Aerobic Ammonium-oxidizing Bacteria Research Articles

Spatial Patterns of Soil Bacterial Communities and N-cycling Functional Groups Along an Altitude Gradient in Datong River Basin

The altitude distribution patterns of soil microorganisms and their driving mechanisms are crucial for understanding the consequences of climate change on terrestrial ecosystems. There is an obvious altitude difference in Datong River Basin in the Qilian Mountains. Two spatial scale transections were set up along the mountain slope (with altitude spanning 1 000 m) and the mainstream direction (with altitude spanning 300-500 m), respectively. The distribution characteristics of the soil bacterial community structure and diversity along the altitude gradients were examined using high-throughput sequencing technology. Based on the FAPROTAX database, the altitude distribution patterns of nitrogen cycling functional groups were analyzed to investigate the major environmental factors influencing the altitude distribution patterns of soil bacterial communities. The findings revealed that:① Soil physicochemical characteristics varied significantly with altitude. The content of total nitrogen (TN) and nitrate nitrogen (NO3-) were positively correlated with the altitude (P < 0.01), whereas the soil bulk density and pH were negatively connected (P < 0.001). ② The abundance of OTU increased significantly along the altitude (P < 0.01), and the richness and diversity indices increased along the altitude, although the trend was not statistically significant (P > 0.05). ③ The predominant bacterial communities were Acidobacteria, Proteobacteria, and Bacteroidetes, and as altitude climbed, their relative abundances varied between increasing, decreasing, and slightly decreasing, respectively. ④ The nitrogen cycling processes involved 13 functional groups, primarily nitrification, aerobic ammonia oxidation, aerobic nitrite oxidation, etc. As the altitude increased, the response law changed, with an increase in the abundance of nitrobacteria (P < 0.01), a slight increase in the abundance of aerobic ammonia-oxidizing bacteria and nitrite-oxidizing bacteria, and a hump-back tendency in bacteria abundance for nitrogen respiration. ⑤ Redundancy analysis revealed that the key determinants influencing soil bacterial populations at the phylum level were altitude, pH, and the content of NH4+. Mantel analysis showed that the dominant groups of soil bacterial nitrogen cycling were all statistically and significantly driven by altitude (P < 0.01). ⑥ The α-diversity of the bacterial community with increasing altitude were both increased along the mountain slope and the mainstream direction, but the soil properties, the abundance of N-cycling functional groups, and the main environmental factors differed. Therefore, it is of great significance to explore the altitude distribution pattern of soil microorganisms at different spatial scales.

Biofilm colonization and succession in a full-scale partial nitritation-anammox moving bed biofilm reactor

BackgroundPartial nitritation-anammox (PNA) is a biological nitrogen removal process commonly used in wastewater treatment plants for the treatment of warm and nitrogen-rich sludge liquor from anaerobic digestion, often referred to as sidestream wastewater. In these systems, biofilms are frequently used to retain biomass with aerobic ammonia-oxidizing bacteria (AOB) and anammox bacteria, which together convert ammonium to nitrogen gas. Little is known about how these biofilm communities develop, and whether knowledge about the assembly of biofilms in natural communities can be applied to PNA biofilms.ResultsWe followed the start-up of a full-scale PNA moving bed biofilm reactor for 175 days using shotgun metagenomics. Environmental filtering likely restricted initial biofilm colonization, resulting in low phylogenetic diversity, with the initial microbial community comprised mainly of Proteobacteria. Facilitative priority effects allowed further biofilm colonization, with the growth of initial aerobic colonizers promoting the arrival and growth of anaerobic taxa like methanogens and anammox bacteria. Among the early colonizers were known ‘oligotrophic’ ammonia oxidizers including comammox Nitrospira and Nitrosomonas cluster 6a AOB. Increasing the nitrogen load in the bioreactor allowed colonization by ‘copiotrophic’ Nitrosomonas cluster 7 AOB and resulted in the exclusion of the initial ammonia- and nitrite oxidizers.ConclusionsWe show that complex dynamic processes occur in PNA microbial communities before a stable bioreactor process is achieved. The results of this study not only contribute to our knowledge about biofilm assembly and PNA bioreactor start-up but could also help guide strategies for the successful implementation of PNA bioreactors.8NUh31NTQCwxAXZWHM2SHSVideo

An effective strategy for rapid nitrite accumulation related to quorum sensing and the impact of quorum quenching enzyme

Nitrite accumulation is crucial in nitrogen removal processes that treat wastewater with a low carbon-to-nitrogen (C/N) ratio. This study investigates the effectiveness of effluent reflux in accelerating nitrite accumulation, considering quorum sensing, and explores the impact of quorum quenching enzymes. The results demonstrate that effluent reflux successfully accelerates the nitrite accumulation rate to 92.2% and increases the abundance of Nitrosomonas from 0.2% to 19.5%, as well as hydroxylamine oxidoreductase (HAO) from 0.285 Eu g−1 SS to 0.475 Eu g−1 SS. The quenching enzyme suppresses the growth of aerobic ammonia-oxidizing bacteria, leading to a decrease in Nitrosomonas abundance to 4.2% and HAO to 0 Eu g−1 SS. However, it enhances denitrification, which consumes nitrite and results in the deterioration of nitrite accumulation. Consequently, the total nitrogen (TN) removal efficiency increases from 15.5% to 43.2%, and the abundance of Hyphomicrobium increases to 33.7% accordingly. Quorum sensing proves to be an effective strategy for regulating ammonia removal and nitrite accumulation, and effluent reflux has a positive effect on enhancing quorum sensing.

Using sulfide as nitrite oxidizing bacteria inhibitor for the successful coupling of partial nitrification-anammox and sulfur autotrophic denitrification in one reactor

The partial nitrification-anammox (PNA) process, as a new “green” biological nitrogen removal technology, is completely autotrophic and has great economic and environmental benefits. However, the applied bottleneck of this process is the inability to inhibit nitrite oxidizing bacteria (NOB) activity and the low total nitrogen removal efficiency (TNRE) due to nitrate accumulation. In this study, the addition of sulfide was utilized to selectively screen NOB while promoting the sulfur autotrophic denitrification (SAD) process in one sequencing batch biofilter granular reactor. The results showed that when the influent concentration of NH4+-N and S2− were 184.6 ± 4.4 mg/L and 153.9 ± 3.1 mg/L, the average TNRE reached 89.6 ± 3.6 %. At this stage, aerobic ammonia oxidizing bacteria (AOB) and anaerobic ammonium oxidation bacteria (AnAOB) showed good activities (specific oxygen uptake rate of AOB and specific anammox activity could reach to 0.09 ± 0.00 mg O2/g SS/h and 0.47 ± 0.06 mg TN/g SS/h, respectively), while NOB activity was significantly suppressed by sulfide, with specific oxygen uptake rate of 0.003 ± 0.00 mg O2/g SS/h, which further demonstrating the feasibility and long-term application of this sustainable and efficient process. Anammox was the predominant nitrogen removal pathway contributing approximately 89.0 % of nitrogen removal. Typical AOB (Nitrosomonas), AnAOB (Candidatus Kuenenia), and SAD bacteria (Thiobacillus, Pseudoxanthomonas, and Limobacter) coexisted in the PNA-SAD system. The syntrophic interactions among sulfur-oxidizing, sulfate-reducing, and anammox bacterial populations of this system were revealed by PICRUST2 and network analysis. Sulfides were most likely oxidized to biologically produced elemental sulfur first and then converted to sulfate via the Sox system. Our study provided a new reaction scheme and theoretical reference for efficient nitrogen and sulfur removal in a single reactor.

Performance and microbial characteristics of completely autotrophic nitrogen removal over nitrite process in different types of reactors

There are various types of reactors for completely autotrophic nitrogen removal over nitrite (CANON) process, but the advantages and disadvantages of different types of reactors and their scope of application still need to be studied. In this paper, high-throughput sequencing technology was used to analyze the microbial characteristics of four CANON reactors with similar and stable influent water quality conditions, and the applicable scenarios of different types of CANON reactors was clarified. At the gene level, the proportions of aerobic ammonia oxidizing bacteria (AerAOB), anaerobic ammonia oxidation bacteria (AnAOB) and denitrifying bacteria in a flocculent sludge sequencing batch reactor (FSBR), a membrane bioreactor (MBR), a biological aerated filter (BAF) and a granular sludge sequencing batch reactor (GSBR) were 16.88 %, 3.49 %, 19.36 % and 29.67 %; 9.51 %, 4.72 %, 5 % and 27.75 %; 7.12 %, 11.5 %, 6.81 % and 13.6 %. The results showed that FSBR was low nitrogen removal load and low stability in practical application. MBR can treat different concentrations of ammonia nitrogen wastewater by controlling the influent load. BAF can treat ammonia nitrogen wastewater with moderate influent load. GSBR has high removal load and can treat high ammonia nitrogen wastewater.

Response of partial nitritation/anammox granules to biodegradable organic input in bacterial community and functions

Biodegradable organic input is considered as a critical factor influencing the stability of the partial nitritation/anammox (PN/A) process for autotrophic nitrogen removal. In this study, the response of PN/A granules to stepwise increasing the organic loading rate (OLR) was investigated in a continuous-flow reactor, according to the nitrogen removal activity, sludge morphology, bacterial community composition and nitrogen cycle pathways. Results show that the increased OLR from 0.47 to 3.36 kg·m−3·d−1 with different influent organic and nitrogen (COD/N) ratios from 0.25 to 1.0 caused the specific nitrogen removal rate of the granules to decrease by 60.9 %, which was accompanied by a looser granular structure and poor settling property. Inorganic feeding was then used to recover the performance of the granules, and the removal of total inorganic nitrogen rose to 78.4 % at a nitrogen loading rate of 3.36 kg·m−3·d−1. Based on high-throughput sequencing, biodegradable organics shaped the bacterial community structure in the granules by enriching symbiotic heterotrophs (such as Chloroflexi, Proteobacteria and Bacteroidetes) and washing out the anammox biomass. Candidatus Kuenenia dominated the anammox biomass, while Candidatus Brocadia was also found, which coordinated with aerobic ammonium-oxidizing bacteria (Nitrosomonas) in the PN/A process. Metagenomic analysis indicates that biodegradable organic stress and performance recovery significantly changed the composition of nitrogen cycle-related genes in the granules, in terms of a lower capacity of anammox and higher risk of nitrous oxide emission. These findings provide insight into the self-adaption strategy of PN/A granules in response to biodegradable organic input into wastewater treatment engineering applications.

Regulating performance of CANON process via adding external quorum sensing signal molecules in membrane bioreactor

In this study, the regulation effect of the external quorum sensing signals, N-dodecanoyl homoserine lactone (C12-HSL) on CANON process were investigated in a membrane bioreactor. C12-HSL significantly enhanced the aerobic ammonia-oxidizing bacteria and improved the ammonia monooxygenase activity to 0.134 from 0.076 μg NO2−-N mg−1 protein min−1, while suppressed anaerobic ammonia-oxidizing bacteria and limited the TN removal to 0.07 from 0.22 kg m−3 d-1. Key enzymes synthesis were enhanced during the operation without C12-HSL addition, enabling the resistance of CANON system to high C12-HSL. As a result, the hydroxylamine oxidoreductase and nitrite reductase activity reached 35.9 EU g−1 SS and 1.28 μg NO2−-N mg−1 protein min−1, respectively; Nitrosomonas and Candidatus Kuenenia, with the abundance as 12.5 % and 22.9 %, cooperatively contributed to the TN removal, which maintained at 0.19 kg m−3 d-1. C12-HSL was profitable for aerobic ammonia oxidation, which could be adopted for regulating the nitrite production rate.

Influence of low air pressure on combined nitritation and anaerobic ammonium oxidation process

At high altitude, wastewater aeration efficiency is low, which is detrimental to nitrification in conventional biological nitrogen removal. The combined partial nitritation and anaerobic ammonium oxidation (CPNA) process requires little oxygen and can be appropriate in low-pressure conditions. As such, in this study, we investigated the effect of air pressure on CPNA using a laboratory-scale reactor. We found that low air pressure promoted the removal of total inorganic nitrogen (TIN), achieving a TIN removal rate of 43,000 mg·N/(kg·VSS·d). The secretion of extracellular polymeric substances under low air pressure was not significantly different from that under ordinary air pressure, indicating no adverse effects on microbial aggregation ability, stability, or settleability. The abundance of aerobic ammonia-oxidizing bacteria (AeAOB) increased from 0.2% to 5.6%, and the activity of anaerobic ammonia-oxidizing bacteria (AnAOB) enhanced, giving AeAOB and AnAOB a competitive advantage over nitrite-oxidizing bacteria, thus forming a microbial community structure favorable to the CPNA process. Our further analysis of the results of batch tests in serum bottles confirmed the positive effect of low air pressure on the anaerobic ammonium oxidation (anammox) process, with a 28.5% ± 1.9% improvement in the specific anammox rate at 70 kPa compared with 100 kPa. AnAOB activity increased, which was reflected in the intracellular heme content increasing from 0.56 ± 0.18 μmol/(g·VSS) at 100 kPa to 2.56 ± 0.20 μmol/(g·VSS) at 70 kPa. We clarified the CPNA-process-promoting effect of low air pressure, which shows potential for nitrogen removal in high-altitude regions.

Microbial response on the first full-scale DEMON® biomass transfer for mainstream deammonification

Sidestream partial nitritation and deammonification (pN/A) of high-strength ammonia wastewater is a well-established technology. Its expansion to the mainstream is, however mainly impeded by poor retention of anaerobic ammonia oxidizing bacteria (AnAOB), insufficient repression of nitrite oxidizing bacteria (NOB) and difficult control of soluble chemical oxygen demand and nitrite levels.At the municipal wastewater treatment plant in Strass (Austria) the microbial consortium was exhaustively monitored at full-scale over one and a half year with regular transfer of sidestream DEMON® biomass and further retention and enrichment of granular anammox biomass via hydrocyclone operation.Routine process parameters were surveyed and the response and evolution of the microbiota was followed by molecular tools, ex-situ activity tests and further, AnAOB quantification through particle tracking and heme measurement.After eight months of operation, the first anaerobic, simultaneous depletion of ammonia and nitrite was observed ex-situ, together with a direction to higher nitrite generation (68% of total NOx-N) as compared to nitrate under aerobic conditions. Our dissolved oxygen (DO) scheme allowed for transient anoxic conditions and had a strong influence on nitrite levels and the NOB community, where Nitrobacter eventually dominated Nitrospira. The establishment of a minor but stable AnAOB biomass was accompanied by the rise of Chloroflexi and distinct emergence of Chlorobi, a trend not seen in the sidestream system. Interestingly, the most pronounced switch in the microbial community and noticeable NOB repression occurred during unfavorable conditions, i.e. the cold winter season and high organic load. Further abatement of NOB was achieved through bioaugmentation of aerobic ammonia oxidizing bacteria (AerAOB) from the sidestream-DEMON® tank. Performance of the sidestream pN/A was not impaired by this operational scheme and the average volumetric nitrogen removal rate of the mainstream even doubled in the second half of the monitoring campaign. We conclude that a combination of both, regular sidestream-DEMON® biomass transfer and granular SRT increase via hydrocyclone operation was crucial for AnAOB establishment within the mainstream.

Metagenomic Insights Into Competition Between Denitrification and Dissimilatory Nitrate Reduction to Ammonia Within One-Stage and Two-Stage Partial-Nitritation Anammox Bioreactor Configurations.

Anaerobic ammonia oxidizing bacteria (Anammox) are implemented in high-efficiency wastewater treatment systems operated in two general configurations; one-stage systems combine aerobic ammonia oxidizing bacteria (AOB) and Anammox within a single aerated reactor, whereas two-stage configurations separate these processes into discrete tanks. Within both configurations heterotrophic populations that perform denitrification or dissimilatory nitrate reduction to ammonia (DNRA) compete for carbon and nitrate or nitrite and can impact reactor performance because DNRA retains nitrogen in the system. Therefore, it is important to understand how selective pressures imposed by one-stage and two-stage reactor configurations impact the microbial community structure and associated nitrogen transforming functions. We performed 16S rRNA gene and metagenomic sequencing on different biomass fractions (granules, flocs, and suspended biomass) sampled from two facilities treating sludge dewatering centrate: a one-stage treatment facility (Chambers Creek, Tacoma, WA) and a two-stage system (Rotterdam, Netherlands). Similar microbial populations were identified across the different samples, but relative abundances differed between reactor configurations and biomass sources. Analysis of metagenome assembled genomes (MAGs) indicated different lifestyles for abundant heterotrophic populations. Acidobacteria, Bacteroidetes, and Chloroflexi MAGs had varying capacity for DNRA and denitrification. Acidobacteria MAGs possessed high numbers of glycosyl hydrolases and glycosyl transferases indicating a role in biomass degradation. Ignavibacteria and Phycosphaerae MAGs contributed to the greater relative abundance of DNRA associated nrf genes in the two-stage granules and contained genomic features suggesting a preference for an anoxic or microoxic niche. In the one-stage granules a MAG assigned to Burkholderiales accounted for much of the abundant denitrification genes and had genomic features, including the potential for autotrophic denitrification using reduced sulfur, that indicate an ability to adapt its physiology to varying redox conditions. Overall, the competition for carbon substrates between denitrifying and DNRA performing heterotrophs may be impacted by configuration specific selective pressures. In one-stage systems oxygen availability in the bulk liquid and the oxygen gradient within granules would provide a greater niche space for heterotrophic populations capable of utilizing both oxygen and nitrate or nitrite as terminal electron acceptors, compared to two-stage systems where a homogeneous anoxic environment would favor heterotrophic populations primarily adapted to anaerobic metabolism.

Characterization of Sludge Morphology and Bacterial Community Evolution in the Rapid Activation of Freeze-stored PN/A Granular Sludge

Inoculating granular sludge is an alternative method for the quick start-up of a high-performance autotrophic nitrogen removal reactor. In order to establish the response relationship between sludge activation and reactor performance, the freeze-stored granular sludge was inoculated into a continuous-flow reactor, and a control strategy of the high loading rate and high hydraulic selective pressure was carried out in this study. As a result, a one-stage partial nitritation/ANAMMOX process was started up in 34 days, and the removal efficiency of total nitrogen was over 83%, with a removal loading rate of total nitrogen of 1.67 kg·(m3·d)-1. During this period, the Image pro-plus software was employed to analyze the evolution of the characteristic dimensions of particles. A good linear positive correlation (R2=0.988) between the projected area of the erythrine zone in the inner layer and the specific nitrogen removal rate of granules was found, which provide a simple method to estimate the activity of the PN/A granules. The results of MiSeq high-throughput sequencing showed that the enrichment of aerobic ammonia-oxidizing bacteria (Nitrosomonas) and the wash-out of heterotrophic bacteria (such as Denitratisoma and Haliangium, etc.) were achieved in the start-up of the reactor. Meanwhile, the improvement in granular compactness was in favor of activating anaerobic ammonia oxidizing bacteria (Candidatus_Kuenenia, abundance>30%) that colonized the inner layer of the granules.

Multiscale modelling of the start-up process of anammox-based granular reactors.

This work proposes a mathematical model on partial nitritation/anammox (PN/A) granular bioreactors, with a particular interest in the start-up phase. The formation and growth of granular biofilms is modelled by a spherical free boundary problem with radial symmetry and vanishing initial value. Hyperbolic PDEs describe the advective transport and growth of sessile species inhabiting the granules. Parabolic PDEs describe the diffusive transport and conversion of soluble substrates, and the invasion process mediated by planktonic species. Attachment and detachment phenomena are modelled as continuous and deterministic fluxes at the biofilm-bulk liquid interface. The dynamics of planktonic species and substrates within the bulk liquid are modelled through ODEs. A simulation study is performed to describe the start-up process of PN/A granular systems and the development of anammox granules. The aim is to investigate the role that the invasion process of anaerobic ammonia-oxidizing (anammox) bacteria plays in the formation of anammox granules and explore how it affects the microbial species distribution of anaerobic ammonia-oxidizing, aerobic ammonia-oxidizing, nitrite-oxidizing and heterotrophic bacteria. Moreover, the model is used to study the role of two key parameters in the start-up process: the anammox inoculum size and the inoculum addition time. Numerical results confirm that the model can be used to simulate the start-up process of PN/A granular systems and to predict the evolution of anammox granular biofilms, including the ecology and the microbial composition. In conclusion, after being calibrated, the proposed model could provide quantitatively reliable results and support the start-up procedures of full-scale PN/A granular reactors.

Millimeter-scale vertical partitioning of nitrogen cycling in hypersaline mats reveals prominence of genes encoding multi-heme and prismane proteins

Microbial mats are modern analogues of the first ecosystems on the Earth. As extant representatives of microbial communities where free oxygen may have first been available on a changing planet, they offer an ecosystem within which to study the evolution of biogeochemical cycles requiring and inhibited by oxygen. Here, we report the distribution of genes involved in nitrogen metabolism across a vertical oxygen gradient at 1 mm resolution in a microbial mat using quantitative PCR (qPCR), retro-transcribed qPCR (RT-qPCR) and metagenome sequencing. Vertical patterns in the presence and expression of nitrogen cycling genes, corresponding to oxygen requiring and non-oxygen requiring nitrogen metabolism, could be seen across gradients of dissolved oxygen and ammonium. Metagenome analysis revealed that genes annotated as hydroxylamine dehydrogenase (proper enzyme designation EC 1.7.2.6, hao) and hydroxylamine reductase (hcp) were the most abundant nitrogen metabolism genes in the mat. The recovered hao genes encode hydroxylamine dehydrogenase EC 1.7.2.6 (HAO) proteins lacking the tyrosine residue present in aerobic ammonia oxidizing bacteria (AOB). Phylogenetic analysis confirmed that those proteins were more closely related to ɛHao protein present in Campylobacterota lineages (previously known as Epsilonproteobacteria) rather than oxidative HAO of AOB. The presence of hao sequences related with ɛHao protein, as well as numerous hcp genes encoding a prismane protein, suggest the presence of a nitrogen cycling pathway previously described in Nautilia profundicola as ancestral to the most commonly studied present day nitrogen cycling pathways.

Storage without nitrite or nitrate enables the long-term preservation of full-scale partial nitritation/anammox sludge

Bioaugmentation with summer harvested sludge during winter could compensate for bacterial activity loss but requires that sludge activity can be restored after storage. This study assesses the effect of temperature and redox adjustment during the storage over 180 days of partial nitritation/anammox (PN/A) granular resp. floccular sludge from potato processing resp. sludge reject water treatment. Anoxic storage conditions (in the presence of nitrite or nitrate and the absence of oxygen) resulted in a loss of 80–100% of the anammox bacteria (AnAOB) activity capacity at 20 °C and 4 °C, while anaerobic conditions (without oxygen, nitrite, and nitrate) lost only 45–63%. Storage at 20 °C was more cost-effective compared to 4 °C, and this was confirmed in the sludge reactivation experiment (20 °C). Furthermore, AnAOB activity correlated negatively with the electrical conductivity level (R2 > 0.85, p < 0.05), so strong salinity increases should be avoided. No significant differences were found in the activity capacity of aerobic ammonia-oxidizing bacteria (AerAOB) under different storage conditions (p > 0.1). The relative abundance of dominant AnAOB (Candidatus Brocadia) and AerAOB genera (Nitrosomonas) remained constant in both sludges. In conclusion, preserving PN/A biomass without cooling and nitrite or nitrate addition proved to be a cost-effective strategy.

Comparisons of the characteristics of partial nitrification sludge exposed to CuO nanoparticles combined with different antibiotics

Nanoparticles (NPs) and antibiotics are currently two of the main emerging pollutants that can significantly impact wastewater treatment processes. In this study, the joint effects of CuO NPs with the antibiotic sulfamethoxazole (SMX) or oxytetracycline (OTC) on partial nitrification (PN) sludge were investigated. After long-term exposure, the gene transcripts abundance of aerobic ammonia-oxidizing bacteria (AOB), specific AOB activity (SAA), and extracellular polymers (EPS) were detected and compared. Both CuO NPs+SMX and CuO NPs+OTC treatment exhibited severe inhibition of the PN sludge, which ultimately decreased SAA to 2.693 (±0.127) and 3.710 (±0.099) mg g−1 SS h−1 from 5.372 mg g−1 SS h−1 under the control conditions, while AOB abundance was 0.622 (±0.071) and 0.817 (±0.088), respectively. EPS, which is a pre-indicator of the reactor performance, was the key factor that enabled the PN sludge in resisting NPs and antibiotics, but the recovery ability of PN sludge after the stress was poor.

Effects of Cold Acclimation on the Activity of Autotrophic Nitrogen Removal in Granular Sludge and Its Bacterial Population Structure

Cold acclimation is an effective approach for improving the nitrogen removal performance and operational stability of partial nitritation/ANAMMOX (PN/A) combined processes at low temperatures. To explore the specific effects of cold acclimation on the characteristics of sludge, differentiations in temperature sensitivity, granular morphology, composition of extracellular polymer substance (EPS), and bacterial community structure between PN/A granular sludges cultivated at medium-high temperature (30℃) and acclimated to low temperature (15℃) were investigated in this study. The results of reaction thermodynamics showed that the nitrogen removal performance of the granules acclimated to low temperature (GL) was significantly higher than that of those cultivated at medium-high temperature (GH) under the low temperature (10-20℃), and the apparent activation energy (Ea) of total inorganic nitrogen removal for the former was decreased by 28.4%. Compared with GH, GL had a smaller average particle size of 25.8% and higher EPS contents of 16.6%, resulting in a significant lower settling property. Based on the high-throughput sequencing results, GL exhibited a higher diversity of bacterial community, and a lower relative abundance ratio (0.04) of aerobic ammonium-oxidizing bacteria (Nitrosomonas) and anaerobic ammonium-oxidizing bacteria (Candidatus_Kuenenia) than 0.34 for GH. It indicated that the PN/A granules held a strong ability to retain slow-growing autotrophic bacteria in the system, even under low temperatures. These findings could provide meaningful references for analyzing the self-adaption mechanisms of PN/A sludge to low temperature conditions and promote the industrial application of combined processes.

A simple and sensitive direct mRNA multiplexed detection strategy for amoA-targeted monitoring of ammonia-oxidizing activity in water environment

Nitrogenous pollutants are the main causes of water eutrophication, which greatly interfere with the balance of the aquatic ecosystem. The biological nitrogen removal using bacteria is the leading technology to control nitrogenous pollutants. The α-subunit (amoA) of ammonia monooxygenase (AMO) of aerobic ammonia-oxidizing bacteria (AOB) is an ammonia-depleting biomarker in nitrogen removal processes. Currently, RT-PCR and metatranscriptome are the general methods for analyzing amoA gene expression, however they can introduce amplification bias and are time consuming. Herein we report a new direct mRNA multiplexed detection strategy based on xMAP array, for analyzing gene expression without reverse transcription and amplification steps. After analyzing all amoA sequences in database, we design a universal probe for AOB with coverage of 79.8% whose high coverage is further confirmed by metatranscriptome. Moreover, we classify AOB into 10 clusters, and then design the specific probes of each cluster. This technique enables single-nucleotide, wide linear dynamic range (5–50,000 amol) and high sensitivity (5 amol with detection volume of 1 µL) detection with using locked-nucleic-acid probes. Particularly, the sensitivity is by far among the highest level for multiplex mRNA detection without amplification. We found that mRNA fragmentation is not necessary in our direct detection, simplifying our strategy. We have successfully detected amoA mRNA semi-quantitatively and determined the predominant cluster in sludge from wastewater treatment plants, which was consistent with RT-PCR and metatranscriptome results. Meanwhile, the ammonia-oxidizing activities of sludge verified the amoA mRNA detection results. In conclusion, we have developed a mRNA direct, simple, specific and sensitive multiplexed detection strategy, which shows great potential for application in gene expression analysis, environmental microbial detection and activity diagnosis of wastewater treatment plants.

Partial nitrification performance and microbial community evolution in the membrane bioreactor for saline stream treatment

Effects of salinity level and gradient on partial nitrification performance, sludge properties and microbial activities were investigated using partial nitrification membrane bioreactors (PN-MBRs). PN-MBRs obtained stable nitrite accumulation rate of 91.1% and ammonia removal of 64.8% at 10 g/L NaCl. 10 g/L NaCl obtained higher oxygen uptake rate than 5 g/L, and promoted the differentiation of ammonium-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria. Salinity increased sludge flocs size, stimulated secretion of extracellular polymeric substances with high carbohydrates contents, but had insignificant impact on sludge settleability and dewaterability. Salt level and gradient were both important for microbial community evolution to salt-tolerant bacteria. PN-MBRs enriched aerobic AOBs (Nitrosomonas and norank_f_Nitrosomonadaceae) and anaerobic AOBs (Candidatus_Kuenenia and Candidatus_Brocadia) for partial nitrification, while salt gradients resulted in different metabolism pathways for nitrification even at the same salinity. Increasing salinity promoted hydroxylamine oxidizer in nitrification process evolving from Candidatus_Kuenenia and Candidatus_Brocadia to aerobic AOBs.

Sludge ratio affects the start-up performance and functional bacteria distribution of a hybrid CANON system

To investigate the effect of sludge ratio on the hybrid CANON system, autotrophic nitrogen removal sludge was inoculated with different granule/floc ratios to initiate the CANON system, and maintained the sludge ratio during the operation process. The start-up performances were compared, and the distribution characteristics of functional bacteria were investigated. The results show that the Equivalent system (granules:flocs = 1:1–1:1.5) successfully started-up on day 19, and the nitrogen removal rate (NRR) reached 0.299 kgN m−3·d−1 on day 63. At the same time, it was less affected by the load shock than High-granules and High-flocs systems. Therefore, the Equivalent system had the strongest start-up performance. The activities of the functional bacteria conformed to spatial heterogeneity, unlike the abundance. With the increased floc proportion, the difference in the activity and abundance of anaerobic ammonium-oxidizing bacteria (AAOB) between the granules and flocs increased, while there was a decrease in the difference in aerobic ammonium-oxidizing bacteria (AOB). However, the abundance of Nitrosomonas in the granules was higher than in the flocs when the proportion of flocs was higher than 50%. This study provides new ideas and insights for the fast start-up of the CANON system and can conform to the varying needs of engineering applications.

Optimization of nitrogen and carbon removal with simultaneous partial nitrification, anammox and denitrification in membrane bioreactor.

In this study, a membrane bioreactor (MBR) was used to achieve both nitrogen and carbon removal by a simultaneous partial nitrification, anammox and denitrification (SNAD) process. During the entire experiment, the intermittent aeration (non-aerobic time : aeration time, min min−1) cycle was controlled by a time-controlled switch, and the aeration rate was controlled by a gas flowmeter, and the optimal operating parameters as determined by response surface methodology (RSM) were a C/N value of 1.16, a DO value of 0.84 mg l−1 and an aerobic time (Tae) of 15.75 min. Under these conditions, the SNAD process achieved efficient and stable nitrogen and carbon removal; the total inorganic nitrogen removal efficiency and chemical oxygen demand removal efficiency were 92.31% and 95.67%, respectively. With the formation of granular sludge, the membrane fouling rate decreased significantly from 35.0 Pa h−1 at SNAD start-up to 19.9 Pa h−1 during stable operation. Fluorescence in situ hybrid analyses confirmed the structural characteristics and the relative ratio of aerobic ammonia-oxidizing bacteria, anaerobic ammonia-oxidizing bacteria and denitrifying bacteria in the SNAD system.