Study of bacterial population dynamics in seed culture developed for ammonia reduction from synthetic wastewater.

The waterbodies have been polluted by various natural and anthropogenic activities. The aquatic waste includes ammonia as one of the most toxic pollutants. Several biological treatment systems involving anoxic and semi anoxic bacteria have been proposed for reducing nitrogen loads from wastewater and increasing the efficiency and cost effectiveness. These bacteria play a vital role in the processes involved in the nitrogen cycle in nature. However, the enrichment, sustainability and identification of bacterial communities for wastewater treatment is an important aspect. Most of the chemolithotrophs are unculturable hence their identification and measurement of abundance remains a challenging task. In this study the different bacteria involved in total nitrogen removal from the wastewater are enriched for 700 days under anoxic condition. The synthetic wastewater containing 0.382 g/L of ammonium chloride was used. Molecular identification of the bacteria involved in various steps of the nitrogen cycle was carried out based on amplification of functional genes and 16S rRNA gene Polymerase chain reaction followed by DNA sequencing. Change in the abundance of chemolithotrophs was studied using qPCR. The mutual growth of various nitrifiers along with anaerobic bacteria were identified by molecular characterisation of DNA at various time intervals with the different genes involved in the nitrogen cycle. Nitrosomonas species like Nitrosomonas europaea were identified throughout the batch scale studies possessing the genes associated with ammonia oxidizing bacteria and nitrite oxidizing bacteria which act as a complete ammonia oxidizer. The uncultured species of Nitrospira and anammox bacteria were also observed which predicts the coexistence of the anammox and comammox bacteria in a batch scale study. The coexistence of the semi anoxic and anoxic bacteria helped in the growth of these bacteria for a longer duration of time. The nitrite produced by the comammox during nitrification can be utilized by anammox as an electron carrier. The other species of denitrifiers like Pseudomonas denitrificans and Aminobacter aminovorans were also observed. It is concluded that the enrichment of semi anoxic and anoxic bacteria was faster with the increase in growth of the bacteria involved in nitrification, comammox, anammox and partial denitrification process. The bacterial growth is enhanced and the efficiency is increased which can be further used in the development of small pilot scale bioreactor for total nitrogen removal.

Feasibility of low-intensity ultrasound treatment with hydroxylamine to accelerate the initiation of partial nitrification and allow operation under intermittent aeration.

Partial nitrification is a key aspect of efficient nitrogen removal, although practically it suffers from long start-up cycles and unstable long-term operational performance. To address these drawbacks, this study investigated the effect of low intensity ultrasound treatment combined with hydroxylamine (NH2OH) on the performance of partial nitrification. Results show that compared with the control group, low-intensity ultrasound treatment (0.10 W/mL, 15 min) combined with NH2OH (5 mg/L) reduced the time required for partial nitrification initiation by 6 days, increasing the nitrite accumulation rate (NAR) and ammonia nitrogen removal rate (NRR) by 20.4% and 6.7%, respectively, achieving 96.48% NRR. Mechanistic analysis showed that NH2OH enhanced ammonia oxidation, inhibited nitrite-oxidizing bacteria (NOB) activity and shortened the time required for partial nitrification initiation. Furthermore, ultrasonication combined with NH2OH dosing stimulated EPS (extracellular polymeric substances) secretion, increased carbonyl, hydroxyl and amine functional group abundances and enhanced mass transfer. In addition, 16S rRNA gene sequencing results showed that ultrasonication-sensitive Nitrospira disappeared from the ultrasound + NH2OH system, while Nitrosomonas gradually became the dominant group. Collectively, the results of this study provide valuable insight into the enhancement of partial nitrification start-up during the process of wastewater nitrogen removal.

Assessing the activity of different plant-derived molecules and potential biological nitrification inhibitors on a range of soil ammonia- and nitrite-oxidizing strains.

IMPORTANCE: Synthetic nitrification inhibitors are routinely used with nitrogen fertilizers to reduce nitrogen losses from agroecosystems, despite having drawbacks like poor efficiency, cost, and entry into the food chain. Plant-derived BNIs constitute a more environmentally conducive alternative. Knowledge on the activity of BNIs to soil nitrifiers is largely based on bioassays with a single Nitrosomonas europaea strain which does not constitute a dominant member of the community of ammonia-oxidizing microorganisms (AOM) in soil. We determined the activity of several plant-derived molecules reported as having activity, including the recently discovered maize-isolated BNI, zeanone, and its natural analog, 2-methoxy-1,4-naphthoquinone, on a range of ecologically relevant AOM and one nitrite-oxidizing bacterial culture, expanding our knowledge on the intrinsic inhibition potential of BNIs toward AOM and highlighting the necessity for a deeper understanding of the effect of BNIs on the overall soil microbiome integrity before their further use in agricultural settings.

Physiological adaptation and population dynamics of a nitrifying sludge exposed to ampicillin.

Antibiotics in wastewater treatment plants can alter the physiological activity and the structure of microbial communities through toxic and inhibitory effects. Physiological adaptation, kinetic, and population dynamics behavior of a nitrifying sludge was evaluated in a sequential batch reactor (SBR) fed with 14.4 mg/L of ampicillin (AMP). The addition of AMP did not affect ammonium consumption (100 mg NH4+-N/L) but provoked nitrite accumulation (0.90 mg NO2--N formed/mg NH4+-N consumed) and an inhibition of up to 67% on the nitrite oxidizing process. After 30 cycles under AMP feeding, the sludge recovered its nitrite oxidizing activity with a high nitrate yield (YNO3-) of 0.87 ± 0.10 mg NO3--N formed/mg NH4+-N consumed, carrying out again a stable and complete nitrifying process. Increases in specific rate of nitrate production (qNO3-) showed the physiological adaptation of the nitrite oxidizing bacteria to AMP inhibition. Ampicillin was totally removed since the first cycle of addition. Exposure to AMP had effects on the abundance of bacterial populations, promoting adaptation of the nitrifying sludge to the presence of the antibiotic and its consumption. Nitrosomonas and Nitrosospira always remained within the dominant genera, keeping the ammonium oxidizing process stable while an increase in Nitrospira abundance was observed, recovering the stability of the nitrite oxidizing process. Burkholderia, Pseudomonas, and Thauera might be some of the heterotrophic bacteria involved in AMP consumption.

Niche Differentiation Among Canonical Nitrifiers and N2O Reducers Is Linked to Varying Effects of Nitrification Inhibitors DCD and DMPP in Two Arable Soils.

The efficacy of nitrification inhibitors (NIs) dicyandiamide (DCD) and 3,4-dimethylpyrazole phosphate (DMPP) varies with soil types. Understanding the microbial mechanisms for this variation may lead to better modelling of NI efficacy and therefore on-farm adoption. This study addressed the response patterns of mineral nitrogen, nitrous oxide (N2O) emission, abundances of N-cycling functional guilds and soil microbiota characteristics, in relation to urea application with or without DCD or DMPP in two arable soils (an alkaline and an acid soil). The inhibition of nitrification rate and N2O emission by NI application occurred by suppressing ammonia-oxidizing bacteria (AOB) abundances and increasing the abundances of nosZI-N2O reducers; however, abundances of ammonia-oxidizing archaea (AOA) were also stimulated with NIs-added in these two arable soils. DMPP generally had stronger inhibition efficiency than DCD, and both NIs' addition decreased Nitrobacter, while increased Nitrospira abundance only in alkaline soil. N2O emissions were positively correlated with AOB and negatively correlated with nosZI in both soils and AOA only in acid soil. Moreover, N2O emissions were also positively correlated with nirK-type denitrifiers in alkaline soil, and clade A comammox in acid soil. Amendment with DCD or DMPP altered soil microbiota community structure, but had minor effect on community composition. These results highlight a crucial role of the niche differentiation among canonical ammonia oxidizers (AOA/AOB), Nitrobacter and Nitrospira, as well as nosZI- and nosZII-N2O reducers in determining the varying efficacies of DCD and DMPP in different arable soils.

Deregulation of Ribosome Biogenesis in Nitrite-Oxidizing Bacteria Leads to Nitrite Accumulation.

Nitrite (NO2-) accumulation caused by nitrite-oxidizing bacteria (NOB) inhibition in nitrification is a double-edged sword, i.e., a disaster in aquatic environments but a hope for innovating nitrogen removal technology in wastewater treatment. However, little information is available regarding the molecular mechanism of NOB inhibition at the cellular level. Herein, we investigate the response of NOB inhibition on NO2- accumulation established by a side-stream free ammonia treatment unit in a nitrifying reactor using integrated metagenomics and metaproteomics. Results showed that compared with the baseline, the relative abundance and activity of NOB in the experimental stage decreased by 91.64 and 68.66%, respectively, directly resulting in a NO2- accumulation rate of 88%. Moreover, RNA polymerase, translation factors, and aa-tRNA ligase were significantly downregulated, indicating that protein synthesis in NOB was interfered during NO2- accumulation. Further investigations showed that ribosomal proteins and GTPases, responsible for bindings between either ribosomal proteins and rRNA or ribosome subunits, were remarkably downregulated. This suggests that ribosome biogenesis was severely disrupted, which might be the key reason for the inhibited protein synthesis. Our findings fill a knowledge gap regarding the underlying mechanisms of NO2- accumulation, which would be beneficial for regulating the accumulation of NO2- in aquatic environments and engineered systems.

Nitrite Oxidation in Wastewater Treatment: Microbial Adaptation and Suppression Challenges.

Microbial nitrite oxidation is the primary pathway that generates nitrate in wastewater treatment systems and can be performed by a variety of microbes: namely, nitrite-oxidizing bacteria (NOB). Since NOB were first isolated 130 years ago, the understanding of the phylogenetical and physiological diversities of NOB has been gradually deepened. In recent endeavors of advanced biological nitrogen removal, NOB have been more considered as a troublesome disruptor, and strategies on NOB suppression often fail in practice after long-term operation due to the growth of specific NOB that are able to adapt to even harsh conditions. In line with a review of the history of currently known NOB genera, a phylogenetic tree is constructed to exhibit a wide range of NOB in different phyla. In addition, the growth behavior and metabolic performance of different NOB strains are summarized. These specific features of various NOB (e.g., high oxygen affinity of Nitrospira, tolerance to chemical inhibitors of Nitrobacter and Candidatus Nitrotoga, and preference to high temperature of Nitrolancea) highlight the differentiation of the NOB ecological niche in biological nitrogen processes and potentially support their adaptation to different suppression strategies (e.g., low dissolved oxygen, chemical treatment, and high temperature). This review implicates the acquired physiological characteristics of NOB to their emergence from a genomic and ecological perspective and emphasizes the importance of understanding physiological characterization and genomic information in future wastewater treatment studies.

Long-Term Exposure and Effects of rGO-nZVI Nanohybrids and Their Parent Nanomaterials on Wastewater-Nitrifying Microbial Communities.

Single nanomaterials and nanohybrids (NHs) can inhibit microbial processes in wastewater treatment, especially nitrification. While existing studies focus on short-term and acute exposures of single nanomaterials on wastewater microbial community growth and function, long-term, low-exposure, and emerging NHs need to be examined. These NHs have distinctly different physicochemical properties than their parent nanomaterials and, therefore, may exert previously unknown effects onto wastewater microbial communities. This study systematically investigated long-term [∼6 solid residence time [(SRT)] exposure effects of a widely used carbon-metal NH (rGO-nZVI = 1:2 and 1:0.2, mass ratio) and compared these effects to their single-parent nanomaterials (i.e., rGO and nZVI) in nitrifying sequencing batch reactors. nZVI and NH-dosed reactors showed relatively unaffected microbial communities compared to control, whereas rGO showed a significantly different (p = 0.022) and less diverse community. nZVI promoted a diverse community and significantly higher (p < 0.05) biomass growth under steady-state conditions. While long-term chronic exposure (10 mg·L-1) of single nanomaterials and NHs had limited impact on long-term nutrient recovery, functionally, the reactors dosed with higher iron content, that is, nZVI and rGO-nZVI (1:2), promoted faster NH4+-N removal due to higher biomass growth and upregulation of amoA genes at the transcript level, respectively. The transmission electron microscopy images and scanning electron microscopy─energy-dispersive X-ray spectroscopy analysis revealed high incorporation of iron in nZVI-dosed biomass, which promoted higher cellular growth and a diverse community. Overall, this study shows that NHs have unique effects on microbial community growth and function that cannot be predicted from parent materials alone.

Nitrite oxidation in oxygen-deficient conditions during landfill leachate treatment.

Until recently, all known nitrite oxidation occurred in oxygen-rich conditions but now the oxidation of nitrite into nitrate within a low oxygen or anoxic environment has been observed in the ocean. However, this phenomenon is rarely reported in wastewater treatments and its mechanism is unknown. In this study, the partial nitrification and nitrite oxidation were conducted in no enough oxygen in order to remove nitrogen from landfill leachate, save energy, and save money. The results show that the NH4+-N removal efficiency was 99.4%. During phase I of the anaerobic sequential batch reactor (ASBR), no change in Chemical Oxygen Demand (COD) and ammonium were detected. The nitrite concentration decreased from 107 ± 3 mg/L to 0.16 mg/L during 96 h of oxygen- deficiency, while NO3--N increased from 152.5 ± 3 mg/L to 253.65 ± 3 mg/L. The main microorganisms involved in this reaction in the ASBR were Nitrite-Oxidizing Bacteria (NOB), including Nitrospira and Nitrolancea, their relative abundances were 3.56% and 0.13%, respectively. The major NOB (Nitrospira) were confirmed by the further metagenomic binning analysis. This finding shows that nitrite oxidation can occur in oxygen-deficient conditions with specific NOB.

Model study on real-time aeration based on nitrite for effective operation of single-stage anammox.

Anaerobic ammonia oxidation (Anammox) is an innovative technology for cost-efficient nitrogen removal without intensive aeration. However, effective control of the competition between nitrite oxidizing bacteria (XNOB) and Anammox bacteria (XANA) for nitrite is a key challenge for broad applications of single-stage Anammox processes in real wastewater treatment. Therefore, a real-time aeration scheme was proposed to determine dissolved oxygen (DO) based on nitrite concentration for effective control of XNOB growth while maintaining the XANA activity in a single-stage Anammox process. In this study, a non-steady state mathematical model was developed and calibrated using previously reported lab-scale Anammox results to investigate the efficiency of the proposed real-time aeration scheme in enhancing the Anammox process. Based on the calibrated model simulation results, DO of about 0.10 mg-O2/L was found to be ideal for maintaining effective nitrite creation by ammonia oxidizing bacteria (XAOB) while slowing down the growth of XNOB. If DO is too low (e.g., 0.01 mg-O2/L or lower), the overall rate of the ammonia removal is limited due to slow growth of XAOB. On the other hand, high DO (e.g., 1.0 mg-O2/L or higher) inhibits the growth of XANA, resulting in dominancy of XAOB and XNOB. According to the simulation results, nitrite concentration was found to be a rate-limiting parameter on effective nitrogen removal in single-stage Anammox processes. We also found that nitrite concentration can be used as a real-time switch for aeration in a single-stage Anammox process. A schematic aeration method based on real-time nitrite concentration was proposed and examined to control the competition between XANA and XNOB. In the model simulation, the XANA activity was successfully maintained because the schematic aeration prevented an outgrowth of XNOB, allowing energy-efficient nitrogen removal using single-stage Anammox processes.

Effects of Different Land Use Types on Active Autotrophic Ammonia and Nitrite Oxidizers in Cinnamon Soils.

Land use types with different disturbance gradients show many variations in soil properties, but the effects of different land use types on soil nitrifying communities and their ecological implications remain poorly understood. Using 13CO2-DNA-based stable isotope probing (DNA-SIP), we examined the relative importance and active community composition of ammonia-oxidizing archaea (AOA) and bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in soils under three land use types, forest, cropland, and greenhouse vegetable soil, representing three interference gradients. Soil net nitrification rate was in the order forest soil > cropland soil > greenhouse vegetable soil. DNA-SIP showed that active AOA outcompeted AOB in the forest soil, whereas AOB outperformed AOA in the cropland and greenhouse vegetable soils. Cropland soil was richer in NOB than in AOA and AOB. Phylogenetic analysis revealed that ammonia oxidation in the forest soil was predominantly catalyzed by the AOA Nitrosocosmicus franklandus cluster within the group 1.1b lineage. The 13C-labeled AOB were overwhelmingly dominated by Nitrosospira cluster 3 in the cropland soil. The active AOB Nitrosococcus watsonii lineage was observed in the greenhouse vegetable soil, and it played an important role in nitrification. Active NOB communities were closely affiliated with Nitrospira in the forest and cropland soils, and with Nitrolancea and Nitrococcus in the greenhouse vegetable soil. Canonical correlation analysis showed that soil pH and organic matter content significantly affected the active nitrifier community composition. These results suggest that land use types with different disturbance gradients alter the distribution of active nitrifier communities by affecting soil physicochemical properties. IMPORTANCE Nitrification plays an important role in the soil N cycle, and land use management has a profound effect on soil nitrifiers. It is unclear how different gradients of land use affect active ammonia-oxidizing archaea and bacteria and nitrite-oxidizing bacteria. Our research is significant because we determined the response of nitrifiers to human disturbance, which will greatly improve our understanding of the niche of nitrifiers and the differences in their physiology.

Light Irradiation Enables Rapid Start-Up of Nitritation through Suppressing nxrB Gene Expression and Stimulating Ammonia-Oxidizing Bacteria.

Nitritation facilitates the application of anaerobic ammonium oxidation (Anammox)-based processes for cost-efficient nitrogen removal from wastewater. This study proposed light irradiation as a novel strategy to rapidly start up nitritation by stimulating both the activities and growth of ammonia-oxidizing bacteria (AOB) while suppressing that of nitrite-oxidizing bacteria (NOB). Batch assays and kinetic model jointly suggested that AOB activity presented an initial increase followed by a decline while NOB decreased continuously throughout the light energy densities applied. Under optimal light energy densities (0.03-0.08 kJ/mg VSS), the highest nitrite accumulation ratio of 70.0% was achieved in sequencing batch reactors with both mainstream online and sidestream offline light treatments when treating real or synthetic municipal wastewater. Light irradiation induced different responses of AOB and NOB, leading to microbial structure optimization. Specifically, the expression of nxrB was downregulated, while the expression of amoA was upregulated under appropriate light irradiation. Moreover, although Nitrosomonas as typical AOB disappeared, the family Nitrosomonadaceae was doubled with enrichment of Ellin6067 and another four Nitrosomonadaceae genera that were only identified in light-treated reactors, thus ensuring AOB predominance and stable nitritation. These findings offer a new approach to rapidly establishing nitritation using light irradiation in municipal wastewater, especially for nitritation/microalgae system.

Relevance of Candidatus Nitrotoga for nitrite oxidation in technical nitrogen removal systems.

Many biotechnological applications deal with nitrification, one of the main steps of the global nitrogen cycle. The biological oxidation of ammonia to nitrite and further to nitrate is critical to avoid environmental damage and its functioning has to be retained even under adverse conditions. Bacteria performing the second reaction, oxidation of nitrite to nitrate, are fastidious microorganisms that are highly sensitive against disturbances. One important finding with relevance for nitrogen removal systems was the discovery of the mainly cold-adapted Cand. Nitrotoga, whose activity seems to be essential for the recovery of nitrite oxidation in wastewater treatment plants at low temperatures, e.g., during cold seasons. Several new strains of this genus have been recently described and ecophysiologically characterized including genome analyses. With increasing diversity, also mesophilic Cand. Nitrotoga representatives have been detected in activated sludge. This review summarizes the natural distribution and driving forces defining niche separation in artificial nitrification systems. Further critical aspects for the competition with Nitrospira and Nitrobacter are discussed. Knowledge about the physiological capacities and limits of Cand. Nitrotoga can help to define physico-chemical parameters for example in reactor systems that need to be run at low temperatures. KEY POINTS: • Characterization of the psychrotolerant nitrite oxidizer Cand. Nitrotoga • Comparison of the physiological features of Cand. Nitrotoga with those of other NOB • Identification of beneficial environmental/operational parameters for proliferation.

Abundance, community structure and diversity of nitrifying bacterial enrichments from low and high saline brackishwater environments.

The study reports diversity in nitrifying microbial enrichments from low (0·5-5‰) and high (18-35‰) saline ecosystems. Microbial community profiling of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) enrichments was analysed by sequencing 16S rRNA and was processed using Mothur pipeline. The α-diversity indices showed the richness of nitrifying bacterial consortia from the high saline environment and were clustering based on the source of the sample. AOB and NOB enrichments from both the environments showed diverse lineages of phyla distributed in both groups with 38 and 34 phyla from low saline and 53 and 40 phyla in high saline sources, respectively. At class level, α- and γ-proteobacteria were found to be more dominant in both the enrichments. AOBs and NOBs in enrichments from low saline environments were dominated by Nitrosomonadaceae, Gallionellaceae (Nitrotoga sp.) and Ectothiorhodospiraceae and Nitrospira, respectively. Though Chromatiaceae were present in both AOB and NOB enrichments, Nitrosoglobus and Nitrosococcus dominated the AOBs while NOBs were dominated by uncultured genera, whereas Rhizobiales were found in both the enrichments. AOBs and NOBs in enrichments from high saline environments were dominated by Nitrospira-like AOBs, Nitrosomonas and Nitrosococcus genera, whereas ammonia-oxidizing archaea (AOA) group included Nitrosopumilus and Nitrososphaera genera comprising and Nitrospirae, respectively. The majority of the genera obtained in both the salinities were found to be either uncultured or unclassified groups. Results of the study suggest that the AOB and NOB consortia have unique and diverse microbes in each of the enrichments, capable of functioning in aquaculture systems practised at different salinities (0-60 ppt).

Use of Newly Designed Primers for Quantification of Complete Ammonia-Oxidizing (Comammox) Bacterial Clades and Strict Nitrite Oxidizers in the Genus Nitrospira.

Complete ammonia-oxidizing (comammox) bacteria play key roles in environmental nitrogen cycling and all belong to the genus Nitrospira, which was originally believed to include only strict nitrite-oxidizing bacteria (sNOB). Thus, differential estimation of sNOB abundance from that of comammox Nitrospira has become problematic, since both contain nitrite oxidoreductase genes that serve as common targets for sNOB detection. Herein, we developed novel comammox Nitrospira clade A- and B-specific primer sets targeting the α-subunit of the ammonia monooxygenase gene (amoA) and a sNOB-specific primer set targeting the cyanase gene (cynS) for quantitative PCR (qPCR). The high coverage and specificity of these primers were checked by use of metagenome and metatranscriptome data sets. Efficient and specific amplification with these primers was demonstrated using various environmental samples. Using the newly designed primers, we successfully estimated the abundances of comammox Nitrospira and sNOB in samples from two chloramination-treated drinking water systems and found that, in most samples, comammox Nitrospira clade A was the dominant type of Nitrospira and also served as the primary ammonia oxidizer. Compared with other ammonia oxidizers, comammox Nitrospira had a higher abundance in process water samples in these two drinking water systems. We also demonstrated that sNOB can be readily misrepresented by an earlier method, calculated by subtracting the comammox Nitrospira abundance from the total Nitrospira abundance, especially when the comammox Nitrospira proportion is relatively high. The new primer sets were successfully applied to comammox Nitrospira and sNOB quantification, which may prove useful in understanding the roles of Nitrospira in nitrification in various ecosystems.IMPORTANCENitrospira is a dominant nitrite-oxidizing bacterium in many artificial and natural environments. The discovery of complete ammonia oxidizers in the genus Nitrospira prevents the use of previously identified primers targeting the Nitrospira 16S rRNA gene or nitrite oxidoreductase (nxr) gene for differential determination of strict nitrite-oxidizing bacteria (sNOB) in the genus Nitrospira and among comammox bacteria in this genus. We designed three novel primer sets that enabled quantification of comammox Nitrospira clades A and B and sNOB with high coverage, specificity, and accuracy in various environments. With the designed primer sets, sNOB and comammox Nitrospira were differentially estimated in drinking water systems, and we found that comammox clade A predominated over sNOB and other ammonia oxidizers in process water samples. Accurate quantification of comammox Nitrospira and sNOB by use of the newly designed primers will provide essential information for evaluating the contribution of Nitrospira to nitrification in various ecosystems.

Evolution of microbial dynamics with the introduction of real seawater portions in a low-strength feeding anammox process.

The salinity effect on anammox bacteria has been widely reported; however, rare studies describe the microbial dynamics of anammox-based process response to the introduction of real seawater at mainstream conditions. In this study, an anammox process at mainstream conditions without pre-enriching anammox bacteria was shifted to the feeds of a synthetic wastewater with a portion of seawater mixture. It achieved over 0.180 kg-N/(m3 day) of nitrogen removal rate with an additional seawater proportion of 20% in the influent. The bacterial biodiversity was significantly increased with the increase of seawater proportions. High relative abundance of anammox bacteria (34.24-39.92%) related to Ca. Brocadia was enriched and acclimated to the saline environment. However, the introduction of seawater caused the enrichment of nitrite-oxidizing Ca. Nitrospira, which was responsible for the deterioration of nitrogen removal efficiency. Possible adaptation metabolisms in anammox bacteria and other nitrogen transforming bacteria are discussed. These results highlight the importance of microbial diversity for anammox process under the saline environments of 20% and 40% seawater composition.

Responses of nitrification performance, triclosan resistome and diversity of microbes to continuous triclosan stress in activated sludge system.

Triclosan (TCS) is commonly found in wastewater treatment plants, which often affects biological treatment processes. The responses of nitrification, antibiotic resistome and microbial community under different TCS concentrations in activated sludge system were evaluated in this study. The experiment was conducted in a sequencing batch reactor (SBR) for 240 days. Quantitative PCR results demonstrated that the abundance of ammonium oxidizing bacteria could be temporarily inhibited by 1 mg/L TCS and then gradually recovered. And the abundances of nitrite oxidizing bacteria (NOB) under 2.5 and 4 mg/L TCS were three orders of magnitude lower than that of seed sludge, which accounted for partial nitrification. When the addition of TCS was stopped, the abundance of NOB increased. The mass balance experiments of TCS demonstrated that the primary removal pathway of TCS changed from adsorption to biodegradation as TCS was continuously added into the SBR system. Moreover, TCS increased the abundance of mexB, indicating the efflux pump might be the main TCS-resistance mechanism. As a response to TCS, bacteria could secrete more protein (PN) than polysaccharide. Three-dimensional excitation-emission matrix revealed that tryptophan PN-like substances might be the main component in PN to resist TCS. High-throughput sequencing found that the relative abundances of Paracoccus, Pseudoxanthomonas and Thauera increased, which could secrete extracellular polymeric substances (EPS). And Sphingopyxis might be the main TCS-degrading bacteria. Overall, TCS could cause partial nitrification and increase the relative abundances of EPS-secreting bacteria and TCS-degrading bacteria.

Low Temperature and Neutral pH Define "Candidatus Nitrotoga sp." as a Competitive Nitrite Oxidizer in Coculture with Nitrospira defluvii.

Nitrification is an essential process for N removal in activated sludge to avoid toxicity of ammonium and nitrite. Besides Nitrospira, "Candidatus Nitrotoga" has been identified as a key nitrite-oxidizing bacterium (NOB) performing the second step of nitrification, nitrite oxidation to nitrate, in wastewater treatment plants (WWTPs). However, the driving forces for the dominance of Nitrotoga in certain plants have often remained unclear and could not be explained solely by temperature effects. In this study, we characterized the physiology of the ammonium-dependent Nitrotoga sp. BS with regard to temperature and pH variations and evaluated its competitiveness against Nitrospira defluvii Both NOB originated from the same WWTP and shared a comparable pH optimum of 7.3. Based on these results, coculturing experiments with these NOB were performed in batch reactors operated at either 17°C or 22°C to compare their abundances under optimal (pH 7.4) or suboptimal (pH 6.4) conditions using 1 mM nitrite. As revealed by quantitative PCR (qPCR), fluorescence in situ hybridization (FISH), and 16S amplicon sequencing, Nitrotoga sp. BS was clearly favored by its optimal growth parameters and dominated over Ns. defluvii at pH 7.4 and 17°C, whereas a pH of 6.4 was more selective for Ns. defluvii Our synthetic communities revealed that niche differentiation of NOB is influenced by a complex interaction of environmental parameters and has to be evaluated for single species.IMPORTANCE "Ca. Nitrotoga" is a NOB of high environmental relevance, but physiological data exist for only a few representatives. Initially, it was detected in specialized niches of low temperature and low nitrite concentrations, but later on, its ubiquitous distribution revealed its critical role for N removal in engineered systems like WWTPs. In this study, we analyzed the competition between Nitrotoga and Nitrospira in bioreactors and identified conditions where the K strategist Ns. defluvii was almost replaced by Nitrotoga sp. BS. We show that the pH value is an important factor that regulates the composition of the nitrite-oxidizing enrichment with a dominance of Nitrotoga sp. BS versus Ns. defluvii at a neutral pH of 7.4 in combination with a temperature of 17°C. The physiological diversity of novel Nitrotoga cultures improves our knowledge about niche differentiation of NOB with regard to functional nitrification under suboptimal conditions.

Complete nitrification: insights into the ecophysiology of comammox Nitrospira.

Nitrification, the oxidation of ammonia via nitrite to nitrate, has been considered to be a stepwise process mediated by two distinct functional groups of microorganisms. The identification of complete nitrifying Nitrospira challenged not only the paradigm of labor division in nitrification, it also raises fundamental questions regarding the environmental distribution, diversity, and ecological significance of complete nitrifiers compared to canonical nitrifying microorganisms. Recent genomic and physiological surveys identified factors controlling their ecology and niche specialization, which thus potentially regulate abundances and population dynamics of the different nitrifying guilds. This review summarizes the recently obtained insights into metabolic differences of the known nitrifiers and discusses these in light of potential functional adaptation and niche differentiation between canonical and complete nitrifiers.

Effect of graphene oxide on the bioactivities of nitrifying and denitrifying bacteria in aerobic granular sludge.

With the widespread application of graphene oxide (GO), it would be inevitably released into wastewater treatment plants (WWTPs) and get involved in the biochemical process. So far, there are controversies on the effects of low GO concentration (0.05-0.1 g/L) on the nitrogen removal process. Therefore, this study essentially investigates any potential effects of GO on wastewater microbial communities functions. In present study, the nitrifying and denitrifying batch tests were introduced to investigate the influence of 0.06 g/L of GO on bacteria. The results showed that GO could be easily combined with the aerobic granular sludge (AGS), and NH4+-N was sharply absorbed, which directly promoted the bioactivities of ammonium oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) and extracellular polymeric substances (EPS) production. The influence of GO on the denitrifying bacteria was negligible, which resulted in the stable EPS production. Furthermore, as inferred from the near maximum chemical reaction rates, there were no obvious changes on the microbial community functions during nitrogen removal process.