanger sequencing of the 16S rRNA genes revealed a disappearance of the original “Ca. Brocadia” and “Ca. Jettenia” taxa, yielding Planctomycetes members with only 94–95% similarity to “Ca. Brocadia anammoxidans” and “Ca. B. caroliniensis”, accounting for 45% of the bacterial FBBR community. The long-term operation of thermophilic anammox reactors and snapshot views on the nitrogen stoichiometry, kinetics and microbial community open up the development path of thermophilic partial nitritation/anammox. A first economic assessment highlighted that treatment of sludge reject water from thermophilic anaerobic digestion of sewage sludge may become attractive.

Anaerobic ammonium oxidation is a major N-sink in aquifer systems around the world.

Global-scale N-oxide contamination of groundwater within aquifers occurs due to the widespread use of N-bearing fertilizers and chemicals, threatening both human and environmental health. However, the conversion of these pollutants in active nitrogen (N) cycling processes in the subsurface biosphere still remains unclear. This study investigates the global occurrence of anaerobic ammonium oxidation (anammox) in aquifers, where anammox was found to be turned on and off between saturated and unsaturated soil horizons, and contributed 36.8-79.5% to N loss in saturated soil horizons, the remainder being due to denitrification which has traditionally been considered the main pathway for removal of N-pollutants from aquifers. Although anammox activity was undetectable in the unsaturated soil horizons, it could potentially be activated by contact with ascending groundwater. High-throughput pyrosequencing analysis identified Candidatus Brocadia anammoxidans as being the most abundant anammox bacterium in the saturated soils investigated. However, the anammox bacterial abundance was determined by the relative richness of Candidatus Jettenia asiatica. Isotopic pairing experiments revealed that coupling anammox with ammonium oxidation and respiratory ammonification enabled the formation of a revised N cycle in aquifer systems, in which respiratory ammonification acted as an important coordinator. Anammox can therefore contribute substantially to aquifer N cycling and its role in remediation of aquifers contaminated with N-oxides may be of global importance.

Anammox start-up strategies: the use of local mixed activated sludge seed versus Anammox seed

The start-up period of Anammox systems is still a big challenge due to the unavailability of large volumes of slowly growing Anammox seed locally in most countries. This study aims to evaluate the effects of seeding strategy on the start-up and enrichment period of Anammox systems by monitoring both process performance and microbial population dynamics. Two different seeding strategies, the use of mixed activated sludge culture from a local STP and the use of enriched Anammox culture transported from abroad, were comparatively studied in SBR systems operated for 410 days. The enriched Anammox seed from abroad inhibited seriously during transportation. Anammox activity re-started after 195 days' recovery period. An active Anammox culture was successfully enriched within 95 days from a local activated sludge source without seeding any Anammox. The Anammox population reached levels of 1011 copies/ng at the end of 410 days' enrichment period. Based on FISH, Ca. Brocadia anammoxidans and Ca. Scalindua species were dominant in the enriched culture. The maximum TNRR was observed as 430 mg N/day. DGGE analyses revealed a drastic change in the microbial community (56%) with Anammox enrichment. Phylogenetic analysis demonstrated a significant decrease in phylotype Proteobacteria and increase in phylotypes Planctomycetes, Chloroflexi and Acidobacteria with enrichment.

Microbial pathways for nitrogen loss in an upland soil.

The distribution and importance of anaerobic ammonium oxidation (anammox) and nitrite-dependent anaerobic methane oxidation (n-damo) have been identified in aquatic ecosystems; their role in agricultural upland soils however has not yet been well investigated. In this study, we examined spatio-temporal distributions of anammox and n-damo bacteria in soil profiles (300cm depth) from an agricultural upland. Monitoring nitrogen (N) conversion activity using isotope-tracing techniques over the course of one year showed denitrification (99.0% N-loss in the winter and 85.0% N-loss in the summer) predominated over anammox (1.0% N-loss in the winter and 14.4% N-loss in the summer) and n-damo (0.6% N-loss in the winter) in surface soils (0-20cm). While below 20cm depth, N-loss was dominated by anammox (79.4±14.3% in the winter and 65.4±12.5% in the summer) and n-damo was not detected. Phylogenetic analysis showed that Candidatus Brocadia anammoxidans dominated the anammox community in the surface soil and Candidatus Brocadia fulgida dominated below 20cm depth. Dissimilatory nitrate reduction to ammonium (DNRA), another nitrite reduction process, was found to play a limited role (4.9±3.5%) in the surface soil compared with denitrification; below 80cm DNRA rates were much higher than rates of anammox and denitrification. Ammonium oxidation was the main source of NO2- above 80cm (70.9±23.3%), the key influencing factor on anammox rates, and nitrate reduction (100%) was the main NO2- source below 80cm. Considering the anammox, n-damo and denitrification rates as a whole in the sampled soil profile, denitrification is still the main N-loss process in upland soils.

Production of nitrate spikes in a model of ammonium biodegradation

Nitrification at the site of a contaminant ammonium plume from a former coal carbonisation plant can be modelled with three competing bacterial populations of Nitrosomonas, Nitrobacter, and Brocadia anammoxidans. Oscillations of chemical species at the site can be explained by a reduced model of ammonium competition between Nitrosomonas and B. anammoxidans which effectively acts as an activator-inhibitor system. Stable oscillations occur in conditions of low nutrient (ammonium) supply and this causes a spatial travelling wave in a borehole profile when diffusion is introduced.

Simulation of simultaneous anammox and denitrification for kinetic and physiological characterization of microbial community in a granular biofilm system

A mathematical model is proposed and validated for biological nitrogen removal in a granular biofilm reactor to describe independent batch processes under anoxic conditions. The activated sludge model consists of anammox and heterotrophic bacteria using a novel stoichiometric matrix. Identifiability of sensitive biokinetic parameters of the model was assessed with regards to observed concentrations of ammonium-, nitrite-, and nitrate-nitrogen. The Chi-squared function was used for the error estimation and the R-squared index (R2) was used for the regression analysis. The results imply that the model can elucidate the interactions of nitrogen converting bacteria in a biofilm system for various feeding characteristics. The calibration results showed satisfactory R2 equal to 0.95, 0.97 and 0.67 for NH4-N, NO2-N and NO3-N respectively. For validation, model simulations were performed under three varying scenarios and R2 was more than 0.9 so that all forecasted values lied within the 95% confidence interval. In addition, the estimated physiologic characterization of two dominant anammox species was discussed upon calibration and validation of the model. The maximum specific bacterial growth rates (μmax) for Candidatus Brocadia anammoxidans and Ca. Scalindua sp. were estimated at 0.0025h−1 and 0.0048h−1 respectively. Decay rate of Ca. Brocadia anammoxidans was estimated at 0.0003h−1 which is 15% higher than decay rate of species belonging to Ca. Scalindua.

Preservation and reactivation of Candidatus Jettenia asiatica and Anammoxoglobus propionicus using different preservative agents

Anaerobic ammonium oxidation (anammox) bacteria have peculiar characteristics that make them difficult to cultivate. The conservation of these microorganisms in culture collections or laboratories requires successful preservation and reactivation techniques. Furthermore, studies have shown that successful reactivation may be preservative dependent. Considering this, the present study aimed to evaluate the preservation and reactivation of anammox consortia enriched from swine manure treatment lagoons, by using different preservative agents at different temperatures: KNO3 (at 4 °C), glycerol (−20 °C, −80 °C), and skimmed cow milk (−20 °C, −80 °C, −200 °C). After 4 months, the biomass was thawed (except for KNO3), and the reestablishment of anammox activity was evaluated by stoichiometric coefficients. Microbial community transformation during the reactivation process was also studied by 16S rDNA sequence analysis. The results showed that the anammox biomass preserved with glycerol or skimmed cow milk at −80 °C recovered activity, while the biomass preserved with other methodologies did not reestablish activity during the studied time (90 days). The bacterial community from the biomass with anammox activity was characterized and showed the presence of Candidatus Brocadia anammoxidans, Candidatus Jettenia asiatica, and Candidatus Anammoxoglobus propionicus. Preservation with skimmed cow milk at −80 °C favored the selection of Candidatus Anammoxoglobus propionicus, while preservation with glycerol at −80 °C was successful for Candidatus Jettenia asiatica. The present study was effective on anammox sludge preservation and reactivation using low-cost processes for anammox cultures preservation, which is important for biomass transport and deammonification reactor start up.

Low-strength wastewater treatment in an anammox UASB reactor: Effect of the liquid upflow velocity

Two-stage systems have been proposed to overcome the drawbacks associated to the implementation of the autotrophic biological nitrogen removal process in the mainstream of urban wastewater treatment plants. In this study, an upflow anammox sludge blanket (UAnSB) reactor was successfully operated for 325days treating a low-strength synthetic influent mimicking mainstream conditions. A nitrogen loading rate of up to 1.8±0.2gNL−1d−1 was achieved at 26°C and the nitrogen removal rate obtained (1.7±0.1gNL−1d−1) resulted considerably higher than most of the previously reported values for systems treating low-strength wastewater at similar temperatures. Fluorescence in situ hybridization analysis showed a high enrichment in the anammox specie Candidatus Brocadia anammoxidans during the whole operation. The evolution of the granule diameter was followed throughout the operation of the UAnSB reactor and a direct correlation of the average granule diameter with the liquid upflow velocity (Vup) was established, being the higher the Vup, the bigger the granules. A stable granule diameter of 790±40μm was achieved by maintaining a Vup of 1.0±0.1mh−1. The low VupS applied avoid the use of effluent recirculation which would present a huge inconvenient to implement UAnSB reactors at real scale, however these low VupS led to external mass transfer problems in the reactor. In spite of the mass transfer limitations, not only a high specific anammox activity (0.26±0.02gNg−1VSd−1) was achieved in the UASB reactor but also a high nitrogen removal (80±3%).

Resuscitation of starved suspended- and attached-growth anaerobic ammonium oxidizing bacteria with and without acetate.

Anammox application for nutrient removal from wastewater is increasing, though questions remain about anammox resilience to fluctuating conditions. Resuscitation of anammox suspended- and attached-growth cultures after 3 months of starvation was studied with and without acetate dosing. Without acetate, the attached-growth culture recovered more quickly than the suspended-growth culture. Suspended-growth cultures recovered more quickly (within 60 days) with weekly and daily acetate dosing than without, but anammox activity and copy numbers decreased with continued acetate addition. All attached-growth cultures recovered within 60 days, but after that activity with acetate dosing was consistently at least 20% lower than that without acetate addition. Ca. Jettenia caeni, Ca. Anammoxoglobus sp., Ca. Brocadia fulgida, Ca. Brocadia anammoxidans, Ca. Brocadia fulgida and Ca. Jettenia asiatica were identified. Acetate addition can significantly accelerate short-term resuscitation of enriched anammox suspended-growth cultures after starvation but may reduce anammox activity over the longer term in suspended- and attached-growth cultures.

Co-occurrence of nitrite-dependent anaerobic ammonium and methane oxidation processes in subtropical acidic forest soils

Anaerobic ammonium oxidation (anammox) and nitrite-dependent anaerobic methane oxidation (n-damo) are two new processes of recent discoveries linking the microbial nitrogen and carbon cycles. In this study, 16S ribosomal RNA (rRNA) gene of anammox bacteria and pmoA gene of n-damo bacteria were used to investigate their distribution and diversity in natural acidic and re-vegetated forest soils. The 16S rRNA gene sequences retrieved featured at least three species in two genera known anammox bacteria, namely Candidatus Brocadia anammoxidans, Candidatus Brocadia fulgida, and Candidatus Kuenenia stuttgartiensis while the pmoA gene amplified was affiliated with two species of known n-damo bacteria Candidatus Methylomirabilis oxyfera and a newly established Candidatus Methylomirabilis sp. According to the results, the diversity of anammox bacteria in natural forests was lower than in re-vegetated forests, but no significant difference was observed in n-damo community between them. Quantitative real-time PCR showed that both anammox and n-damo bacteria were more abundant in the lower layer (10-20cm) than the surface layer (0-5cm). The abundance of anammox bacteria varied from 2.21×10(5) to 3.90×10(6) gene copies per gram dry soil, and n-damo bacteria quantities were between 1.69×10(5) and 5.07×10(6) gene copies per gram dry soil in the two different layers. Both anammox and n-damo bacteria are reported for the first time to co-occur in acidic forest soil in this study, providing a more comprehensive information on more defined microbial processes contributing to C and N cycles in the ecosystems.

Start-up performance of Anammox process in a fixed bed reactor (FBR) filled with honeycomb-like polypropylene carriers

Novel honeycomb-like carriers, made of polypropylene, were applied to enhance biomass retention capacity so as to improve Anammox start-up performance in a fixed bed reactor (FBR). The reactor was operated for 3 months. On day 45, Anammox activity appeared. After 61 days' operation, the removal efficiencies of ammonium and nitrite were both over 91% based on 70 mg N L(-1) of the influent ammonium and influent nitrite, indicating that a remarkable Anammox activity was attained. A final specific Anammox activity of 0.12 g NH4(+)-N gVSS(-1) d(-1) was reached (VSS: volatile suspended solids). The FBR showed a good capacity for resisting shock loading and was more able to resist shock loading of nitrogen concentration than resist hydraulic shock loading. Phylogenetic analysis showed that Candidatus Brocadia anammoxidans' and Candidatus Kuenenia stuttgartiensis' were detected in the mature biofilm, and Candidatus Brocadia anammoxidans' was the dominant Anammox strain. Candidatus Kuenenia stuttgartiensis' played a positive role in the reactor performance, as it could consumed nitrite quickly and efficiently so as to avoid an adverse effect of temporary nitrite accumulation. The results showed that the honeycomb-like carriers were suitable for start-up of Anammox.

Anaerobic ammonium oxidation in the old trickling filters at Daspoort Wastewater Treatment Works

The century-old trickling filters at the Daspoort Wastewater Treatment Works in Pretoria (Gauteng, South Africa) are known for their remarkable removal of nitrogen from municipal wastewater. Our study was conducted to identify the microbiological processes responsible for this phenomenon and to establish whether anammox bacteria may be involved. An aerobic and anaerobic bench top reactor, both inoculated with biofilm-covered stones from one of the filters, were spiked with ammonia-nitrogen (NH4+-N) and nitrite-nitrogen (NO2.-N). These reactors were subsequently monitored by conducting stoichiometric analyses of chemical oxygen demand (COD), NH4+-N, NO2.-N, and nitrate-nitrogen (NO3.-N). In the aerobic reactor, the COD concentration decreased over the 56 h batch reaction period and nitrification was revealed by a decrease in NH4+-N and NO2.-N concentrations. However, the NO3.-N concentration showed no notable decrease. In contrast, in the anaerobic reactor the concentrations of COD, NH4+-N, NO2¡¥-N, NO3¡¥-N, as well as total nitrogen decreased during the batch reaction period. The decrease of both the NH4+-N and NO2.-N concentrations, the latter to zero under anaerobic conditions, suggested that, in addition to heterotrophic denitrification, anaerobic ammonium oxidation (anammox) may also occur in the trickling filter biofilm. This was highlighted by the observation that ammonium removal in the anaerobic reactor stopped as soon as the nitrite concentration became zero. The ratio of nitrite:ammonium removal was 1.33 on average, which conforms to anammox behaviour. Gene sequence analysis was used to test for the possible presence of anammox bacteria in the trickling filter biofilm. Genomic DNA was extracted from trickling filter humus sludge and the polymerase chain reaction (PCR) was used to amplify taxonomically informative 16S rRNA gene sequences, using primers specific for selected anammox species. Subsequent sequence analysis, including using the online Basic Local Alignment Search Tool (BLAST), and constructing a phylogenic tree using a heuristic search strategy for Maximum Parsimony analysis, confirmed the presence of an anammox bacterium closely related to Candidatus ¡®Brocadia anammoxidans¡¯ and Candidatus ¡®Brocadia fulgida¡¯ on the biofilm-covered stones of the Daspoort trickling filters.Keywords: Daspoort, trickling filter, anammox, nitrification, denitrification, stoichiometric analyses, polymerase chain reaction

Aerobic and anaerobic ammonium-oxidising bacterial enrichment from municipal solid waste

Leachate from the municipal solid waste (MSW) landfills contains high concentration of ammoniacal nitrogen that is a major toxic pollutant that has a great threat to environment. Among the processes available for the removal of ammoniacal nitrogen, one process is a combination of partial nitrification and anaerobic ammonium oxidation (anammox) process. It requires aerobic ammonium-oxidising bacteria (AOB) and anaerobic ammonium-oxidising bacteria (AnAOB). This paper presents the feasibility of enriching the AOB and AnAOB in 100-mL and 2.5-L batch reactors from fresh and mined MSW and leachate under aerobic and anaerobic conditions with varying feed-to-seed ratio. The AOB and AnAOB activity was monitored by measuring the intermediates such as hydroxylamine and hydrazine along with variations in ammoniacal nitrogen, nitrite nitrogen, and nitrate nitrogen concentrations in the reactor contents. The formation of intermediates such as hydroxylamine and hydrazine and ammoniacal nitrogen transformation data confirmed the enrichment of AOB and AnAOB. Further, AOB and AnAOB were validated by most probable number test and scanning electron microscopy analysis, respectively. DNA extraction, polymerase chain reaction amplification, and sequencing analysis authenticated the AnAOB as Candidatus Brocadia anammoxidans.

Sequentially aerated membrane biofilm reactors for autotrophic nitrogen removal: microbial community composition and dynamics

Membrane-aerated biofilm reactors performing autotrophic nitrogen removal can be successfully applied to treat concentrated nitrogen streams. However, their process performance is seriously hampered by the growth of nitrite oxidizing bacteria (NOB). In this work we document how sequential aeration can bring the rapid and long-term suppression of NOB and the onset of the activity of anaerobic ammonium oxidizing bacteria (AnAOB). Real-time quantitative polymerase chain reaction analyses confirmed that such shift in performance was mirrored by a change in population densities, with a very drastic reduction of the NOB Nitrospira and Nitrobacter and a 10-fold increase in AnAOB numbers. The study of biofilm sections with relevant 16S rRNA fluorescent probes revealed strongly stratified biofilm structures fostering aerobic ammonium oxidizing bacteria (AOB) in biofilm areas close to the membrane surface (rich in oxygen) and AnAOB in regions neighbouring the liquid phase. Both communities were separated by a transition region potentially populated by denitrifying heterotrophic bacteria. AOB and AnAOB bacterial groups were more abundant and diverse than NOB, and dominated by the r-strategists Nitrosomonas europaea and Ca. Brocadia anammoxidans, respectively. Taken together, the present work presents tools to better engineer, monitor and control the microbial communities that support robust, sustainable and efficient nitrogen removal.