Nitrifying Community Structure Research Articles

Activity and community structure of nitrifiers and denitrifiers in nitrogen-polluted rivers along a latitudinal gradient

Nitrogen (N) cycling in rivers is particularly active and dynamic due to excess nutrient inputs worldwide. However, the multidimensional spatial patterns of the activity and community structure of N-cycling microorganisms in rivers remain unclear, limiting our understanding of river ecological functions, especially N removal capacity. Here, we measured the nitrification and denitrification rates and identified nitrifying and denitrifying microorganisms using high-throughput sequencing of archaeal amoA, bacterial amoA, nirK, and nirS genes in channel sediments, riparian rhizosphere soils, and riparian bulk soils of 30 N-polluted rivers across China. Results showed that in the lateral dimension, nitrification rates in sediments did not differ significantly from those in rhizosphere and bulk soils, but denitrification rates were higher in sediments than in bulk soils. However, the archaeal amoA gene abundance in sediments was considerably lower than that in rhizosphere and bulk soils, and bacterial amoA gene abundance in sediments was greater than that in rhizosphere soils. In the vertical dimension, both nitrification and denitrification rates in riparian bulk soils decreased with soil depth, and topsoils harbored more nitrifying and denitrifying microbes than subsoils. Denitrification but not nitrification rates increased with latitude and altitude but decreased with increasing mean annual temperature and precipitation. Overall, these results provide new insights into the multidimensional spatial patterns of river N cycling at a large scale, which is crucial to evaluating the N removal function of global rivers.

Comammox plays a functionally important role in the nitrification of rice paddy soil with different nitrogen fertilization levels

Chemoautotrophic ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA), and the recently discovered complete ammonia oxidizers (comammox) catalyze ammonia oxidation; however, quantifying their individual gross ammonia oxidation activities in complex soil ecosystems remains elusive. In this study, we investigated the nitrifying population and community dynamics in response to fertilization in paddy soil under long-term treatment with different urea concentrations (0, 100, 200, and 300 kg N ha−1; designated N0, N100, N200, and N300, respectively). The community structure of nitrifiers, including comammox, was changed by long-term N fertilizers input, which also enhanced AOB abundance and comammox clade A/B abundance ratio, whereas it did not affect AOA abundance. Nevertheless, the microbial population sizes and community compositions remained largely similar following short-term fertilization throughout one rice-growing season. Furthermore, by employing a 15NO3− pool dilution technique in combination with chlorate and 1-octyne inhibition, we conducted a soil microcosm incubation without substrate supplementation to distinguish the relative contribution of comammox to gross ammonia oxidation from those of AOA and AOB. Comammox exhibited higher nitrification activity in soils treated with more N fertilizers, with the contribution increasing from 34 % (N0) to 75 % (N300), although its abundance was not dominant. This could be attributed to the repeated N-based fertilization, which reduced the soil pH, thereby decreasing the soil-free ammonia concentration. In contrast, the contribution of AOA declined from 57 % (N0) to 19 % (N300), while that of AOB only accounted for a minor fraction (<25 %) despite being in high abundance. Altogether, our findings provide evidence that the activity of nitrifying guilds is more valuable than their abundance and provide insights into the potential functional significance of comammox in the nitrification process in rice paddy systems.

Incorporating straw into intensively farmed cropland soil can reduce N2O emission via inhibition of nitrification and denitrification pathways

Straw incorporation (SI) combined with N fertilizer has been shown to affect soil N2O emission and N-related functional microbes in agriculture. However, the responses of N2O emission, community structure of nitrifiers and denitrifiers and related microbial functional genes to straw management strategies in the winter wheat season in China remain unclear. Here, we conducted a two-season experiment in a winter wheat field in Ningjing County, northern China, to examine four treatments: no fertilizer with (N0S1) and without maize straw (N0S0); N fertilizer with (N1S1) and without maize straw (N1S0), and their effects on N2O emissions, soil chemical parameters, crop yield, as well as the dynamics of nitrifying and denitrifying microbial communities. We found that seasonal N2O emissions decreased by 7.1–11.1% (p < 0.05) in N1S1 as compared to N1S0, without significant difference between N0S1 and N0S0. In combination with N fertilization, SI increased the yield by 2.6–4.3%, altered the microbial community composition, increased Shannon and ACE indexes, and decreased the abundance of AOA (9.2%), AOB (32.2%; p < 0.05), nirS (35.2%; p < 0.05), nirK (21.6%; p < 0.05) and nosZ (19.2%). However, in the absence of N fertilizer, SI promoted the major genera of Nitrosavbrio (AOB), unclassifiied_Gammaproteobacteria, Rhodanobacter (nirS), Sinorhizobium (nirK), which strongly correlated positively with N2O emissions. Thereby, a negative interaction effect between SI and N fertilizer on AOB and nirS emphasized that SI could offset the increase of N2O emission caused by fertilization. Soil moisture and NO3− concentration were the major factors affecting N-related microbial community structure. Our study reveals that SI suppressed N2O emission significantly and simultaneously decreased the abundance of N-related functional genes and altered denitrifying bacterial community composition. We conclude that SI helps to enhance yield and alleviate fertilizer-induced environmental costs in intensively farmed fields in northern China.

Partial Nitrification Characteristics of an Immobilized Carrier in Municipal Wastewater under Low-Temperature Shock: The Role of the Nitrifying Bacterial Community Structure

To analyze the effects of the nitrifying bacterial community structure on the partial nitrification (PN) characteristics in a PN-immobilized carrier in municipal wastewater under low-temperature shock, two PN-immobilized carriers with different nitrifying bacterial communities were investigated. The E1-immobilized carrier contained a high abundance of ammonia-oxidizing bacteria (AOB; 38.59%), and the E2-immobilized carrier had a low AOB abundance of 4.78%. The results of experiments with different dissolved oxygen (DO) concentrations showed that the oxygen-limited environment inside the immobilized carrier, generated by the high AOB abundance, was critical for achieving PN. The nitrite accumulation rate (NAR) decreased from 90.0–93.9% to 84.2–88.3% for the E1-immobilized carrier and from 86.0–90.4% to 81.7–85.8% for the E2-immobilized carrier under low-temperature shock (the temperature suddenly decreased from 25 ± 1 °C to 15 ± 1 °C). The decrease in the ammonia oxidation rate due to the decreased AOB activity led to a decrease in NAR. Moreover, NOB abundance in the E2-immobilized carrier increased because of the destruction of the oxygen-limiting region in the immobilized carrier due to the low AOB abundance. Increasing the abundance of AOB in the PN-immobilized carrier could reduce the adverse effects from the low-temperature shock. The results of this study can be used to further develop immobilization technology for efficient PN in mainstream wastewater treatment.

Distinct Nitrification Rates and Nitrifiers in Needleleaf and Evergreen Broadleaf Forest Soils.

Research on niche specialization in the microbial communities of ammonia oxidizers is important for assessing the consequences of vegetation shift on nitrogen (N) cycling. In this study, soils were sampled from three tree stands (needleleaf, mixed, and evergreen broadleaf) from the Hannam experimental forest in South Korea in spring (May 2019), summer (August 2019), autumn (November 2019), and winter (January 2020). Quantitative polymerase chain reaction (qPCR) and high-throughput sequencing were used to measure the abundance and community structure of various nitrifiers: ammonia-oxidizing archaea and bacteria (AOA and AOB, respectively) as well as complete ammonia oxidizers (comammox). Nitrification rates and total ammonia oxidizer abundance were significantly higher in needleleaf forest soil than those in other forest stands, and they were lowest in evergreen broadleaf forest soil. Comammox clade B was most abundant in needleleaf and evergreen broadleaf forest soils, while AOA were significantly more abundant in mixed forest soil. The abundances of comammox clade B and AOA were negatively correlated with dissolved organic carbon. Phylogenetic analysis showed that NT-alpha and NS-gamma-2.3.2 were the most abundant AOA lineages in all the samples. The seasonal of AOA, AOB, and comammox varied with the sites, suggesting the need to examine the combinations of environmental factors when considering the effects of seasonal changes in the environment. Overall, the results suggest that potential vegetation shifts in forest ecosystems might affect nitrification activities by regulating the abundance and community structure of ammonia oxidizers.

Effects of Ionized Water Addition on Soil Nitrification Activity and Nitrifier Community Structure

Water ionization is an efficient physical water treatment technology, and crop water and nutrient use efficiencies can be improved using ionized water for irrigation. In order to explore the effect of ionized water on soil nitrification and nitrifying microorganisms, we conducted a laboratory soil incubation experiment with the addition of ionized water and ordinary water under different soil water contents (equal to 30%, 60%, 100% and 175% of the field capacity, θFC). During the soil incubation, we analyzed soil inorganic nitrogen transformation, ammonia oxidation gene abundances and nitrifying microbial community structure. The results showed that, no matter adding ordinary water or ionized water, the soil nitrification rate and the abundance of ammonia oxidizing bacteria in the 100%θFC treatment were significantly higher than those in other water conditions, while the abundance of ammonia oxidizing archaea was not affected by the soil water content. With the same soil water content, the nitrification rate of ionized water treatment was stronger than that of the ordinary water treatment. Although the absolute abundance of ammonia-oxidizing microorganisms in ionized water treatment was significantly lower than that of ordinary water (p &lt; 0.05), the relative abundance of some dominant nitrifying microbial genera in the ionized water treatment was significantly higher (p &lt; 0.05). The dominant genera may play a key role in the nitrification process. The results show that ionized water irrigation can significantly promote the nitrification of silt loam soil, especially under 100%θFC conditions, and may regulate soil nitrification by affecting some dominant nitrifying microorganisms. This study provides a theoretical basis for understanding the biological regulation mechanism of ionized water irrigation on soil nutrient transformation and for application of ionized water to field irrigation.

Modification of nitrifying microbial community via DC electrical field application

The performance and operational stability of nitrifying-based treatment processes are closely associated with microbial community structures and species-to-species interactions. In this work, we investigated the effect of intermittent direct current (DC) on the change of nitrifying community structure and process performance in a membrane electro-bioreactor (MEBR). Microbial populations and dynamics were characterized using a fluorescent in situ hybridization (FISH) technique. The outcomes demonstrated that the MEBR system achieved substantial nitrification rates compared to a conventional membrane bioreactor (MBR). The MEBR enhanced the removal of NH3+−N to almost 99%. FISH analysis indicated a dominance of different nitrifying bacteria species in both reactors, leading to differences in nitrification and NH3+−N removal performance. In robust MEBR system, rapid nitrifiers, such as AOB (Nitrosomonas spp.) and NOB (Nitrobacter spp.) were dominat. In contrast, slower nitrifier consortiums like AOB (Nitrosospira spp.) and NOB (Nitrospira spp.) occupied the control MBR. Taken together, the development of fast and robust nitrifying community can lead to extra savings in capital and operational costs of the WWTPs, suggesting the adoption of MEBR oxic/anoxic process. Moreover, a better understanding of biological processes taking place in the new MEBR system and microbial environmental preferences would be valuable for process control and optimization, as well as beneficial for designing bioaugmentation processes, in order to avoid potential washout of target bacteria not adapted to real conditions in bioreactors. Such bio-electrically enhanced processes presented in this work can be used in a new WWTP design or retrofitting existing treatment plants.

Sustained organic loading disturbance favors nitrite accumulation in bioreactors with variable resistance, recovery and resilience of nitrification and nitrifiers

Sustained disturbances are relevant for environmental biotechnology as they can lead to alternative stable states in a system that may not be reversible. Here, we tested the effect of a sustained organic loading alteration (food-to-biomass ratio, F:M, and carbon-to-nitrogen ratio, C:N) on activated sludge bioreactors, focusing on the stability of nitrification and nitrifiers. Two sets of replicate 5-L sequencing batch reactors were operated at different, low and high, F:M (0.19–0.36 mg COD/mg TSS/d) and C:N (3.5–6.3 mg COD/mg TKN) conditions for a period of 74 days, following 53 days of sludge acclimation. Recovery and resilience were tested during the last 14 days by operating all reactors at low F:M and C:N (henceforth termed F:M–C:N). Stable nitrite accumulation (77%) was achieved through high F:M–C:N loading with a concurrent reduction in the abundance of Nitrospira. Subsequently, only two of the three reactors experiencing a switch back from high to low F:M–C:N recovered the nitrite oxidation function, with an increase in Nitrobacter as the predominant NOB, without a recovery of Nitrospira. The AOB community was more diverse, resistant and resilient than the NOB community. We showed that functional recovery and resilience can vary across replicate reactors, and that nitrification recovery need not coincide with a return to the initial nitrifying community structure.

An efficient oxic-anoxic process for treating low COD/N tropical wastewater: Startup, optimization and nitrifying community structure

In this study, we assessed and optimized a low-dissolved-oxygen oxic-anoxic (low-DO OA) process to achieve a low-cost and sustainable solution for wastewater treatment systems in the developing tropical countries treating low chemical oxygen demand-to-nitrogen ratio (COD/N) wastewater. The low-DO OA process attained complete ammonia removal and the effluent nitrate nitrogen (NO3–N) was below 0.3 mg/L. The recommended hydraulic retention time and sludge retention time (SRT) were 16 h and 20 days, respectively. The 16S rRNA sequencing data revealed that long SRT (20 days) encouraged the growth of nitrite-oxidizing bacteria (NOB) affiliated with “Candidatus Nitrospira defluvii”. Comammox made up 10–20% of the Nitrospira community. NOB and comammox related to Nitrospira were enriched at long SRT (20 days) to achieve good low-DO nitrification performance. The low-DO OA process was efficient and has simpler design than conventional processes, which are keys for sustainable wastewater treatment systems in the developing countries treating low COD/N wastewater.

Geospatial variation in co-occurrence networks of nitrifying microbial guilds.

Microbial communities transform nitrogen (N) compounds, thereby regulating the availability of N in soil. The N cycle is defined by interacting microbial functional groups, as inorganic N‐products formed in one process are the substrate in one or several other processes. The nitrification pathway is often a two‐step process in which bacterial or archaeal communities oxidize ammonia to nitrite, and bacterial communities further oxidize nitrite to nitrate. Little is known about the significance of interactions between ammonia‐oxidizing bacteria (AOB) and archaea (AOA) and nitrite‐oxidizing bacterial communities (NOB) in determining the spatial variation of overall nitrifier community structure. We hypothesize that nonrandom associations exist between different AO and NOB lineages that, along with edaphic factors, shape field‐scale spatial patterns of nitrifying communities. To address this, we sequenced and quantified the abundance of AOA, AOB, and Nitrospira and Nitrobacter NOB communities across a 44‐hectare site with agricultural fields. The abundance of Nitrobacter communities was significantly associated only with AOB abundance, while that of Nitrospira was correlated to AOA. Network analysis and geostatistical modelling revealed distinct modules of co‐occurring AO and NOB groups occupying disparate areas, with each module dominated by different lineages and associated with different edaphic factors. Local communities were characterized by a high proportion of module‐connecting versus module‐hub nodes, indicating that nitrifier assemblages in these soils are shaped by fluctuating conditions. Overall, our results demonstrate the utility of network analysis in accounting for potential biotic interactions that define the niche space of nitrifying communities at scales compatible to soil management.

Ammonia oxidizers and nitrite-oxidizing bacteria respond differently to long-term manure application in four paddy soils of south of China

Nitrification plays an important role in the soil nitrogen (N) cycle, and fertilizer application may influence soil nitrifiers' abundance and composition. However, the effect of long-term manure application in paddy soils on nitrifying populations is poorly understood. We chose four long-term manure experimental fields in the south of China to study how the abundance and community structure of nitrifiers would change in response to long-term manure application using quantitative PCR and Miseq sequencing analyses. Our results showed that manure application significantly increased ammonia oxidizing archaea (AOA) abundance at the ChangSha (CS) and NanChang (NC) sites, while the abundance of ammonia oxidizing bacteria (AOB) represented 4.8- and 12.8- fold increases at the JiaXing (JX) and YingTan (YT) sites, respectively. Miseq sequencing of 16S rRNA genes indicated that manure application altered the community structure of nitrifying populations, especially at the NC and YT sites. The application of manure significantly changed AOA and nitrite oxidizing bacteria (NOB) community structures but not those of AOB, suggesting that AOA and NOB may be more sensitive to manures. Variation partitioning analysis (VPA) and redundancy analysis (RDA) indicated that soil pH, TN, NO3−-N and water content were the main factors in shaping nitrifying communities. These findings suggest that nitrifiers respond diversely to manure application, and soil physiochemical properties play an important role in determining nitrifiers' abundance and communities with long-term manure addition.

Effect of Flow Rate Increase on the Performance of a Pilot-Scale Biological Nutrient Removal Reactor

AbstractNutrient removal in two identical pilot-scale reactors is assessed at influent flow rates of 30, 45, and 60 L/min. The bacteria community structure of nitrifiers and denitrifiers and the p...

Low temperature effects on nitrification and nitrifier community structure in V-ASP for decentralized wastewater treatment and its improvement by bio-augmentation.

The vegetation-activated sludge process (V-ASP) has been proved to be an environment-friendly decentralized wastewater treatment system with extra esthetic function and less footprint. However, the effects of low temperature on the treatment performance of V-ASP and related improvement methods are rarely investigated, up to now. In this work, the effect of low temperature on nitrification in V-ASP was comprehensively investigated from overall nitrification performance, substrate utilization kinetics, functional enzymatic activities, and microbial community structure shift by comparison with conventional ASP. Bio-augmentation methods in terms of single-time nitrifier-enriched biomass dosage were employed to improve nitrification efficiency in bench- and full-scale systems. The experiment results demonstrated that the NH4+-N removal efficiency in V-ASP system decreased when the operational temperature decreased from 30 to 15°C, and the decreasing extent was rather smaller compared to ASP, as well as ammonium and nitrite oxidation rates and enzymatic activities, which indicated the V-ASP system possesses high resistance to low temperature. With direct dosage of 1.6mg nitrifier/gSS sludge, the nitrification efficiency in V-ASP was enhanced dramatically from below 50% to above 90%, implying that bio-augmentation was effective for V-ASP whose enzymatic activities and microbial communities were both also improved. The feasibility and effectiveness of bio-augmentation was further confirmed in a full-scale V-ASP system after a long-term experiment which is instructive for the practical application.

Long term operation at low dissolved oxygen conditions results in Comammox dominated nitrifying community structure

Long term operation at low dissolved oxygen conditions results in Comammox dominated nitrifying community structure

Impacts of continuous excessive fertilization on soil potential nitrification activity and nitrifying microbial community dynamics in greenhouse system

Sampling and analysis of greenhouse soils were conducted in Shouguang, China, to study continuous excessive fertilization effect on nitrifying microbial community dynamics in greenhouse environment. Potential nitrification activity (PNA), abundance, and structure of nitrifying microbial communities as well as the correlations with soil properties were investigated. Short-term excessive fertilization increased soil nutrient contents and the diversity of nitrifying microbial communities under greenhouse cultivation. However, the abundance and diversity of nitrifying communities decreased greatly due to the increase of soil acidity and salinity after 14 years of high fertilization in greenhouse. There was a significant positive correlation between soil PNA and the abundance of ammonia-oxidizing bacteria (AOB) but not that of ammonia-oxidizing archaea (AOA) in topsoil (0–20 cm) when pH ≥7. Soil PNA and AOB were strongly influenced by soil pH. The groups of Nitrososphaeraceae, Nitrosomonadaceae, and Nitrospiraceae were predominant in the AOA, AOB, and nitrite-oxidizing bacteria (NOB) communities, respectively. Nitrifying community structure was significantly correlated with soil electrical salinity (EC), organic carbon (OC), and nitrate nitrogen (NO3 −–N) content by redundancy analysis (RDA). Nitrification was predominated by AOB in greenhouse topsoil with high fertilizer loads. Soil salinity, OC, NO3 −–N content, and pH affected by continuous excessive fertilization were the major edaphic factors in shaping nitrifying community structure in greenhouse soils.

Editorial: Linking Ecosystem Function to Microbial Diversity.

Editorial: Linking Ecosystem Function to Microbial Diversity.

Dynamics of the diversity and structure of the overall and nitrifying microbial community in activated sludge along gradient copper exposures.

Diversity and composition of the microbial community, especially the nitrifiers, are essential to the treatment efficiency of wastewater in activated sludge systems. Heavy metals commonly present in the wastewater influent such as Cu can alter the community structure of nitrifiers and lower their activity. However, the dynamics of microbial community along a gradient of metal exposure have largely been unexplored, partially due to the limitations in traditional molecular methods. This study explored the dynamics regarding the diversity and community structures of overall and nitrifying microbial communities in activated sludge under intermittent Cu gradient loadings using Illumina sequencing. We created a new local nitrifying bacterial database for sequence BLAST searches. High Cu loadings (>10.9mg/L) impoverished microbial diversity and altered the microbial community. Overall, Proteobacteria was the predominant phylum in the activated sludge system, in which Zoogloea, Thauera, and Dechloromonas (genera within the Rhodocyclaceae family of the Beta-proteobacteria class) were the dominant genera in the presence of Cu. The abundance of unclassified bacteria at the phylum level increased substantially with increasing Cu loadings. Nitrosomonas and Nitrospira were the predominant nitrifiers. The nitrifying bacterial community changed through increasing abundance and shifting to Cu-tolerant species to reduce the toxic effects of Cu. Our local nitrifying bacterial database helped to improve the resolution of bacterial identification. Our results provide insights into the dynamics of microbial community in response to various metal concentrations in activated sludge systems and improve our understanding regarding the effect of metals on wastewater treatment efficiency.

Impact of plant harvest management on function and community structure of nitrifiers and denitrifiers in a constructed wetland.

Plant harvest is one of the most important management practices in constructed wetlands. In this study, we evaluated the impact of harvesting Phragmites australis (Cav.) Trin. ex Steudel on the activity and community structure of nitrifiers and denitrifiers in a free-water surface constructed wetland. The nitrifiers were targeted using bacterial and archaeal-amoA that encode ammonia monooxygenase, and the denitrifiers were targeted using nirK and nirS that encode the nitrite reductase. The community structures were evaluated using denaturing gradient gel electrophoresis. The potential nitrification and nitrate reduction rates were shown to be significantly higher in the harvested plant rhizosphere than in a non-harvested control plot. The potential nitrification rate positively correlated with the potential nitrate reduction rate and influenced the community structure of nirK. In addition, plant canopy developed differently after harvest and simultaneously changed the microclimate beneath the plant community. These results suggest that plant harvest management could change subsequent plant development and associated microenvironments, thereby affecting the function and community structure of nitrifiers and denitrifiers. Our study highlights the importance of plant harvest management within constructed wetlands to regulate the functions of nitrification and denitrification.

Integration of microbial fuel cell techniques into activated sludge wastewater treatment processes to improve nitrogen removal and reduce sludge production

Bioelectrochemical systems are emerging for wastewater treatment, yet little is known about how well they can be integrated with current wastewater treatment processes. In this bench-scale study, the microbial fuel cell (MFC) technique was incorporated into the Modified Ludzack–Ettinger (MLE) process (phase I) and later with the membrane bioreactor (MBR) process (phase II) to evaluate the performance of MFC assisted wastewater treatment systems (i.e., MLE–MFC and MBR–MFC). There was no significant difference in the effluent NH4+–N concentration between the systems integrating MFC and the open circuit controls. The average effluent COD concentration was significantly lower in the MLE–MFC, but it did not change much in the MBR–MFC because of the already low COD concentrations in MBR operation. The MLE–MFC and MBR–MFC systems increased the NO3−–N removal efficiencies by 31% (±12%) and 20% (±12%), respectively, and reduced sludge production by 11% and 6%, respectively, while generating an average voltage of 0.13 (±0.03) V in both systems. Analysis of the bacterial specific oxygen uptake rate, the sludge volume index, and ammonia-oxidizing bacterial population (dominated by Nitrosomonas through terminal restriction fragment length polymorphism analysis) indicated that there was no significant difference in sludge activity, settling property, and nitrifying community structure between the MFC assisted systems and the open circuit controls. The results suggest that the wastewater treatment systems could achieve higher effluent water quality and lower sludge production if it is integrated well with MFC techniques.

Analysis and succession of nitrifying bacteria community structure in sequencing biofilm batch reactor

To reveal the succession procedure of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) community structure in sequencing biofilm batch reactor (SBBR), the molecular biological techniques of denaturing gradient gel electrophoresis (DGGE), cloning, and real-time PCR were applied. DGGE showed that the structural diversity of the bacterial community increased during the biofilm formation period, and some kinds of populations had been highly preponderant consistently. The results of cloning and sequencing revealed that Nitrosomonas was the dominant species. The real-time PCR analysis indicated that the amount of the AOB increased significantly after the cultivation period, and the NOB gradually decreased. The AOB content on the 25th day was 17 times that of the 6th day. It also showed the biofilm formed successfully with accumulating nitrite and prepared to achieve the achievement of simultaneous nitrification and denitrification in SBBR. Furthermore, the ammonia-oxidizing rate was in correspondence with the NH4 (+)-N removal efficiency.