Anammox Organisms Research Articles

The anammox coupled partial-denitrification process in an integrated granular sludge and fixed-biofilm reactor developed for mainstream wastewater treatment: Performance and community structure

This study describes an integrated granular sludge and fixed-biofilm (iGB) reactor innovatively designed to carry out the anammox/partial-denitrification (A/PD) process for nitrogen removal with mainstream municipal wastewater. The iGB-A/PD reactor consists of anammox granules inoculated in the lower region of reactor and an acclimated fixed-biofilm positioned in the upper region. Compared to the other reported A/PD systems for mainstream wastewater treatment, this iGB-A/PD reactor is notable due to its higher quality effluent with a total inorganic nitrogen (TIN) of ∼3 mg•L−1 and operation at a high nitrogen removal rate (NRR) of 0.8 ± 0.1 kg-N•m−3•d−1. Reads-based metatranscriptomic analysis found that the expression values of hzsA and hdh, key genes associated with anammox, were much higher than other functional genes on nitrogen conversion, confirming the major roles of the anammox bacteria in nitrogen bio-removal. In both regions of the reactor, the nitrate reduction genes (napA/narG) had expression values of 56–99 RPM, which were similar to that of the nitrite reduction genes (nirS/nirK). The expression reads from genes for dissimilatory nitrate reduction to ammonium (DNRA), nrfA and nirB, were unexpectedly high, and were over the half of the levels of reads from genes required for nitrate reduction. Kinetic assays confirmed that the granules had an anammox activity of 16.2 g-NH4+-N•kg−1-VSS•d−1 and a nitrate reduction activity of 4.1 g-N•kg−1-VSS•d−1. While these values were changed to be 4.9 g- NH4+-N•kg−1-VSS•d−1and 4.3 g-N•kg−1-VSS•d−1 respectively in the fixed-biofilm. Mass flux determination found that PD and DNRA was responsible for ∼50% and ∼25% of nitrate reduction, respectively, in the whole reactor, consistent with high effluent quality and treatment efficiency via a nitrite loop. Metagenomic binning analysis revealed that new and unidentified anammox species, affiliated with Candidatus Brocadia, were the dominant anammox organisms. Myxococcota and Planctomycetota were the principal organisms associated with the PD and DNRA processes, respectively.

Activities and metabolic versatility of distinct anammox bacteria in a full-scale wastewater treatment system

Anaerobic ammonium oxidation (anammox) is a key N2-producing process in the global nitrogen cycle. Major progress in understanding the core mechanism of anammox bacteria has been made, but our knowledge of the survival strategies of anammox bacteria in complex ecosystems, such as full-scale wastewater treatment plants (WWTPs), remains limited. Here, by combining metagenomics with in situ metatranscriptomics, complex anammox-driven nitrogen cycles in an anoxic tank and a granular activated carbon (GAC) biofilm module of a full-scale WWTP treating landfill leachate were constructed. Four distinct anammox metagenome-assembled genomes (MAGs), representing a new genus named Ca. Loosdrechtii, a new species in Ca. Kuenenia, a new species in Ca. Brocadia, and a new strain in “Ca. Kuenenia stuttgartiensis”, were simultaneously retrieved from the GAC biofilm. Metabolic reconstruction revealed that all anammox organisms highly expressed the core metabolic enzymes and showed a high metabolic versatility. Pathways for dissimilatory nitrate reduction to ammonium (DNRA) coupled to volatile fatty acids (VFAs) oxidation likely assist anammox bacteria to survive unfavorable conditions and facilitate switches between lifestyles in oxygen fluctuating environments. The new Ca. Kuenenia species dominated the anammox community of the GAC biofilm, specifically may be enhanced by the uniquely encoded flexible ammonium and iron acquisition strategies. The new Ca. Brocadia species likely has an extensive niche distribution that is simultaneously established in the anoxic tank and the GAC biofilm, the two distinct niches. The highly diverse and impressive metabolic versatility of anammox bacteria revealed in this study advance our understanding of the survival and application of anammox bacteria in the full-scale wastewater treatment system.

Purification of the key enzyme complexes of the anammox pathway from DEMON sludge.

Anaerobic Ammonium Oxidation ("anammox") is a bacterial process in which nitrite and ammonium are converted into nitrogen gas and water, yielding energy for the cell. Anammox is an important branch of the global biological nitrogen cycle, being responsible for up to 50% of the yearly nitrogen removal from the oceans. Strikingly, the anammox process uniquely relies on the extremely reactive and toxic compound hydrazine as a free intermediate. Given its global importance and biochemical novelty, there is considerable interest in the enzymes at the heart of the anammox pathway. Unfortunately, obtaining these enzymes in sufficiently large amounts for biochemical and structural studies is problematic, given the slow growth of pure cultures of anammox bacteria when high cell densities are required. However, the anammox process is being applied in wastewater treatment to remove nitrogenous waste in processes like DEamMONification (DEMON). In plants using such processes, which rely on a combination of aerobic ammonia-oxidizers and anammox organisms, kilogram amounts of anammox bacteria-containing sludge are readily available. Here, we report a protein isolation protocol starting from anammox cells present in DEMON sludge from a wastewater treatment plan that readily yields pure preparations of key anammox proteins in the tens of milligrams, including hydrazine synthase HZS and hydrazine dehydrogenase (HDH), as well as hydroxylamine oxidoreductase (HAO). HDH and HAO were active and of sufficient quality for biochemical studies and for HAO, the crystal structure could be determined. The method presented here provides a viable way to obtain materials for the study of proteins not only from the central anammox metabolism but also for the study of other exciting aspects of anammox bacteria, such as for example, their unusual ladderane lipids.

Dissimilatory nitrate reduction processes and corresponding nitrogen loss in tidal flow constructed wetlands

Nitrate reduction processes play significant roles in the retention and removal of nitrogen from water in anaerobic soils of constructed wetlands. The microbial mechanisms regulating nitrate reduction in artificial tidal wetlands remain poorly understood. The current study explored three microbial nitrate reduction processes in two tidal wetland mesocosms, PA (planted with Phragmites australis) and NP (unplanted) based on molecular and stable isotopic analyses. Potential rates of anaerobic ammonium oxidation (anammox), dissimilatory nitrate reduction to ammonium (DNRA) and denitrification ranged between 2.54 and 8.77, 0.12–0.93 and 40.11–387.94 nmol N2 g−1 ds (dry soil) d−1, with relatively higher values obtained in PA than NP. Denitrification contributed to 93.78–97.71% of total nitrate reduction, compared to 1.99–5.94% and 0.23–0.40% via anammox and DNRA, as 94.04–98.00% of nitrogen gas was produced through denitrification, with the rest via anammox. The absolute abundances of bacterial 16S rRNA and nitrogen functional genes were 3.22–9.11 × 109 and 0.8 × 104–1.1 × 108 copies g−1 ds, respectively. Proteobacteria, Chloroflexi, Acidobacteria, Nitrospirae, and Actinobacteria were the dominated phylum in artificial tidal wetlands, accounting for over 70%. The relative abundances of denitrification and anammox organisms varied from 0.97% to 3.60% and from 0.02% to 0.14%, respectively. Pearson correlation analyses indicate that nitrate played a vital role in the competition between DNRA and denitrification, while nitrite was the major factor regulating anammox. Collectively, these findings improve our understanding of nitrate removal mechanisms in tidal flow constructed wetlands, providing a sustainable solution to the nitrate pollution problem in the Yangtze River Estuary.

Impact of mixing intensity on dissolved oxygen half-velocity constants in a sidestream deammonification environment

Abstract A partial nitritation/anammox (PN/A) process was operated at two different mixing intensities to quantify the extent to which diffusional limitations impact process rates. At a steady-state operation, the total inorganic nitrogen removal efficiency in the bench-scale sequencing batch reactors was found to increase as mixing intensity decreased (62 and 84% for average velocity gradient (G) values of 15 and 5.3 s−1, respectively). The half-velocity constants with respect to bulk-phase dissolved oxygen (DO) concentration for ammonia-oxidizing bacteria (AOB) and anaerobic ammonium-oxidizing (anammox) organisms were estimated on the basis of nitrogen removal rates that were observed in activity tests. The activity tests were conducted over a range of bulk-phase DO concentrations. The best-fit values were estimated to be 0.68 ± 0.34 and 0.54 ± 0.56 mg O2/L for G values of 15 and 5.3 s−1, respectively. The AOB values were not statistically different (p = 0.19) between mixing conditions which were consistent with AOB dominating the surface of granules. The best-fit values were estimated to be 0.13 ± 0.09 and 0.55 ± 0.40 mg O2/L for G values of 15 and 5.3 s−1, respectively, and were statistically different . The results demonstrated that mixing conditions should be considered when designing PN/A processes and provide quantitative results that can be employed to improve models of these processes. This article has been made Open Access thanks to the kind support of CAWQ/ACQE (https://www.cawq.ca).

Conversion Pathways of Substrates in Sulfate-Reducing Ammonia Oxidation System

The phenomenon of simultaneous transformation of ammonium and sulfate under the conditions of inoculating ANAMMOX culture has gotten the attention of researchers. However, there are some problems and doubts reported in the related literature. In this study, the characteristics of ammonium and sulfate synchronous transformation were investigated in a CFSTR via inoculation with ANAMMOX culture. Under the condition of oxygen removal and non-filling, in the unfilled sealed fermentation tank, the average conversion of NH4+-N was 50.8 mg·L-1 while that of sulfate-sulfur was 4.5 mg·L-1. Elemental analysis results showed that the observed yellow solid was not elemental sulfur but rather iron-containing compounds. However, no obvious change of ammonium was observed when using a filled sealed batch reactor. Only sulfate transformed significantly, and the transformation rate was affected by the inoculation biomass. Under these two conditions, the ORP in the reactor was completely different. This is an indication that the synchronous transformation of ammonium and sulfate observed in both our study and other related studies is probably not a process mediated by ANAMMOX organisms, in which sulfate acted as the electron acceptor to oxidize ammonium. Actually, ammonium and sulfate transformation were completely independent: ammonium oxidation is due to the micro oxygen environment created by the reactor operation form, whereas sulfate conversion is attributed to the sulfate heterotrophic reduction that results from the organic matter release via microbial decay. This transformation can clarify and explain the problems and doubts reported in the related research.

Contribution of Anammox to Nitrogen Removal in Two Temperate Forest Soils.

Anaerobic ammonium oxidation with nitrite reduction to dinitrogen (termed anammox) has been reported to be an important process for removing fixed nitrogen (N) in marine ecosystems and in some agricultural and wetland soils. However, its importance in upland forest soils has never been quantified. In this study, we evaluated the occurrence of anammox activity in two temperate forest soils collected from northeastern China. With (15)N-labeled NO3 (-) incubation, we found that the combined potential of the N2 production rates of anammox and codenitrification ranged from 0.01 ± 0.01 to 1.2 ± 0.18 nmol N per gram of soil per hour, contributing 0.5% to 14.4% of the total N2 production along the soil profile. Denitrification was the main pathway of N2 production and accounted for 85.6% to 99.5% of the total N2 production. Further labeling experiments with (15)NH4 (+) and (15)NO2 (-) indicated that codenitrification was present in the mixed forest soil. Codenitrification and anammox accounted for 2% to 12% and 1% to 7% of the total N2 production, respectively. Two anammox species, "Candidatus Brocadia fulgida" and "Candidatus Jettenia asiatica," were detected in this study but in very low abundance (as indicated by the hzsB gene). Our results demonstrated that the anammox process occurs in forest soils, but the contribution to N2 loss might be low in these ecosystems. More research is necessary to determine the activities of different N2 releasing pathways in different forest soils. In this study, we examined the anammox activity in temperate upland forest soils using the (15)N isotope technique. We found that the anammox process contributed little to the N2 production rate in the studied forest soil. Two anammox organisms, "Candidatus Brocadia fulgida" and "Candidatus Jettenia asiatica," were detected. In addition, we found that codenitrification was another N2 production pathway in forest soils. Our results could contribute to the understanding of soil gaseous N losses and microbial controls in forest soils.

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 (MWWpre-treated), amended with nitrite, was proven in three parallel reactors. The reactors were operated at total N concentrations in the range 5–20 mgN∙L−1, as expected for MWW. Anammox activities up to 465 mgN∙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 mgN∙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, MWWpre-treated had a direct impact on process performance. Changing the influent from synthetic medium to MWWpre-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.

Impact of coexistence of flocs and biofilm on performance of combined nitritation-anammox granular sludge reactors

Nitrogen (N) removal from high-strength wastewater can be accomplished in single-stage combined nitritation-anammox reactors with suspended growth biomass composed of floccular sludge, granular sludge, or of any mix of these 2 different sludge fractions. To date, the influence of floccular biomass on granular sludge reactor performance and stability has not been investigated experimentally or numerically. To address this knowledge gap, two 1D multi-species models were developed in Aquasim to assess the importance of small levels of flocs in putatively granular sludge combined nitritation-anammox reactors for different bulk oxygen concentrations and organics loads. The models included the growth and decay of aerobic ammonium-oxidizing organism (AOO), nitrite-oxidizing organisms (NOO), heterotrophic organisms (OHO), and anammox organisms (AMO) in exclusively granular sludge reactors, and in granular sludge reactors with small levels (∼5% of total biomass) of flocs. While maximum N removal efficiencies were similar for both model structures, floc addition led to a lower optimal dissolved oxygen concentration (DO) as well as a narrower maximum N removal peak, suggesting that small levels of floccular material may decrease process robustness to bulk oxygen changes. For some DO levels, this led to drastic efficiency drops. Furthermore, floc addition also led to substantial segregation in activity and microbial population distribution, with AOO, NOO and OHO concentrated in flocs and AMO concentrated in granules. Increased organic loading (COD:N = 4:3) improved maximum N removal efficiency in both model structures, but yielded substantially different predictions for optimal DO setpoint and process robustness to variations in DO. Taken together, our results indicate that even small levels of floccular biomass in biofilm reactors can have profound implications for reactor performance and optimization and for segregation of linked microbial processes, and suggest that the common practice of neglecting small levels of floccular material in biofilm models and in practice may lead to erroneous predictions.

Diversity and distribution of planktonic anaerobic ammonium-oxidizing bacteria in the Dongjiang River, China

Anaerobic ammonium-oxidizing (anammox) process has recently been recognized as an important pathway for removing fixed nitrogen (N) from aquatic ecosystems. Anammox organisms are widely distributed in freshwater environments. However, little is known about their presence in the water column of riverine ecosystems. Here, the existence of a diverse anammox community was revealed in the water column of the Dongjiang River by analyzing 16S rRNA and hydrazine oxidation (hzo) genes of anammox bacteria. Phylogenetic analyses of hzo genes showed that Candidatus Jettenia related clades of anammox bacteria were dominant in the river, suggesting the ecological microniche distinction from freshwater/estuary and marine anammox bacteria with Ca. Brocadia and Kuenenia genera mainly detected in freshwater/estuary ecosystems, and Ca. Scalindua genus mainly detected in marine ecosystems. The abundance and diversity of anammox bacteria along the river were both significantly correlated with concentrations of NH4+-N based on Pearson and partial correlation analyses. Redundancy analyses showed the contents of NH4+-N, NO3−-N and the ratio of NH4+-N to NO2−-N significantly influenced the spatial distributions of anammox bacteria in the water column of the Dongjiang River. These results expanded our understanding of the distribution and potential roles of anammox bacteria in the water column of the river ecosystem.

Deammonification process start-up after enrichment of anammox microorganisms from reject water in a moving-bed biofilm reactor

Deammonification via intermittent aeration in biofilm process for the treatment of sewage sludge digester supernatant (reject water) was started up using two opposite strategies. Two moving-bed biofilm reactors were operated for 2.5 years at 26 (±0.5)°C with spiked influent (and hence free ammonia (FA)) addition. In the first start-up strategy, an enrichment of anammox biomass was first established, followed by the development of nitrifying biomass in the system (R1). In contrast, the second strategy aimed at the enrichment of anammox organisms into a nitrifying biofilm (R2). The first strategy was most successful, reaching higher maximum total nitrogen (TN) removal rates over a shorter start-up period. For both reactors, increasing FA spiking frequency and increasing effluent concentrations of the anammox intermediate hydrazine correlated to decreasing aerobic nitrate production (nitritation). The bacterial consortium of aerobic and anaerobic ammonium oxidizing bacteria in the bioreactor was determined via denaturing gel gradient electrophoresis, polymerase chain reaction and pyrosequencing. In addition to a shorter start-up with a better TN removal rate, nitrite oxidizing bacteria (Nitrospira) were outcompeted by spiked ammonium feeding from R1.

Going for mainstream deammonification from bench to full scale for maximized resource efficiency

A three-pronged coordinated research effort was undertaken by cooperating utilities at three different experimental scales investigating bioaugmentation, enrichment and performance of anammox organisms in mainstream treatment. Two major technological components were applied: density-based sludge wasting by a selective cyclone to retain anammox granules and intermittent aeration to repress nitrite oxidizers. This paper evaluates process conditions and operation modes to direct more nitrogen to the resource-saving metabolic route of deammonification.

Effects of Organic Carbon Source, COD/N Ratio and Temperature on Anammox Organisms

Effects of Organic Carbon Source, COD/N Ratio and Temperature on Anammox Organisms

Diversity of microbial communities in an attached-growth system using Bauxsol™ pellets for wastewater treatment

Columns of Bauxsol™ pellets were used in a field experiment as biomass support particle for wastewater microbial communities. The attached microbial community structure was analysed using denaturing gradient gel electrophoresis (DGGE), targeting the 16S rDNA gene's V3 region. DGGE profiles showed that the type and composition of support particles used (i.e. Bauxsol™ pellets or gravel) had a significant impact on the attached bacterial communities (64% dissimilarity). In addition, ecological indices revealed a more heterogeneous bacterial community structure on the Bauxsol™ pellets. TOC/TN ratios post-experiment (6.5–9.3) suggested a good level of biological activity (i.e. active biofilm) in the Bauxsol™ columns. Moreover, Bauxsol™ pellets were mostly made of inorganic carbon, suggesting insoluble carbonate biomineralisation. Polymerase chain reaction (PCR) amplification of specific marker genes (i.e. bacterial and archaeal amoA genes, nosZ gene, and hzo gene) were used to identify the presence of attached bacterial communities associated with nitrogen transformation. The results along with geochemical data (i.e. up to 50% nitrogen removal) revealed co-existence of ammonia-oxidising bacteria, denitrifiers, and anammox organisms. This study conclusively demonstrates that microbial communities are well-adapted to Bauxsol™ pellets and bacterial communities involved in the nitrogen cycle are present.

Inhibitory effects of free ammonia on Anammox bacteria

Anammox bacteria can effectively treat high ammonia and nitrite concentrations under anoxic environments. However, the presence of high ammonia and nitrite concentrations may cause free ammonia and nitrous acid inhibition at high pH and temperature environments. In this study, the inhibitory effect of free ammonia on Anammox bacteria was investigated in a lab-scale upflow fixed-bed reactor with Kaldnes biofilm carriers. Results of continuous operation showed that inhibition was not observed in the Anammox reactor when the free ammonia concentration gradually increased up to 150mg/L. However, Anammox activity suddenly dropped to 10 % when the free ammonia concentration reached to 190mg/L. Nevertheless, high influent ammonia and nitrite concentrations up to 1,500 and 500mg/L, respectively, did not noticeably inhibit the Anammox activity. Gradually decreasing Anammox activity was also supported by fluorescent in situ hybridization (FISH) analysis. FISH and 16S rRNA gene analysis results revealed that main Anammox organisms were phylogenetically related to Candidatus Kuenenia stuttgartiensis, Candidatus Jettenia asiatica and Candidatus Brocadia anammoxidans.

The effect of nitrite inhibition on the anammox process

The negative effect of nitrite on anammox activity has been reported widely during the past decade. Although the adverse effect is clear, conflicting reports exist on the level at which it occurs and its reversible/irreversible nature. An in depth study on nitrite inhibition therefore was performed in which the influence of environmental factors was evaluated. Anammox activity was measured in anammox granules by continuously monitored standardized manometric batch tests extending the interpretation by evaluation of lag times, maximum conversion rates during the tests and substrates/product conversion ratios. The granules where obtained from a one-stage anammox reactor, the dominant anammox organisms belonged to the Brocadia type. The observed 50% activity inhibition for nitrite (IC50) was 0.4 g N L−1. The activity recovered fully after removal of the nitrite. Conversion in fresh medium after exposure to up to 6 g NO2−–N L−1 for 24 h showed less then 60% loss of activity. Presence of ammonium during nitrite (2 g N L−1) exposure resulted in a stronger loss of activity after nitrite exposure (50% and 30% in presence and absence of ammonium respectively). Presence of oxygen during nitrite incubation led to a maximum activity reduction of 32%. The recovery after exposure indicates that the adverse effect of nitrite is reversible and thus inhibitory rather than toxic in nature. Similarities between exposure at three different pH-values indicate that nitrite rather than nitrous acid is the actual inhibiting compound.

The depth-specific significance and relative abundance of anaerobic ammonium-oxidizing bacteria in estuarine sediments (Medway Estuary, UK)

Variations in the overall and depth-specific significance of anammox were measured using (15) N isotope experiments in both bioirrigated and undisturbed sediments of the Medway Estuary, UK. This was performed over two surveys, alongside FISH experiments, to identify and track shifts in the relative abundance of anammox organisms with depth. In Survey 1 (initially screening for the presence of anammox), the potential for anammox (ra) decreased from 32% upstream to 6% downstream. In Survey 2, depth-specific values of ra varied between a maximum of 37% upstream and a minimum of 4% downstream. This was linked to a small population of anammox organisms accounting for <1-8% of total bacteria with depth in Survey 1 and <1-3% in Survey 2. The relationship between the relative abundance of anammox cells and the potential contribution of anammox to total N(2) production did not however correlate. In Survey 2, infaunal disruption of the sediment substrata, and concomitant fluctuations of O(2) over depth, did not appear to inhibit the potential for anammox, even at the most bioturbated site. Moreover, deficits detected in the retrieval of (15) N gas from denitrification in Survey 2 may imply potential links between dissimilatory nitrate reduction to ammonium and anammox in estuarine sediments.

ANAMMOX BACTERIA – UNIQUE MICROORGANISMS OF NITROGEN CYCLE

There were presented the data on the microorganisms with the ability to ANAMMOX process taken part in the nitrogen cycle in nature. These unique organisms are the representatives members of the type Planctomycetes. The were described the structure of anammox bacteria, capable to use anaerobic ammonium and nitrite for power generation due to the presence of specific organella in them – anammoxosome. The biochemical cycle of transformations of ammonium and nitrite with the release of molecular nitrogen due to the presence of membrane anammoxosome ladderan lipids was studied. In the review there were displayed the value of anammox organisms in the nitrogen cycle in nature and significance of these organisms for biotechnology of sewage purification from large concentrations of ammonium at low energy consumption.

Prevalence of Anaerobic Ammonium-Oxidizing Bacteria in Contaminated Groundwater

Anaerobic ammonium-oxidizing (anammox) bacteria perform an important step in the global nitrogen cycle: anaerobic oxidation of ammonium and reduction of nitrite to form dinitrogen gas (N(2)). Anammox organisms appear to be widely distributed in natural and artificial environments. However, their roles in groundwater ammonium attenuation remain unclear and only limited biomarker-based data confirmed their presence prior to this study. We used complementary molecular and isotope-based methods to assess anammox diversity and activity occurring at three ammonium-contaminated groundwater sites: quantitative PCR, denaturing gradient gel electrophoresis, sequencing of 16S rRNA genes, and (15)N-tracer incubations. Here we show that anammox performing organisms were abundant bacterial community members. Although all sites were dominated by Candidatus Brocadia-like sequences, the community at one site was particularly diverse, possessing four of five known genera of anammox bacteria. Isotope data showed that anammox produced up to 18 and 36% of N(2) at these sites. By combining molecular and isotopic results we have demonstrated the diversity, abundance, and activity of these autotrophic bacteria. Our results provide strong evidence for their important biogeochemical role in attenuating groundwater ammonium contamination.

Investigation of aerobic and anaerobic ammonium-oxidising bacteria presence in a small full-scale wastewater treatment system comprised by UASB reactor and three polishing ponds

This work applied PCR amplification method and Fluorescence in situ hybridisation (FISH) with primers and probes specific for the anammox organisms and aerobic ammonia-oxidising beta-Proteobacteria in order to detect these groups in different samples from a wastewater treatment system comprised by UASB reactor and three polishing (maturation) ponds in series. Seven primer pairs were used in order to detect Anammox bacteria. Positive results were obtained with three of them, suggesting that Anammox could be present in polishing pond sediments. However, Anammox bacteria were not detected by FISH, indicating that they were not present in sediment samples, or they could be present but below FISH detection limit. Aerobic ammonia- and nitrite-oxidising bacteria were verified in water column samples through Most Probable Number (MPN) analysis, but they were not detected in sediment samples by FISH. Ammonia removal efficiencies occurred systematically along the ponds (24, 32, and 34% for polishing pond 1, 2, and 3, respectively) but the major reaction responsible for this removal is still unclear. Some nitrification might have occurred in water samples because some nitrifying bacteria were present. Also Anammox reaction might have occurred because Anammox genes were detected in the sediments, but probably this reaction was too low to be noticed. It is important also to consider that some of the ammonia removal observed might be related to NH(3) stripping, associated with the pH increase resulting from the intensive photosynthetic activity in the ponds (mechanism under investigation). Therefore, it can be concluded that more than one mechanism (or reaction) might be involved in the ammonia removal in the polishing ponds investigated in this study.