Low-abundant but highly transcriptionally active uncharacterised Nitrosomonas drive ammonia-oxidation in the Brouage mudflat, France.

Exploring differences in nitrification within adjacent sedimentary structures of ridges and runnels on the Brouage mudflat, France, we quantified Potential Nitrification Rates (PNR) alongside amoA genes and transcripts. PNR was lower in ridges (≈1.7 fold-lower) than runnels, despite higher (≈1.8 fold-higher) ammonia-oxidizing bacteria (AOB) abundance. However, AOB were more transcriptionally active in runnels (≈1.9 fold-higher). Sequencing of amoA genes and transcripts revealed starkly contrasting profiles with transcripts from ridges and runnels dominated (≈91 % in ridges and ≈98 % in runnels) by low abundant (≈4.6 % of the DNA community in runnels and ≈0.8 % in ridges) but highly active phylotypes. The higher PNR in runnels was explained by higher abundance of this group, an uncharacterised Nitrosomonas sp. cluster. This cluster is phylogenetically similar to other active ammonia-oxidizers with worldwide distribution in coastal environments indicating its potential, but previously overlooked, contribution to ammonia oxidation globally. In contrast DNA profiles were dominated by highly abundant but low-activity clusters phylogenetically distinct from known Nitrosomonas (Nm) and Nitrosospira (Ns). This cluster is also globally distributed in coastal sediments, primarily detected as DNA, and often classified as Nitrosospira or Nitrosomonas. We therefore propose to classify this cluster as Ns/Nm. Our work indicates that low abundant but highly active AOB could be responsible for the nitrification globally, while the abundant AOB Ns/Nm may not be transcriptionally active, and as such account for the lack of correlation between rate processes and gene abundances often reported in the literature. It also raises the question as to what this seemingly inactive group is doing?

RNA extraction protocol from low-biomass bacterial Nitrosomonas europaea and Nitrobacter winogradskyi cultures for whole transcriptome studies.

RNA-sequencing for whole transcriptome analysis requires high-quality RNA in adequate amounts, which can be difficult to generate with low-biomass-producing bacteria where sample volume is limited. We present an RNA extraction protocol for low-biomass-producing autotrophic bacteria Nitrosomonas europaea and Nitrobacter winogradskyi cultures. We describe steps for sample collection, lysozyme-based enzymatic lysis, and a commercial silica-column-based RNA extraction. We then detail evaluation of RNA yield and quality for downstream applications such as RNA-Seq. For complete details on the use and execution of this protocol, please refer to Verbeelen et al.1.

Characterisation of bacteria representing a novel Nitrosomonas clade: Physiology, genomics and distribution of missing ammonia oxidizer.

Members of the genus Nitrosomonas are major ammonia oxidizers that catalyse the first step of nitrification in various ecosystems. To date, six subgenus-level clades have been identified. We have previously isolated novel ammonia oxidizers from an additional clade (unclassified cluster 1) of the genus Nitrosomonas. In this study, we report unique physiological and genomic properties of the strain PY1, compared with representative ammonia-oxidising bacteria (AOB). The apparent half-saturation constant for total ammonia nitrogen and maximum velocity of strain PY1 were 57.9 ± 4.8 µM NH3 + NH4 + and 18.5 ± 1.8 µmol N (mg protein)-1 h-1 , respectively. Phylogenetic analysis based on genomic information revealed that strain PY1 belongs to a novel clade of the Nitrosomonas genus. Although PY1 contained genes to withstand oxidative stress, cell growth of PY1 required catalase to scavenge hydrogen peroxide. Environmental distribution analysis revealed that the novel clade containing PY1-like sequences is predominant in oligotrophic freshwater. Taken together, the strain PY1 had a longer generation time, higher yield and required reactive oxygen species (ROS) scavengers to oxidize ammonia, compared with known AOB. These findings expand our knowledge of the ecophysiology and genomic diversity of ammonia-oxidising Nitrosomonas.

Nitrosomonas supralitoralis sp. nov., an ammonia-oxidizing bacterium from beach sand in a supralittoral zone.

A betaproteobacterial chemolithotrophic ammonia-oxidizing bacterium designated APG5T was isolated from supralittoral sand of the Edmonds City Beach, WA, USA. Growth was observed at 10-35 °C (optimum, 30 °C), pH 5-9 (optimum, pH 8) and ammonia concentrations as high as 100 mM (optimum, 1-30 mM NH4Cl). The strain grows optimally in a freshwater medium but tolerates up to 400 mM NaCl. It is most closely related to 'Nitrosomonas ureae' (96.7% 16S rRNA and 92.4% amoA sequence identity). The 3.75-Mbp of AGP5T draft genome contained a single rRNA operon and all necessary tRNA genes and has the lowest G+C content (43.5%) when compared to the previously reported genomes of reference strains in cluster 6 Nitrosomonas. Based on an average nucleotide identity of 82% with its closest relative ('N. ureae' Nm10T) and the suggested species boundary of 95-96%, a new species Nitrosomonas supralitoralis sp. nov. is proposed. The type strain of Nitrosomonas supralitoralis is APG5T (= NCIMB 14870T = ATCC TSD-116T).

Growth of nitrite-oxidizing Nitrospira and ammonia-oxidizing Nitrosomonas in marine recirculating trickling biofilter reactors.

Aerobic ammonia and nitrite oxidation reactions are fundamental biogeochemical reactions contributing to the global nitrogen cycle. Although aerobic nitrite oxidation yields 4.8-folds less Gibbs free energy (∆Gr ) than aerobic ammonia oxidation in the NH4 + -feeding marine recirculating trickling biofilter reactors operated in the present study, nitrite-oxidizing and not ammonia-oxidizing Nitrospira (sublineage IV) outnumbered ammonia-oxidizing Nitrosomonas (relative abundance; 53.8% and 7.59% respectively). CO2 assimilation efficiencies during ammonia or nitrite oxidation were 0.077 µmol-14 CO2 /µmol-NH3 and 0.053-0.054 µmol-14 CO2 /µmol-NO2 - respectively, and the difference between ammonia and nitrite oxidation was much smaller than the difference of ∆Gr . Free-energy efficiency of nitrite oxidation was higher than ammonia oxidation (31%-32% and 13% respectively), and high CO2 assimilation and free-energy efficiencies were a determinant for the dominance of Nitrospira over Nitrosomonas. Washout of Nitrospira and Nitrosomonas from the trickling biofilter reactors was also examined by quantitative PCR assay. Normalized copy numbers of Nitrosomonas amoA were 1.5- to 1.7-folds greater than Nitrospira nxrB and 16S rRNA gene in the reactor effluents. Nitrosomonas was more susceptible for washout than Nitrospira in the trickling biofilter reactors, which was another determinant for the dominance of Nitrospira in the trickling biofilter reactors.

Envisioning role of ammonia oxidizing bacteria in bioenergy production and its challenges: a review.

Ammonia oxidizing bacteria (AOB) play a key role in the biological oxidation of ammonia to nitrite and mark their significance in the biogeochemical nitrogen cycle. There has been significant development in harnessing the ammonia oxidizing potential of AOB in the past few decades. However, very little is known about the potential applications of AOB in the bioenergy sector. As alternate sources of energy represent a thrust area for environmental sustainability, the role of AOB in bioenergy production becomes a significant area of exploration. This review highlights the role of AOB in bioenergy production and emphasizes the understanding of the genetic make-up and key cellular biochemical reactions occurring in AOB, thereby leading to the exploration of its various functional aspects. Recent outcomes in novel ammonia/nitrite oxidation steps occurring in a model AOB - Nitrosomonas europaea propel us to explore several areas of environmental implementation. Here we present the significant role of AOB in microbial fuel cells (MFC) where Nitrosomonas sp. play both anodic and cathodic functions in the generation of bioelectricity. This review also presents the potential role of AOB in curbing fuel demand by producing alternative liquid fuel such as methanol and biodiesel. Herein, the multiple roles of AOB in bioenergy production namely: bioelectricity generation, bio-methanol, and biodiesel production have been presented.

Competition between Ammonia-Oxidizing Archaea and Bacteria from Freshwater Environments.

In the environment, nutrients are rarely available in a constant supply. Therefore, microorganisms require strategies to compete for limiting nutrients. In freshwater systems, ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) compete with heterotrophic bacteria, photosynthetic microorganisms, and each other for ammonium, which AOA and AOB utilize as their sole source of energy and nitrogen. We investigated the competition between highly enriched cultures of AOA (AOA-AC1) and AOB (AOB-G5-7) for ammonium. Based on the amoA gene, the newly enriched archaeal ammonia oxidizer in AOA-AC1 was closely related to Nitrosotenuis spp., and the bacterial ammonia oxidizer in AOB-G5-7, Nitrosomonas sp. strain Is79, belonged to the Nitrosomonas oligotropha group (Nitrosomonas cluster 6a). Growth experiments in batch cultures showed that AOB-G5-7 had higher growth rates than AOA-AC1 at higher ammonium concentrations. During chemostat competition experiments under ammonium-limiting conditions, AOA-AC1 dominated the cultures, while AOB-G5-7 decreased in abundance. In batch cultures, the outcome of the competition between AOA and AOB was determined by the initial ammonium concentrations. AOA-AC1 was the dominant ammonia oxidizer at an initial ammonium concentration of 50 µM, and AOB-G5-7 was dominant at 500 µM. These findings indicate that during direct competition, AOA-AC1 was able to use ammonium that was unavailable to AOB-G5-7, while AOB-G5-7 dominated at higher ammonium concentrations. The results are in strong accordance with environmental survey data suggesting that AOA are mainly responsible for ammonia oxidation under more oligotrophic conditions, whereas AOB dominate under eutrophic conditions. IMPORTANCE Nitrification is an important process in the global nitrogen cycle. The first step, ammonia oxidation to nitrite, can be carried out by ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). In many natural environments, these ammonia oxidizers coexist. Therefore, it is important to understand the population dynamics in response to increasing ammonium concentrations. Here, we study the competition between AOA and AOB enriched from freshwater systems. The results demonstrate that AOA are more abundant in systems with low ammonium availabilities and that AOB are more abundant when the ammonium availability increases. These results will help to predict potential shifts in the community composition of ammonia oxidizers in the environment due to changes in ammonium availability.

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.

Stoichiometric and kinetic characterization of an acid-tolerant ammonia oxidizer 'Candidatus Nitrosoglobus'.

Recently, acidic (i.e. pH<5) nitrification in activated-sludge is attracting attention because it enables stable nitritation (NH4+ â†’ NO2-), and enhances sludge reduction and stabilization. However, the key acid-tolerant ammonia oxidizers involved are poorly understood. In this study, we performed stoichiometric and kinetic characterization of a new acid-tolerant ammonia-oxidizing bacterium (AOB) belonging to gamma-proteobacterium, Candidatus Nitrosoglobus. Ca. Nitrosoglobus was cultivated in activated-sludge in a laboratory membrane bioreactor over 200 days, with a relative abundance of 55.1 ± 0.5% (indicated by 16S rRNA gene amplicon sequencing) at the time of the characterization experiments. Among all known nitrifiers, Ca. Nitrosoglobus bears the highest resistance to nitrite, low pH, and free nitrous acid (FNA). These traits define Ca. Nitrosoglobus as an adversity-strategist that tends to prosper in acidic activated-sludge, where the low pH (< 5.0) and high levels of FNA (at parts per million levels) sustained and inhibited all other nitrifiers. In contrast, in the conventional pH-neutral activated-sludge process, Ca. Nitrosoglobus is less competitive with canonical AOB (e.g. Nitrosomonas) due to the relatively slow specific growth rate and low affinities to both oxygen and total ammonia. These results advance our understanding of acid-tolerant ammonia oxidizers, and support further development of the acidic activated-sludge process in which Ca. Nitrosoglobus can play a critical role.

Deep amoA amplicon sequencing reveals community partitioning within ammonia-oxidizing bacteria in the environmentally dynamic estuary of the River Elbe.

The community composition of betaproteobacterial ammonia-oxidizing bacteria (ß-AOB) in the River Elbe Estuary was investigated by high throughput sequencing of ammonia monooxygenase subunit A gene (amoA) amplicons. In the course of the seasons surface sediment samples from seven sites along the longitudinal profile of the upper Estuary of the Elbe were investigated. We observed striking shifts of the ß-AOB community composition according to space and time. Members of the Nitrosomonas oligotropha-lineage and the genus Nitrosospira were found to be the dominant ß-AOB within the river transect, investigated. However, continuous shifts of balance between members of both lineages along the longitudinal profile were determined. A noticeable feature was a substantial increase of proportion of Nitrosospira-like sequences in autumn and of sequences affiliated with the Nitrosomonas marina-lineage at downstream sites in spring and summer. Slightly raised relative abundances of sequences affiliated with the Nitrosomonas europaea/Nitrosomonas mobilis-lineage and the Nitrosomonas communis-lineage were found at sampling sites located in the port of Hamburg. Comparisons between environmental parameters and AOB-lineage (ecotype) composition revealed promising clues that processes happening in the fluvial to marine transition zone of the Elbe estuary are reflected by shifts in the relative proportion of ammonia monooxygenase sequence abundance, and hence, we propose ß-AOB as appropriate indicators for environmental dynamics and the ecological condition of the Elbe Estuary.

Lost in translation: the quest for Nitrosomonas cluster 7-specific amoA primers and TaqMan probes.

The choice of primer and TaqMan probes to quantify ammonia-oxidizing bacteria (AOB) in environmental samples is of crucial importance. The re-evaluation of primer pairs based on current genomic sequences used for quantification of the amoA gene revealed (1) significant misrepresentations of the AOB population in environmental samples, (2) and a lack of perfect match primer pairs for Nitrosomonas europaea and Nitrosomonas eutropha. We designed two new amoA cluster 7-specific primer pairs and TaqMan probes to quantify N. europaea (nerF/nerR/nerTaq) and N. eutropha (netF/netR/netTaq). Specificity and quantification biases of the newly designed primer sets were compared with the most popular primer pair (amoA1f/amoA2r) using DNA from various AOB cultures as individual templates as well as DNA mixtures and environmental samples. Based on the qPCR results, we found that the newly designed primer pairs and the most popular one performed similarly for individual templates but differed for the DNA mixtures and environmental samples. Using the popular primer pair introduced a high underestimation of AOB in environmental samples, especially for N. eutropha. Thus, there is a strong need for more specific primers and probes to understand the occurrence and competition between N. europaea and N. eutropha in different environments.

Efficient transition from partial nitritation to partial nitritation/Anammox in a membrane bioreactor with activated sludge as the sole seed source.

A lab-scale membrane bioreactor (MBR) was employed to carry out the partial nitritation/Anammox (PN/A) process from conventional activated sludge. Seed sludge was cultivated under microaerobic conditions for 10 days before seeding into the MBR. The bacterial community was analyzed on the basis of cloning and sequencing of 16S rRNA gene. Relative slow ammonia oxidation rates (3.2-13.0 mgN/L/d) were established in the microaerobic cultivation period. In the continuous MBR operation, the nitritation was achieved in the first 16 days and the reactor produced a balanced ratio between ammonia and nitrite which favored the proliferation of Anammox bacteria. Efficient transition from PN to PN/A was achieved in two months which was supported by appearance of reddish spots on the reactor inner wall and the concurrent consumption of ammonium and nitrite. The PN/A performed a robust and high-rate nitrogen removal capability and achieved a peak nitrogen removal of 1.81 kg N/m3/d. 16S rRNA gene-based analysis indicated that "Nitrosomonas sp." and "Candidatus Jettenia sp." accounted for ammonia oxidation and nitrogen depletion, respectively. Denitratisoma facilitated denitrification in the reactor. The present study suggested that a pre-cultivation of seed sludge under microaerobic conditions assists fast realization of PN and further convoyed efficient transition from PN to PN/A. Knowledge gleaned from this study is of significance to initiation, operation, and control of MBR-PN/As.

Transcriptome Analysis of the Ammonia-Oxidizing Bacterium Nitrosomonas mobilis Ms1 Reveals Division of Labor between Aggregates and Free-living Cells.

Bacteria change their metabolic states to increase survival by forming aggregates. Ammonia-oxidizing bacteria also form aggregates in response to environmental stresses. Nitrosomonas mobilis, an ammonia-oxidizing bacterium with high stress tolerance, often forms aggregates mainly in wastewater treatment systems. Despite the high frequency of aggregate formation by N. mobilis, its relationship with survival currently remains unclear. In the present study, aggregates were formed in the late stage of culture with the accumulation of nitrite as a growth inhibitor. To clarify the significance of aggregate formation in N. mobilis Ms1, a transcriptome analysis was performed. Comparisons of the early and late stages of culture revealed that the expression of stress response genes (chaperones and proteases) increased in the early stage. Aggregate formation may lead to stress avoidance because stress response genes were not up-regulated in the late stage of culture during which aggregates formed. Furthermore, comparisons of free-living cells with aggregates in the early stage of culture showed differences in gene expression related to biosynthesis (ATP synthase and ribosomal proteins) and motility and adhesion (flagella, pilus, and chemotaxis). Biosynthesis genes for growth were up-regulated in free-living cells, while motility and adhesion genes for adaptation were up-regulated in aggregates. These results indicate that N. mobilis Ms1 cells adapt to an unfavorable environment and grow through the division of labor between aggregates and free-living cells.

Microbial driven reduction of N2O and NH3 emissions during composting: Effects of bamboo charcoal and bamboo vinegar.

In this study, we systematically analyzed the microbial-driven effects of bamboo charcoal (BC) and bamboo vinegar (BV) on reducing NH3 and N2O emissions during aerobic composting. The results showed that BC and BV improved the nitrogen conversion and compost quality, but the combined BC + BV treatment obtained the best improvements. The BC, BV, and BC + BV treatments reduced the NH3 emissions by 14.35%, 17.90%, and 29.83%, respectively, and the N2O emissions by 44.83%, 55.96%, and 74.53%. BC and BV reduced the NH3 and N2O emissions during composting by controlling ammonia oxidation, where napA, nirK, and nosZ served as useful indicators of the N2O emissions from compost, especially the nirK gene. The dominant nitrifying and denitrifying bacteria belonged to Proteobacteria, and the changes in environmental factors during composting significantly affected the succession of the nitrifying and denitrifying bacterial communities. Nitrosomonas was a key nitrifying bacterial genus in the mesophilic composting period, and BC and BV may have reduced the NH3 emissions by enhancing its conversion to NH4+-N by Nitrosomonas. In addition, norank_p__environmental_samples, unclassified_k__norank_d__Bacteria, and unclassified_p__Proteobacteria were jointly responsible for driving the production of N2O during the compost maturity stage.

A Physiological and Genomic Comparison of Nitrosomonas Cluster 6a and 7 Ammonia-Oxidizing Bacteria.

Ammonia-oxidizing bacteria (AOB) within the genus Nitrosomonas perform the first step in nitrification, ammonia oxidation, and are found in diverse aquatic and terrestrial environments. Nitrosomonas AOB were grouped into six defined clusters, which correlate with physiological characteristics that contribute to adaptations to a variety of abiotic environmental factors. A fundamental physiological trait differentiating Nitrosomonas AOB is the adaptation to either low (cluster 6a) or high (cluster 7) ammonium concentrations. Here, we present physiological growth studies and genome analysis of Nitrosomonas cluster 6a and 7 AOB. Cluster 6a AOB displayed maximum growth rates at ≤ 1 mM ammonium, while cluster 7 AOB had maximum growth rates at ≥ 5 mM ammonium. In addition, cluster 7 AOB were more tolerant of high initial ammonium and nitrite concentrations than cluster 6a AOB. Cluster 6a AOB were completely inhibited by an initial nitrite concentration of 5 mM. Genomic comparisons were used to link genomic traits to observed physiological adaptations. Cluster 7 AOB encode a suite of genes related to nitrogen oxide detoxification and multiple terminal oxidases, which are absent in cluster 6a AOB. Cluster 6a AOB possess two distinct forms of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and select species encode genes for hydrogen or urea utilization. Several, but not all, cluster 6a AOB can utilize urea as a source of ammonium. Hence, although Nitrosomonas cluster 6a and 7 AOB have the capacity to fulfill the same functional role in microbial communities, i.e., ammonia oxidation, differentiating species-specific and cluster-conserved adaptations is crucial in understanding how AOB community succession can affect overall ecosystem function.

Analysis of Bacterial Communities in Partial Nitritation and Conventional Nitrification Systems for Nitrogen Removal.

This work studied the microbial community in partial nitritation and complete nitrification processes, which were applied to treat the low Carbon Nitrogen ratio wastewater. The phospholipid fatty acid and quantitative PCR analysis showed that the sludge circulating ratio of 75% resulted in a good microbial growth and a higher abundance of ammonia oxidizing bacteria relative to the nitrite oxidizing bacteria. The Betaproteobacteria were observed to compose the most abundant sludge bacterial groups in the two processes, based on phylogenetic analysis. The phylogenetic analysis of both 16S rRNA and amoA gene indicated that the Nitrosomonas sp. were the dominant ammonia oxidizing bacteria in the partial nitritation process. The relative abundance of nitrite oxidizing bacteria, such as Nitrobacter sp. and Nitrospira sp., were significantly lower in the partial nitritation system over the complete nitrification system. The abundance of Planctomycetes was higher in the partial nitritation process, indicating the anammox reaction occurred in the partial nitritation system. These results suggested the nitrite accumulation rate of circulating ratios 75% was the highest, with an average of 92%,and a possibility to treat the low Carbon Nitrogen ratio wastewater using the partial nitritation/anammox process.

Single-stage autotrophic nitrogen removal process at high loading rate: granular reactor performance, kinetics, and microbial characterization.

For the possible highest performance of single-stage combined partial nitritation/anammox (PNA) process, a continuous complete-mix granular reactor was operated at progressively higher nitrogen loading rate. The variations in bacterial community structure of granules were also characterized using high-throughput pyrosequencing, to give a detail insight to the relationship between reactor performance and functional organism abundance within completely autotrophic nitrogen removal system. In 172 days of operation, a superior total nitrogen (TN) removal rate over 3.9 kg N/(m3/day) was stable implemented at a fixed dissolved oxygen concentration of 1.9 mg/L, corresponding to the maximum specific substrate utilization rate of 0.36/day for TN based on the related kinetics modeling. Pyrosequencing results revealed that the genus Nitrosomonas responsible for aerobic ammonium oxidation was dominated on the granule surface, which was essential to offer the required niche for the selective enrichment of anammox bacteria (genus Candidatus Kuenenia) in the inner layer. And the present of various heterotrophic organisms with general functions, known as fermentation and denitrification, could not be overlooked. In addition, it was believed that an adequate excess of ammonium in the bulk liquid played a key role in maintaining process stability, by suppressing the growth of nitrite-oxidizing bacteria through dual-substrate competitions.

Distribution and genetic diversity of microbial populations in the pilot-scale biofilter for simultaneous removal of ammonia, iron and manganese from real groundwater.

A pilot-scale biofilter treating real groundwater was developed in this study, which showed that ammonia, iron and manganese were mainly removed at 0.4, 0.4 and 0.8 m of the filter bed, respectively, and the corresponding removal efficiencies were 90.82%, 95.48% and 95.90% in steady phase, respectively. The variation of microbial populations in the biofilter during start-up process was also investigated using high-throughput pyrosequencing (HTP). Results indicated that the main functional microbes for ammonia, iron and manganese removal were Nitrosomonas, Crenothrix and Crenothrix, respectively, which was mainly distributed at 0.8, 0, and 0.8 m of the filter bed with a corresponding abundance of 8.7%, 28.12% and 11.33% in steady phase, respectively. Kinds of other bacteria which may be related to methane, hydrogen sulfide and organic matter removal, were also found. In addition, small part of archaea was also detected, such as Candidatus Nitrososphaera, which plays a role in nitritation.

Screening and optimizing of inhibitors for ammonia-oxidizing bacteria in sediments of malodorous river.

The proper use of selective ammonia-oxidizing archaea (AOA) and/or ammonia-oxidizing bacteria (AOB) inhibitors is critical to distinguish AOA and AOB contribution. In this research, three inhibitors including ampicillin, dicyandiamide (DCD), and allylthiourea (ATU) were examined mainly focusing on inhibiting dosage, adaptability, and effects. The results showed that the optimized inhibitory dosage of ampicillin, DCD, and ATU was separately 1.5 g L-1, 1 mM, and 25 µM. Among the three inhibitors, ATU exhibited the strongest and persistent inhibition effects and resulted in up to 90% inhibition in the AOB-enriched culture. The seemingly weakening inhibiting effects of ATU in the simulated river systems can be attributed to the involved role of AOA, the uneven spatial distribution of ATU, and protection by sediment structure in complex malodorous rivers. The high-throughput pyrosequencing analysis showed the AOB-related genus Nitrosomonas and Nitrosococcus were mostly affected by ATU in the enrichments and the river systems, respectively. The inhibition of ATU was realized mainly by reducing the abundance and activity of AOB. The decrease of the ratio of AOB/AOA amoA gene copy numbers after addition of ATU further confirmed the inhibiting effectiveness of ATU in complex microbial community of malodorous rivers.

Application of hierarchical oligonucleotide primer extension (HOPE) to assess relative abundances of ammonia- and nitrite-oxidizing bacteria.

BACKGROUND: Establishing an optimal proportion of nitrifying microbial populations, including ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), complete nitrite oxidizers (comammox) and ammonia-oxidizing archaea (AOA), is important for ensuring the efficiency of nitrification in water treatment systems. Hierarchical oligonucleotide primer extension (HOPE), previously developed to rapidly quantify relative abundances of specific microbial groups of interest, was applied in this study to track the abundances of the important nitrifying bacterial populations. RESULTS: The method was tested against biomass obtained from a laboratory-scale biofilm-based trickling reactor, and the findings were validated against those obtained by 16S rRNA gene-based amplicon sequencing. Our findings indicated a good correlation between the relative abundance of nitrifying bacterial populations obtained using both HOPE and amplicon sequencing. HOPE showed a significant increase in the relative abundance of AOB, specifically Nitrosomonas, with increasing ammonium content and shock loading (p < 0.001). In contrast, Nitrosospira remained stable in its relative abundance against the total community throughout the operational phases. There was a corresponding significant decrease in the relative abundance of NOB, specifically Nitrospira and those affiliated to comammox, during the shock loading. Based on the relative abundance of AOB and NOB (including commamox) obtained from HOPE, it was determined that the optimal ratio of AOB against NOB ranged from 0.2 to 2.5 during stable reactor performance. CONCLUSIONS: Overall, the HOPE method was developed and validated against 16S rRNA gene-based amplicon sequencing for the purpose of performing simultaneous monitoring of relative abundance of nitrifying populations. Quantitative measurements of these nitrifying populations obtained via HOPE would be indicative of reactor performance and nitrification functionality.