Start-up and inhibition analysis of the Anammox process seeded with anaerobic granular sludge

The longer start-up period of the Anammox process is due to the very low cellular yield and growth rates of Anammox bacteria. Nitrite inhibition is considered to be the key factor in the instability of the Anammox process during the operation. However, little attention was paid to the inhibitory effect of pH and free ammonia. This paper presents start-up and inhibition analysis of an Anammox biofilm reactor seeded with anaerobic granular sludge. Results showed that the start-up period could be divided into the sludge lysis phase, lag phase, propagation phase, stationary phase and inhibition phase. Optimization control could be implemented correspondingly to accelerate the start-up of Anammox bioreactors. Effluent pH increased to 8.7–9.1 when the nitrogen removal rate was higher than 1,200 mg l⁻¹ day⁻¹. The free ammonia concentration was accompanied with a higher level of 64–73 mg l⁻¹. Inhibitory effects of high pH and free ammonia on Anammox bacteria contributed to the destabilization of the Anammox bioreactor during the first 125 days with influent KHCO₃ of 0.5 g l⁻¹. Increasing the suffering capacity in the inlet by dosing 1.25 g KHCO₃ l⁻¹ effectively reduced the pH variation, and the nitrogen removal performance of the reactor was further developed.     

Reactivation of effluent granular sludge from a high-rate Anammox reactor after storage

In this study, effluent sludge from a high-rate Anammox reactor was used to re-start new Anammox reactors for the reactivation of Anammox granular sludge. Different start-up strategies were evaluated in six upflow anaerobic sludge blanket (UASB) reactors (R₁–R₆) for their effect on nitrogen removal performance. Maximal nitrogen removal rates (NRRs) greater than 20 kg N/m³/day were obtained in reactors R₃–R₅, which were seeded with mixed Anammox sludge previously stored for approximately 6 months and 1 month. A modified Boltzmann model describing the evolution of the NRR fit the experimental data well. An amount of sludge added to the UASB reactor or decreasing the loading rate proved effective in relieving the substrate inhibition and increasing the NRR. The modified Stover–Kincannon model fit the nitrogen removal data in the Anammox reactors well, and the simulation results showed that the Anammox process has great nitrogen removal potential. The observed inhibition in the Anammox reactors may have been caused by high levels of free ammonia. The sludge used to seed the reactors did not settle well; sludge flotation was observed even after the reactors were operated for a long time at a floating upward velocity (F_s) of greater than 100 m/h. The settling sludge, however, exhibited good settling properties. Scanning electron microscopy showed that the Anammox granules consisted mainly of spherical and elliptical bacteria with abundant filaments on their surface. Hollows in the granules were also present, which may have contributed to sludge floatation.     

Effect of seed sludge microbial community and activity on the performance of anaerobic reactors during the start-up period

In this study, two laboratory-scale anaerobic batch reactors started up with different inoculum sludges and fed with the same synthetic wastewater were monitored in terms of performance and microbial community shift by denaturant gradient gel electrophoresis fingerprinting and subsequent cloning, sequencing analysis in order to reveal importance of initial quality of inoculum sludge for operation of anaerobic reactors. For this purpose, two different seed sludge were evaluated. In Reactor1 seeded with a sludge having less diverse microbial community (19 operational taxonomic unit (OTU’s) for Bacterial and 8 OTU’s for Archaeal community, respectively) and a methanogenic activity of 150 ml CH₄ g TVS⁻¹ day⁻¹, a chemical oxygen demand (COD) removal efficiency of 78.8 ± 4.17% was obtained at a substrate to microorganism (S/X) ratio of 0.38. On the other hand, Reactor2, seeded with a sludge having a much more diverse microbial community (24 OTU’s for Bacterial and 9 OTU’s for Archaeal communities, respectively) and a methanogenic activity, 450 ml CH₄ g TVS⁻¹ day⁻¹, operated in the same conditions showed a better start-up performance; a COD removal efficiency of over 98% at a S/X ratio of 0.53. Sequence analysis of Seed2 revealed the presence of diverse fermentative and syntrophic bacteria, whereas excised bands of Seed1 related to fermentative and sulfate/metal-reducing bacteria. This study revealed that a higher degree of bacterial diversity, especially the presence of syntrophic bacteria besides the abundance of key species such as methanogenic Archaea may play an important role in the performance of anaerobic reactors during the start-up period.     

Enrichment and granulation of Anammox biomass started up with methanogenic granular sludge

A sequencing batch reactor (SBR) seeded with methanogenic granular sludge was started up to enrich Anammox (Anaerobic Ammonium Oxidation) bacteria and to investigate the feasibility of granulation of Anammox biomass. Research results showed that hydraulic retention time (HRT) was an important factor to enrich Anammox bacteria. When the HRT was controlled at 30 days during the initial cultivation, the SBR reactor presented Anammox activity at t = 58 days. Simultaneously, the methanogenic granular sludge changed gradually from dust black to brown colour and its diameter became smaller. At t = 90 days, the Anammox activity was further improved. NH₄⁺-N and NO₂⁻N were removed simultaneously with higher speed and the maximum removal rates reached 14.6 g NH₄⁺-N /(m³ _reactor·day) and 6.67 g NO₂⁻-N /(m³ _reactor·day), respectively. Between t = 110 days and t = 161 days, the nitrogen load was increased to a HRT of 5 days (70 mg/l NH₄⁺ and 70 mg/l NO₂), the removal rates of ammonium and nitrite were 60.6% and 62.5% respectively. The sludge changed to red and formed Anammox granulation with high nitrogen removal activity.     

Nitrogen source effects on the denitrifying anaerobic methane oxidation culture and anaerobic ammonium oxidation bacteria enrichment process

The co-culture system of denitrifying anaerobic methane oxidation (DAMO) and anaerobic ammonium oxidation (Anammox) has a potential application in wastewater treatment plant. This study explored the effects of permutation and combination of nitrate, nitrite, and ammonium on the culture enrichment from freshwater sediments. The co-existence of NO₃ ⁻, NO₂ ⁻, and NH₄ ⁺ shortened the enrichment time from 75 to 30 days and achieved a total nitrogen removal rate of 106.5 mg/L/day on day 132. Even though ammonium addition led to Anammox bacteria increase and a higher nitrogen removal rate, DAMO bacteria still dominated in different reactors with the highest proportion of 64.7% and the maximum abundance was 3.07 ± 0.25 × 10⁸ copies/L (increased by five orders of magnitude) in the nitrite reactor. DAMO bacteria showed greater diversity in the nitrate reactor, and one was similar to M. oxyfera; DAMO bacteria in the nitrite reactor were relatively unified and similar to M. sinica. Interestingly, no DAMO archaea were found in the nitrate reactor. This study will improve the understanding of the impact of nitrogen source on DAMO and Anammox co-culture enrichment.

     

流加菌种对厌氧氨氧化工艺的影响

厌氧氨氧化工艺具有很高的容积氮去除速率,现已成功应用于污泥压滤液等含氨废水的脱氮处理,容积氮去除速率高达9.5 kg/(m3·d)。但由于厌氧氨氧化菌为自养型细菌,生长缓慢,对环境条件敏感,致使厌氧氨氧化工艺启动时间过长,运行容易失稳,并且不适合处理有机含氨废水和毒性含氨废水,极大地限制了该工艺的进一步推广应用。为了克服厌氧氨氧化工艺实际应用中存在的问题,结合发酵工业中常用的菌种流加技术,提出了一种新型的菌种流加式厌氧氨氧化工艺,研究了该新型工艺在厌氧氨氧化工艺的启动过程、稳定运行以及处理有机含氨废水和毒性含氨废水等方面的应用情况。结果表明,通过向反应器内补加优质厌氧氨氧化菌种,可提高厌氧氨氧化菌数量及其在菌群中的比例,强化厌氧氨氧化功能。据此研发的菌种流加式厌氧氨氧化工艺不仅可以实现快速启动,而且可以稳定运行,并突破了有机物和毒物所致的运行障碍,拓展了厌氧氨氧化工艺的应用范围。     

Anammox反应器启动过程中颗粒污泥性状变化特性

以厌氧颗粒污泥作为接种物,通过185 d的运行,成功启动了上流式厌氧氨氧化污泥床(Upflow anaerobic sludge blanket,UASB)反应器。反应器的进水氨氮与亚硝氮浓度分别提升至224 mg/L和255 mg/L,容积氮去除速率提升至3.76 kg/(m3·d)。采用红外光谱、扫描电镜和透射电镜等对厌氧氨氧化颗粒污泥的性状进行观察,发现颗粒污泥在启动过程中经历了污泥颗粒裂解到污泥颗粒重组的过程,且厌氧氨氧化颗粒污泥表面含有丰富的官能团,说明厌氧氨氧化颗粒污泥可能具有良好的吸附性能。采用宏基因组测序的方法对启动前后颗粒污泥的生态结构进行分析,发现原接种污泥优势菌群(变形菌门、厚壁菌门、拟杆菌门)丰度大幅减少,厌氧氨氧化菌所属的浮霉状菌门丰度则由1.59%提升到23.24%。     

Sludge wash-out as strategy for Anammox process start-up

In this study sludge wash-out was evaluated as a strategy to start-up the Anammox process in order to establish it in a shorter period of time. Sludge from a domestic wastewater treatment plant (WTP) was used to seed two (RI and RII) anaerobic sequencing batch reactors (ASBR). During the start-up period RI was operated as a continuous stirred tank reactor (CSTR) using a dilution rate of 0.2 d⁻¹, which promoted the sludge wash-out. After this period, the remaining sludge was retained in the reactor. The reactor RII was operated as an ASBR throughout the study period with a high cell retention. The performance of the two reactors in terms of nitrogen removal was compared over a period of 380 days. During the last RI operation phase the specific nitrogen removal rate increased exponentially, attaining values of 85 mg N/g TSS d. However, a rate of 190 mg N/g TSS d in the batch test under optimal conditions was achieved. The specific nitrogen removal rate remained almost constant for RII with a mean value of 6 mg N/g TSS d being observed during the operation period. The rate for the RII batch test was 20 mg N/g TSS d. These results confirm that the higher total suspended solids (TSS) in RII (reactor with high cell retention) was not effective in terms of N removal improvement. Anammox-like bacteria were found using fluorescence in situ hybridization (FISH) in reactor RI after 225 days and a new Anammox species was identified.     

NOB suppression and adaptation strategies in the partial nitrification–Anammox process for a poultry manure anaerobic digester

Poultry manure contains high levels of ammonia, which result in a suboptimal bioconversion to methane in anaerobic digesters (AD). A simultaneous process of nitrification, Anammox and denitrification (SNAD) in a continuous granular bubble column reactor to treat the anaerobically digested poultry manure was implemented. Thus, two strategies to achieve high efficiencies were proposed in this study: (1) ammonia overload to suppress nitrite oxidizing bacteria (NOB) and (2) gradual adaptation of the partial nitrification–Anammox (PN–A) biomass to organic matter. During the NOB-suppression stage, microbial and physical biomass characterizations were performed and the NOB abundance decreased from 31.3% to 3.3%. During the adaptation stage, with a nitrogen loading rate of 0.34 g L⁻¹ d⁻¹, a hydraulic retention time of 1.24 d and an influent COD/N ratio of 2.63 ± 0.02, a maximum ammonia and total nitrogen removal of 100% and 91.68% were achieved, respectively. The relative abundances of the aerobic and the anaerobic ammonia-oxidizing bacteria were greater than 35% and 40% respectively, during the study. These strategies provided useful design tools for the efficient removal of nitrogen species in the presence of organic matter.     

Anaerobic ammonium oxidation process in an upflow anaerobic sludge blanket reactor with granular sludge selected from an anaerobic digestor

The purpose of this work was to evaluate the development of the anammox process by the use of granular sludge selected from a digestion reactor as a potential seed source in a lab-scale UASB (upflow anaerobic sludge blanket) reactor system. The reactor was operated for approximately 11 months and was fed by synthetic wastewater. After 200 days of feeding with NH₄ ⁺ and NO₂ ⁻ as the main substrates, the biomass showed steady signs of ammonium consumption, resulting in over 60% of ammonium nitrogen removal. This report aims to present the results and to more closely examine what occurs after the onset of anammox activity, while the previous work described the start-up experiment and the presence of anammox bacteria in the enriched community using the fluorescencein situ hybridization (FISH) technique. By the last month of operation, the consumed NO₂ ⁻N/NH₄ ⁺-N ratio in the UASB reactor was close to 1.32, the stoichiometric ratio of the anammox reaction. The obtained results from the influentshutdown test suggested that nitrite concentration would be one key parameter that promotes the anammox reaction during the start-up enrichment of anammox bacteria from granular sludge. During the study period, the sludge color gradually changed from black to red-brownish.     

Rapid startup and high rate nitrogen removal from anaerobic sludge digester liquor using a SNAP process

In this study, a single-stage autotrophic nitrogen removal reactor, packed with a novel acrylic fiber biomass carrier material (Biofix), was applied for nitrogen removal from sludge digester liquor. For rapid start-up, conventional activated sludge was added to the reactor soon after the attachment of anammox biomass on the Biofix carriers, which allowed conventional activated sludge to form a protective layer of biofilm around the anammox biomass. The Nitrogen removal efficiency reached 75% within 1 week at a nitrogen loading rate of 0.46 kg-N/m³/day for synthetic wastewater treatment. By the end of the synthetic wastewater treatment period, the maximum nitrogen removal rate had increased to 0.92 kg-N/m³/day at a nitrogen loading rate of 1.0 kg-N/m³/day. High nitrogen removal rate was also achieved during the actual raw digester liquor treatment with the highest nitrogen removal rate being 0.83 kg-N/m³/day at a nitrogen loading rate of 0.93 kg-N/m³/day. The thick biofilm on Biofix carriers allowed anammox bacteria to survive under high DO concentration of 5–6 mg/l resulting in stable and high nitrogen removal performance. FISH and CLSM analysis demonstrated that anammox bacteria coexisted and surrounded by ammonium oxidizing bacteria.     

Identification of bacteria coexisting with anammox bacteria in an upflow column type reactor

Anammox process has attracted considerable attention in the recent years as an alternative to conventional nitrogen removal technologies. In this study, a column type reactor using a novel net type acrylic fiber (Biofix) support material was used for anammox treatment. The Biofix reactor was operated at a temperature of 25°C (peak summer temperature, 31.5°C). During more than 340 days of operation for synthetic wastewater treatment, the nitrogen loading rates of the reactor were increased to 3.6 kg-N/m³/d with TN removal efficiencies reaching 81.3%. When the reactor was used for raw anaerobic sludge digester liquor treatment, an average TN removal efficiency of 72% was obtained with highest removal efficiency of 81.6% at a nitrogen loading rate of 2.2 kg-N/m³/d. Results of extracellular polymeric substances (EPS) quantification revealed that protein was the most abundant component in the granular sludge and was found to be almost twice than that in the sludge attached to the biomass carriers. The anammox granules in the Biofix reactor illustrated a dense morphology substantiated by scanning electron microscopy and EPS results. The results of DNA analyses indicated that the anammox strain KSU-1 might prefer relatively low nutrient levels, while the anammox strain KU2 strain might be better suited at high nutrient concentration. Other types of bacteria were also identified with the potential of consuming dissolved oxygen in the influent and facilitating survival of anammox bacteria under aerobic conditions.     

Anammox start-up in sequencing batch biofilm reactors using different inoculating sludge

In this study, anammox bacteria were rapidly enriched in sequencing batch biofilm reactors (SBBRs) with different inoculations. The activated sludge taken from a sequencing batch reactor was used and inoculated to SBBR1, while SBBR2 was seeded with stored anaerobic sludge from an upflow anaerobic fixed bed (2-year stored at 5–15 °C). Nitrogen removal performance, anammox activity, biofilm characteristics and variation of the microbial community were evaluated. The maximum total nitrogen loading rate (NLR) of SBBR1 gradually reached to 1.62 kg?N/(m³/day) with a removal efficiency higher than 88 % and the NLR of SBBR2 reached to 1.43 kg?N/(m³/day) with a removal efficiency of 86 %. SBBR2 was more stable compared to SBBR1. These results, combined with molecular techniques such as scanning electron microscope, fluorescence in situ hybridization, and terminal restriction fragment length polymorphism, indicated that different genera of anammox bacteria became dominant. This research also demonstrates that SBBR is a promising bioreactor for starting up and enriching anammox bacteria.     

中试厌氧氨氧化反应器的启动与调控

研究了中试厌氧氨氧化(Anaerobic ammonium oxidation,Anammox)反应器的启动性能。结果表明,以硝化反硝化污泥、短程硝化污泥、厌氧絮体污泥和厌氧颗粒污泥混合接种,经过255d的运行,可在常温下(5oC～27oC)成功启动中试Anammox反应器,反应器的基质氮去除速率可达1.30kg/(m3·d)。厌氧氨氧化是致碱反应,厌氧氨氧化成为反应器内的主导反应后,进水pH宜控制在厌氧氨氧化适宜范围的偏低水平(6.8左右)。亚硝酸盐既是Anammox菌的基质,也是抑制剂,控制进水亚硝酸盐浓度(13～36mg/L)有助于厌氧氨氧化反应。菌种是生物反应器的功能之源,向中试装置投加少量厌氧氨氧化污泥(投加比2%),可大大加速中试Anammox反应器的启动进程。     

Nitrogen removal performance and operation strategy of anammox process under temperature shock

Sequencing batch reactors were used to study anaerobic ammonium oxidation (anammox) process under temperature shock. Both long-term (15–35 °C) and short-term (10–50 °C) temperature effects on nitrogen removal performance were performed. In reactor operation test, the results indicated that ammonium removal rate decreased from 0.35 kg/(m³ day) gradually to 0.059 kg/(m³ day) when temperature dropped from 35 to 15 °C. Although bacteria morphology was not modified, sludge settling velocity decreased with decreasing temperature. In batch test, apparent activation energy (E_a) increased with decreasing temperature, which suggested the activity decrease of anaerobic ammonium oxidizing bacteria (AAOB). Low temperature inhibited AAOB and weakened nitrogen removal performance. The cardinal temperature model with inflection was first used to describe temperature effect on anammox process. Simulated results revealed that anammox reaction could occur at 10.52–50.15 °C with maximum specific anammox activity of 0.50 kg/(kg day) at 36.72 °C. The cold acclimatization of AAOB could be achieved and glycine betaine could slightly improve nitrogen removal performance at low temperature.     

Phosphorus removal characteristics of granular and flocculent sludge in SBR

Aerobic granulation technology has become a novel biotechnology for wastewater treatment. However, the distinct properties and characteristics of phosphorus removal between granules and flocculent sludge are still sparse in enhanced biological phosphorus removal process. Two identical sequencing batch reactors (SBRs) were operated to compare phosphorus removal performance with granular sludge (R1) and flocculate activated sludge (R2). Results indicated that the start-up period was shorter in R2 than R1 for phosphorus removal, which made R2 reach the steady-state condition on day 21, while R1 was on day 25, and R2 released and took up more phosphorus than R1. As a result, the phosphorus removal was around 90% in R2 while 80% in R1 at the steady-state system. The special phosphorus release rate and special phosphorus uptake rate were 8.818 mg P/g volatile suspended solids (VSS)/h and 9.921 mg P/g VSS/h in R2, which were consistently greater than those (0.999 and 3.016 mg P/g VSS/h) in R1. The chemical oxygen demand removal in two reactors was similar. The granular SBR had better solid-separation performance and higher removal efficiency of NH₄⁺–N than flocculent SBR. Denaturing gradient gel electrophoresis of PCR-amplified 16S rDNA fragment analysis revealed that the diversity and the level of phosphorus-accumulating bacteria in flocculent sludge were much more than those in the granular sludge.     

High-rate nitrogen removal by the Anammox process at ambient temperature

The purpose of this study is to investigate the nitrogen removal performance of the anaerobic ammonium oxidation (Anammox) process and the microbial community that enables the Anammox system to function well at ambient temperatures. A reactor with a novel spiral structure was used as the gas-solid separator. The reactor was fed with synthetic inorganic wastewater composed mainly of NH₄⁺-N and NO₂⁻-N, and operated for 92 days. Stable nitrogen removal rates (NRR) of 16.3 and 17.5 kg-N m⁻³ d⁻¹ were obtained at operating temperatures of 33 ± 1 and 23 ± 2 °C, respectively. To our knowledge, such a high NRR at ambient temperatures has not been reported previously. In addition, the experiments presented herein confirm that high influent NO₂⁻-N concentration of 460 mg L⁻¹ did not noticeably inhibit the Anammox activity. Furthermore, the freshwater Anammox bacterium KU2, which was identified as the dominant bacterial species in the consortium by 16S rRNA gene analysis, is considered to be responsible for the stable nitrogen removal performance at ambient temperatures.     

Enrichment of Anammox from Activated Sludge and Its Application in the CANON Process

A microbial culture capable of actively oxidizing ammonium to dinitrogen gas in the absence of oxygen, using nitrite as the electron acceptor, was enriched from local activated sludge (Western Australia) in <14 weeks. The maximum anaerobic ammonium oxidation (i.e., anammox) activity achieved by the anaerobic culture was 0.26 mmol NH ₄ ⁺ (g biomass)⁻¹ h⁻¹ (0.58 kg total-N m⁻³ day⁻¹). Qualitative FISH analysis (fluorescence in situ hybridization) confirmed the phylogenetic position of the enriched microorganism as belonging to the order Planctomycetales, in which all currently identified anammox strains fall. Preliminary FISH analysis suggests the anammox strain belongs to the same phylogenetic group as the Candidatus ‘Brocadia anammoxidans’ strain discovered in the Netherlands. However, there are quite a few differences in the target sites for the more specific probes of these organisms and it is therefore likely to represent a new species of anammox bacteria. A small amount of aerobic ammonium-oxidizing biomass was inoculated into the anammox reactor (10% v/v) to initiate completely autotrophic nitrogen removal over nitrite (the CANON process) in chemostat culture. The culture was always under oxygen limitation and no organic carbon was added. The CANON reactor was operated as an intermittently aerated system with 20 min aerobiosis and 30 min anaerobiosis, during which aerobic and anaerobic ammonium oxidation were performed in sequential fashion, respectively. Anammox was not inhibited by repeated intermittent exposure to oxygen, allowing sustained, completely autotrophic ammonium removal (0.08 kg N m⁻³ day⁻¹) for an extended period of time.     

Research on soybean protein wastewater treatment by the integrated two-phase anaerobic reactor

The start-up tests of treating soybean protein wastewater by the integrated two-phase anaerobic reactor were studied. The results showed that the soybean protein wastewater could be successfully processed around 30 days when running under the situation of dosing seed sludge with the influent of approximately 2000 mg/L and an HRT of 40 h. When the start-up was finished, the removal rate of COD by the reactor was about 80%. In the zone I, biogas mainly revealed carbon dioxide (CO₂) and hydrogen (H₂). Methane was the main component in the zone 2 which ranged from 53% to 59% with an average of 55%. The methane content in biogas increased from the zone I to II. It indicated that the methane-producing capacity of the anaerobic sludge increased. It was found that the uniquely designed two-phase integrated anaerobic reactor played a key role in treating soybean protein wastewater. The acidogenic fermentation bacteria dominated in the zone I, while methanogen became dominant in the zone II. It realized the relatively effective separation of hydrolysis acidification and methanogenesis process in the reactor, which was benefit to promote a more reasonable space distribution of the microbial communities in the reactor. There were some differences between the activities of the sludge in the two reaction zones of the integrated two-phase anaerobic reactor. The activity of protease was higher in the reaction zone I. And the coenzyme F₄₂₀ in the reaction zone II was twice than that in the reaction zone I, which indicated that the activity of the methanogens was stronger in the reaction zone II.     

Rapid start-up and performance of denitrifying granular sludge in an upflow sludge blanket (USB) reactor treating high concentration nitrite wastewater

Denitrifying granular sludge reactor holds better nitrogen removal efficiency than other kinds of denitrifying reactors, while this reactor commonly needs seeding anaerobic granular sludge and longer period for start-up in practice, which restricted the application of denitrifying granular sludge reactor. This study presented a rapid and stable start-up method for denitrifying granular sludge. An upflow sludge blanket (USB) reactor with packings was established with flocculent activated sludge for treatment of high concentration nitrite wastewater. Results showed mature denitrifying granular sludge appeared only after 15 days with highest nitrogen removal rate of 5.844 kg N/(m³ day), which was much higher than that of compared anoxic sequencing batch reactor (ASBR). No significant nitrite inhibition occurred in USB and denitrification performance was mainly influenced by hydraulic retention time, influent C/N ratio and internal reflux ratio. Hydraulic shear force created by upflow fluid, shearing of gaseous products and stable microorganisms adhesion on the packings might be the reasons for rapid achievement of granular sludge. Compared to inoculated sludge and ASBR, remarkable microbial communitiy variations were detected in USB. The dominance of Proteobacteria and Bacteroidetes and enrichment of species Pseudomonas_stutzeri should be responsible for the excellent denitrification performance, which further verified the feasibility of start-up method.