New insights into syntrophic ethanol oxidation: Effects of operational modes and solids retention times.

Anaerobic ethanol oxidation relies on syntrophic interactions among functional microorganisms to become thermodynamically feasible. Different operational modes (sequencing batch reactors, SBRs, and continuous flow reactors, CFRs) and solids retention times (SRT, 25 days and 10 days) were employed in four ethanol-fed reactors, named as SBR25d, SBR10d, CFR25d, and CFR10d, respectively. System performance, syntrophic relationships, microbial communities, and metabolic pathways were examined. During the long-term operation, 2002.7 ± 56.0 mg COD/L acetate was accumulated in CFR10d due to the washout of acetotrophic methanogens. Microorganisms with high half-saturation constants were enriched in reactors of 25-day SRT. Moreover, ethanol oxidizing bacteria and acetotrophic methanogens with high half-saturation constants could be acclimated in SBRs. In SBRs, Syner-01 and Methanothrix dominated, and the low SRT of 10 days increased the relative abundance of Geobacter to 38.0%. In CFRs, the low SRT of 10 days resulted in an increase of Desulfovibrio among syntrophic bacteria, and CFR10d could be employed in enriching hydrogenotrophic methanogens like Methanobrevibacter.

Mechanistic understanding of acclimation and energy metabolism of acetoclastic methanogens under different substrate to microorganism ratios.

Mechanistic understanding of acetoclastic methanogenesis is pivotal for optimizing anaerobic digestion for efficient methane production. In this study, two different operational modes, continuous flow reactor (CFR) and sequencing batch reactor (SBR), accompanied with solids retention times (SRT) of 10 days (SBR10d and CFR10d) and 25 days (SBR25d and CFR25d) were implemented to elucidate their impacts on microbial communities and energy metabolism of methanogens in acetate-fed systems. Microbial community analysis revealed that the relative abundance of Methanosarcina (16.0%-46.0%) surpassed Methanothrix (3.7%-22.9%) in each reactor. SBRs had the potential to enrich both Methanothrix and Methanosarcina. Compared to SBRs, CFRs had lower total relative abundance of methanogens. Methanosarcina exhibited a superior enrichment in reactors with 10-day SRT, while Methanothrix preferred to be acclimated in reactors with 25-day SRT. The operational mode and SRT were also observed to affect the distribution of acetate-utilizing bacteria, including Pseudomonas, Desulfocurvus, Mesotoga, and Thauera. Regarding enzymes involved in energy metabolism, Ech and Vho/Vht demonstrated higher relative abundances at 10-day SRT compared to 25-day SRT, whereas Fpo and MtrA-H showed higher relative abundances in SBRs than those in CFRs. The relative abundance of genes encoding ATPase harbored by Methanothrix was higher than Methanosarcina at 25-day SRT. Additionally, the relative abundance of V/A-type ATPase (typically for methanogens) was observed higher in SBRs compared to CFRs, while the F-type ATPase (typically for bacteria) exhibited higher relative abundance in CFRs than that in SBRs.

Operational mode and powdered activated carbon promoting syntrophic propionate oxidation during anaerobic digestion of complex organic substances.

Operational mode and powdered activated carbon (PAC) are key factors facilitating microbial syntrophy and interspecies electron transfer during anaerobic digestion, consequently benefiting process stability and efficient methanogenesis. In this study, continuous-flow reactor (CFR) and sequencing batch reactor (SBR), with and without the addition of PAC, respectively, were operated to examine their effects on system performance and methanogenic activity. Based on the cycle-test result, the PAC-amended CFR (CFRPAC) recorded both the highest methane yield (690.1 mL/L) and the maximum CH4 production rate (28.8 mL/(L·h)), while SBRs exhibited slow methanogenic rates. However, activity assays indicated that SBRs were beneficial for organics removal in batch experiments fed with peptone. Taxonomic and functional analysis confirmed that CFRs were optimal for proliferating oligotrophs (e.g., Geobacter) and SBRs were more suitable for copiotrophs (e.g., Desulfobulbus). Metagenomic analysis revealed that CFRs had efficient acetate metabolic pathways from propionate and ethanol, whereas SBRs did not, resulting in the buildup of propionate. Furthermore, Methanobacterium and Methanothrix were acclimated to the different operational conditions, while acetoclastic Methanosarcina and hydrogenotrophic Methanolinea were acclimated in SBRs (5.1-13.4%) and CFRs (0.3-1.7%), respectively. This study confirmed the enhancement of microbial syntrophy by the addition of PAC as well as the acclimation of electroactive bacteria (e.g., Geobacter) with complex organic substances.

New insights into the effect of ethanol and volatile fatty acids proportions on methanogenic activities and pathways.

During anaerobic digestion, methanogenic activities and pathways can be affected by intermediates. Here, the effects of intermediates acetate, propionate, and ethanol on methanogenesis were investigated. Four anaerobic sequencing batch reactors were acclimated with propionate (ASBR_P), ethanol/propionate (ASBR_EP), acetate/propionate (ASBR_AP), and ethanol/acetate/propionate (ASBR_EAP). Ethanol was the easiest one to be biodegraded, thereby enhancing the maximum methane production rate and shortening the lag phase, while the longest acclimation time and lowest methane production rate were observed in ASBR_P. Different microbial communities and syntrophic patterns existed in four reactors. Desulfovibrio and Geobacter were the dominant ethanol-oxidizing bacteria in ASBR_EP and ASBR_EAP, respectively. Both Desulfovibrio and Geobacter possessed the potential of extracellular electron transfer, which might be the advantage of ethanol dosage for enhancing methanogenesis through direct interspecies electron transfer. Methanosarcina was enriched in ASBR_P and ASBR_AP, while Methanosaeta in ASBR_EP and ASBR_EAP. Genes responsible for acetoclastic methanogenesis were significantly enriched in ASBR_EAP, possibly resulting in the highest methanogenic activity from acetate. Results from this study will advance the optimization of practical anaerobic systems, which can be achieved by regulating the intermediates with different fermenting pathways.

Review of characteristics of anammox bacteria and strategies for anammox start-up for sustainable wastewater resource management.

Wastewater management has experienced different stages, including pollutant removal, resource recovery, and water nexus. Within these stages, anaerobic ammonia oxidation-based biotechnology can be incorporated for nitrogen removal, which can help achieve sustainable wastewater management, such as reclamation and ecologization of wastewater. Here, the physiology, metabolism, reaction kinetics and microbial interactions of anammox bacteria are discussed, and strategies to start-up the anammox system are presented. Anammox bacteria are slow growers with a high doubling time and a low reaction rate. Although most anammox bacteria grow autotrophically, some types can grow mixotrophically. The reaction stoichiometric coefficients can be affected by loading rates and other biological reactions. Microbial interactions also contribute to enhanced biological nitrogen removal and promote activities of anammox bacteria. The start-up of the anammox process is the key aspect for its practical application, which can be realized through seed selection, system stimulation, and biomass concentration enhancement.

Impacts of environmental factors on microbial diversity, distribution patterns and syntrophic correlation in anaerobic processes.

Anaerobic processes are widely used for treating high-strength organic wastewater. Understanding the ecological patterns of the microorganisms involved and the effect of environmental factors on microbial community are important to manage the performance of anaerobic processes. Microbial communities of 12 anaerobic sludge samples acclimated under different environmental conditions were investigated. Genera detected from these anaerobic sludge samples generally presented three distribution patterns: frequently detected with high abundance, frequently detected with low abundance and occasionally detected with permanently low abundance. The type of feed stock was one of the most important process parameters affecting the shape of microbial community (e.g., Syntrophus, Methylomonas and Methylobacillus). Dye wastewater (Bacteroides) and the supplement of conductive materials (genus T78) were also found to shape the microbial community. Some syntrophic bacteria and methanogens were rare in many anaerobic samples. However, correlation analysis suggested that rare genera are potential syntrophic partners and are responsible for syntrophic methanogenesis.

Comprehensive assessment of system performance in a full-scale wastewater treatment plant with an anaerobic/anoxic/aerobic membrane bioreactor combined with the ozonation process.

The system performance, economic cost and environmental impact of a full-scale anaerobic/anoxic/aerobic/membrane bioreactor (3AMBR) combined with the ozonation process were evaluated. The 3AMBR/ozonation process removed biochemical oxygen demand (BOD), chemical oxygen demand (COD), suspended solids, NH4-N and total phosphorus efficiently, with removal percentages above 94%, while the total nitrogen removal percentage was only 70%. The multiple linear regression analysis showed that hydraulic retention time (HRT) had a significant effect on nitrogen removal. A low HRT benefited nitrogen removal. Ferrous sulfate dosage close to the optimal value and a high mixed liquid suspended solid could enhance the phosphorus removal. The electricity cost accounted for 88% of the total economic costs. Greenhouse gas (GHG) emissions from the BOD oxidation and endogenous decay accounted for more than 50% of total emissions. The second largest GHG emission source was electricity consumption, accounting for 41%. The key to reduce the eutrophication was to enhance nitrogen removal. The composite cost of the 3AMBR/ozonation process was 251 CNY/t CODeq removed, among which economic cost accounted for 82.5%, while environmental impact cost accounted for a small proportion.

Nitrifiers activity and community characteristics under stress conditions in partial nitrification systems treating ammonium-rich wastewater.

Long-term exposure of nitrifiers to high concentrations of free ammonia (FA) and free nitrous acid (FNA) may affect nitrifiers activity and nitrous oxide (N2O) emission. Two sequencing batch reactors (SBRs) were operated at influent ammonium nitrogen (NH4-N) concentrations of 800mg/L (SBRH) and 335mg/L (SBRL), respectively. The NH4-N removal rates in SBRH and SBRL were around 2.4 and 1.0g/L/day with the nitritation efficiencies of 99.3% and 95.7%, respectively. In the simulated SBR cycle, the N2O emission factors were 1.61% in SBRH and 2.30% in SBRL. N2O emission was affected slightly by FA with the emission factor of 0.22%-0.65%, while N2O emission increased with increasing FNA concentrations with the emission factor of 0.22%-0.96%. The dominant ammonia oxidizing bacteria (AOB) were Nitrosomonas spp. in both reactors, and their relative proportions were 38.89% in SBRH and 13.36% in SBRL. Within the AOB genus, a species (i.e., operational taxonomic unit [OTU] 76) that was phylogenetically identical to Nitrosomonas europaea accounted for 99.07% and 82.04% in SBRH and SBRL, respectively. Additionally, OTU 215, which was related to Nitrosomonas stercoris, accounted for 16.77% of the AOB in SBRL.

Determination of quorum-sensing signal substances in water and solid phases of activated sludge systems using liquid chromatography-mass spectrometry.

The detection of acyl homoserine lactones (AHLs) in activated sludge is essential for clarifying their function in wastewater treatment processes. An LC-MS/MS method was developed for the detection of AHLs in both the aqueous and solid phases of activated sludge. In addition, the effects of proteases and extracellular polymeric substances (EPS) on the detection of AHLs were evaluated by adding protease inhibitors and extracting EPS, respectively. Recoveries of each AHL were improved by adding 50µL of protease inhibitor, and recoveries were also improved from 0 to 56.9% to 24.2%-105.8% by EPS extraction. Applying the developed method to determine the type and concentration of AHLs showed that C4-HSL, C6-HSL, C8-HSL and 3-oxo-C8-HSL were widely detected in a suspended activated sludge system. The dominant AHL was C8-HSL, with a highest concentration of 304.3ng/L. C4-HSL was mainly distributed in the aqueous phase, whereas C6-HSL, C8-HSL and 3-oxo-C8-HSL were preferentially distributed in the sludge phase.

Enhanced system performance by dosing ferroferric oxide during the anaerobic treatment of tryptone-based high-strength wastewater.

Conductive materials can facilitate syntrophic methane (CH4) production by improving direct interspecies electron transfer. The effect of a conductive material, ferroferric oxide (Fe3O4), on the anaerobic treatment of tryptone-based high-strength wastewater was investigated in two anaerobic sequencing batch reactors (ASBRs). One was the control reactor without amendment of Fe3O4, and the other was the Fe3O4-amended reactor. In an ASBR reaction cycle, the dosage of Fe3O4 increased the CH4 production rate by 12.2%, shortened the methanogenic lag phase by 32.3%, and improved the COD removal rate by 8.1%. During anaerobic treatment, the dosage of Fe3O4 not only enhanced the CH4 production from acetate but also facilitated the hydrolysis/acidification process fed by tryptone. Maximum electron transport activities were increased by 40.4, 137.3, and 64.6% in the processes of the ASBR reaction cycle, the CH4 production from acetate, and the hydrolysis/acidification of tryptone, respectively. Among all tested quorum sensing signal substances, N-octanoyl-L-homoserine lactone was dominant in both ASBRs, with the highest concentration in the biomass and the lowest concentration in the water phase. In addition, the concentration of signal substance was high in the ASBR cycle and low in the CH4 production from acetate and the hydrolysis/acidification of tryptone. Proteiniclasticum and vadinCA02 were dominant acidogens, and their relative abundances increased during the long-term operation with Fe3O4 dosing. Methanosarcina was the predominant methanogen, contributing to interspecies electron transfer.

Characterization of heavy metal desorption from road-deposited sediment under acid rain scenarios.

Road-deposited sediments (RDS) on urban impervious surfaces are important carriers of heavy metals. Dissolved heavy metals that come from RDS influenced by acid rain, are more harmful to urban receiving water than particulate parts. RDS and its associated heavy metals were investigated at typical functional areas, including industrial, commercial and residential sites, in Guangdong, Southern China, which was an acid rain sensitive area. Total and dissolved heavy metals in five particle size fractions were analyzed using a shaking method under acid rain scenarios. Investigated heavy metals showed no difference in the proportion of dissolved fraction in the solution under different acid rain pHs above 3.0, regardless of land use. Dissolved loading of heavy metals related to organic carbon content were different in runoff from main traffic roads of three land use types. Coarse particles (>150µm) that could be efficiently removed by conventional street sweepers, accounted for 55.1%-47.1% of the total dissolved metal loading in runoff with pH3.0-5.6. The obtained findings provided a significant scientific basis to understand heavy metal release and influence of RDS grain-size distribution and land use in dissolved heavy metal pollution affected by acid rain.

Removal of nitrogen and phosphorus from the secondary effluent in tertiary denitrifying biofilters combined with micro-coagulation.

Effective control of nitrogen and phosphorus in secondary effluent can reduce or avoid the eutrophication of receiving water bodies. Two denitrifying biofilters (DNBFs) packed with different sizes of quartz sands combined with micro-coagulation were operated for simultaneous removal of nitrogen and phosphorus from the secondary effluent. The quartz sand size in one DNBF was 2-4 mm (DNBFS), and in the other was 4-6 mm (DNBFL). In both DNBFs, methanol was used as the electron donor and different organic carbon to nitrogen (C/N) ratios were applied. Under C/N ratios of 1.5, 1.25, and 0.75 g/g, the nitrate nitrogen (NO3(-)-N) removal percentages were 73%, 77%, and 50% in DNBFS, and 43%, 25%, and 21% in DNBFL; the effluent total phosphorus concentrations were 0.15, 0.14, and 0.18 mg/L in DNBFS, and 0.29, 0.35, and 0.24 mg/L in DNBFL. The performance of both biofilters was quite stable within a backwashing cycle. The NO3(-)-N reduction rates were 1.31, 1.10, and 0.48 mg/(L·min) in DNBFS, and 0.97, 0.27, and 0.10 mg/(L·min) in DNBFL. For biomass detached from both biofilters, their denitrifying activities were similar. Biofilm biomass in DNBFS was higher than that in DNBFL, inducing a high denitrification efficiency in DNBFS.

Aerobic N2O emission for activated sludge acclimated under different aeration rates in the multiple anoxic and aerobic process.

Nitrous oxide (N2O) is a potent greenhouse gas that can be emitted during biological nitrogen removal. N2O emission was examined in a multiple anoxic and aerobic process at the aeration rates of 600mL/min sequencing batch reactor (SBRL) and 1200mL/min (SBRH). The nitrogen removal percentage was 89% in SBRL and 71% in SBRH, respectively. N2O emission mainly occurred during the aerobic phase, and the N2O emission factor was 10.1% in SBRL and 2.3% in SBRH, respectively. In all batch experiments, the N2O emission potential was high in SBRL compared with SBRH. In SBRL, with increasing aeration rates, the N2O emission factor decreased during nitrification, while it increased during denitrification and simultaneous nitrification and denitrification (SND). By contrast, in SBRH the N2O emission factor during nitrification, denitrification and SND was relatively low and changed little with increasing aeration rates. The microbial competition affected the N2O emission during biological nitrogen removal.

Denitrification and biofilm growth in a pilot-scale biofilter packed with suspended carriers for biological nitrogen removal from secondary effluent.

Tertiary denitrification is an effective method for nitrogen removal from wastewater. A pilot-scale biofilter packed with suspended carriers was operated for tertiary denitrification with ethanol as the organic carbon source. Long-term performance, biokinetics of denitrification and biofilm growth were evaluated under filtration velocities of 6, 10 and 14 m/hr. The pilot-scale biofilter removed nitrate from the secondary effluent effectively, and the nitrate nitrogen (NO3-N) removal percentage was 82%, 78% and 55% at the filtration velocities of 6, 10 and 14 m/hr, respectively. At the filtration velocities of 6 and 10 m/hr, the nitrate removal loading rate increased with increasing influent nitrate loading rates, while at the filtration velocity of 14 m/hr, the removal loading rate and the influent loading rate were uncorrelated. During denitrification, the ratio of consumed chemical oxygen demand to removed NO3-N was 3.99-4.52 mg/mg. Under the filtration velocities of 6, 10 and 14 m/hr, the maximum denitrification rate was 3.12, 4.86 and 4.42 g N/(m2·day), the half-saturation constant was 2.61, 1.05 and 1.17 mg/L, and the half-order coefficient was 0.22, 0.32 and 0.24(mg/L)1/2/min, respectively. The biofilm biomass increased with increasing filtration velocity and was 2845, 5124 and 7324 mg VSS/m2 at filtration velocities of 6, 10 and 14 m/hr, respectively. The highest biofilm density was 44 mg/cm3 at the filtration velocity of 14 m/hr. Due to the low influent loading rate, biofilm biomass and thickness were lowest at the filtration velocity of 6m/hr.

Denitrifying kinetics and nitrous oxide emission under different copper concentrations.

Denitrifying activities and nitrous oxide (N2O) emission during denitrification can be affected by copper concentrations. Different denitrifiers were acclimated in sequencing batch reactors with acetate or methanol as the electron donor and nitrate as the electron acceptor. The effect of copper concentrations on the denitrifying activity and N2O emission for the acclimated denitrifiers was examined in batch experiments. Denitrifying activities of the acclimated denitrifiers declined with increasing copper concentrations, and the copper concentration exhibited a higher effect on denitrifiers acclimated with acetate than those acclimated with methanol. Compared with the control without the addition of copper, at the copper concentration of 1 mg/L, the acetate utilization rate reduced by 89% for acetate-acclimated denitrifiers, while the methanol utilization rate only reduced by 15% for methanol-acclimated denitrifiers. Copper also had different effects on N2O emission during denitrification carried out by various types of denitrifiers. For the acetate-acclimated denitrifiers, N2O emission initially increased and then decreased with increasing copper concentrations, while for the methanol-acclimated denitrifiers, N2O emission decreased with increasing copper concentrations.

Powdered activated carbon facilitated degradation of complex organic compounds and tetracycline in stressed anaerobic digestion systems.

Tetracycline exerts an inhibitory effect on anaerobic digestion, inducing stressed microbial activities and even system failure. Continuous-flow reactors (CFRs) and sequencing batch reactors (SBRs) were employed along with the dosage of powdered activated carbon (PAC) to enhance tetracycline removal during anaerobic digestion of complex organic compounds. PAC increased the maximum methane production rate by 15.6% (CFRs) and 13.8% (SBRs), and tetracycline biodegradation by 24.4% (CFRs) and 19.2% (SBRs). CFRs showed higher tetracycline removal and methane production rates than SBRs. Geobacter was enriched in CFRs, where Methanothrix was enriched with the addition of PAC. Desulfomicrobium harbored abundant propionate degradation-related genes, significantly correlating with tetracycline removal. The genes encoding carbon dioxide reduction in Methanothrix along with the detection of Geobacter might indicate direct interspecies electron transfer for methanogenesis in CFRs and PAC-added reactors. The study offers new insights into anaerobic digestion under tetracycline-stressed conditions and strategies for optimizing tetracycline removal.

Deciphering anaerobic ethanol metabolic pathways shaped by operational modes.

Efficient anaerobic digestion requires the syntrophic cooperation among diverse microorganisms with various metabolic pathways. In this study, two operational modes, i.e., the sequencing batch reactor (SBR) and the continuous-flow reactor (CFR), were adopted in ethanol-fed systems with or without the supplement of powdered activated carbon (PAC) to examine their effects on ethanol metabolic pathways. Notably, the operational mode of SBR and the presence of CO2 facilitated ethanol metabolism towards propionate production. This was further evidenced by the dominance of Desulfobulbus, and the increased relative abundances of enzymes (EC: 1.2.7.1 and 1.2.7.11) involved in CO2 metabolism in SBRs. Moreover, SBRs exhibited superior biomass-based rates of ethanol degradation and methanogenesis, surpassing those in CFRs by 53.1% and 22.3%, respectively. Remarkably, CFRs with the extended solids retention time enriched high relative abundances of Geobacter of 71.7% and 70.4% under conditions with and without the addition of PAC, respectively. Although both long-term and short-term PAC additions led to the increased sludge conductivity and a reduced methanogenic lag phase, only the long-term PAC addition resulted in enhanced rates of ethanol degradation and propionate production/degradation. The strategies by adjusting operational mode and PAC addition could be adopted for modulating the anaerobic ethanol metabolic pathway and enriching Geobacter.

Unacclimated activated sludge improved nitrate reduction and N2 selectivity in iron filling/biochar systems.

Iron (Fe)-based denitrification is a proven technology for removing nitrate from water, yet challenges such as limited pH preference range and low N2 selectivity (reduction of nitrate to N2) persist. Adding biochar (BC) can improve the pH preference range but not N2 selectivity. This study aimed to improve nitrate reduction and N2 selectivity in iron filling/biochar (Fe/BC) systems with a simplified approach by coupling unacclimated microbes (M) in the system. Factors such as initial pH, Fe/BC ratio, and Fe/BC dosage on nitrate removal efficiency and N2 selectivity were evaluated. Results show that the introduction of microbes significantly enhanced nitrate removal and N2 selectivity, achieving 100 % nitrate removal and 79 % N2 selectivity. The Fe/BC/M system exhibited efficient nitrate reduction at pH of 2-10. Moreover, the Fe/BC/M system demonstrated an improved electrochemical active surface area (ECSA), lower electron transfer resistance and lower corrosion potential, leading to enhanced nitrate reduction. The high i0 value in Fe/BC/M system means more Hads could be generated, thus improving the N2 selectivity. This study provides valuable insights into a novel approach for effective nitrate removal, offering a potential solution to the environmental challenges posed by excessive nitrate in wastewater, surface water and ground water.

Effect of ammonium on nitrous oxide emission during denitrification with different electron donors.

Nitrous oxide (N2O) emission during denitrification is receiving intensive attention due to its high potential to cause greenhouse effects. In this study, denitrifiers were acclimated in sequencing batch reactors with methanol or acetate as the electron donor and nitrate as the electron acceptor. The effects of ammonium on N2O emission were examined in batch experiments with various electron donors. With the addition of ammonium, N2O emission increased under all the examined conditions compared to experiments without ammonium addition. With different electron donors, the highest ratio of N2O emission to the removed oxidized nitrogen was 0.70% for methanol, 5.34% for acetate, and 34.79% for polyhydroxybutyrate.

Quorum sensing responses of r-/K-strategists Nitrospira in continuous flow and sequencing batch nitrifying biofilm reactors.

A better understanding of r-/K-strategists nitrifiers will help to balance the design and operation of bioprocesses for efficient pollution removal from wastewater. The objectives of study were to investigate the nitrite oxidation biokinetics, biofilm property, microbial community and quorum sensing (QS) of nitrifying biofilm in a continuously flow reactor (CFR) and a sequencing batch reactor (SBR). Results showed that nitrite-oxidizing bacteria were estimated to have a nitrite half saturation constant of 76.23 and 224.73 µM in CFR and SBR, respectively. High-throughput and metagenomic sequencing results showed that Nitrospira and Candidatus Nitrospira defluvii were the dominated nitrite-oxidizing taxa performing nitrite oxidation in both reactors. Nitrifying biofilm developed in CFR and SBR showed obviously different properties. Biofilm in SBR had an obviously higher ratio of polysaccharide and protein in extracellular polymeric substances, and higher thickness than in CFR. Metagenomics and chemical analysis revealed various types of acyl-homoserine lactone (AHL) circuit genes (e.g., luxI, lasI, hdtS) and four types of AHL signaling substances (e.g., C6-HSL, C8-HSL, C10-HSL and 3-oxo-C10-HSL) in nitrifying biofilm. The concentrations of these AHLs in biomass and water phases were obviously higher in SBR than that in CFR. Together, AHLs-based QS might affect the formation of nitrifying biofilm and thus contribute to the different biokinetics of Nitrospira in CFR and SBR. Our insights may reveal the molecular mechanism of Nitrospira for different biokinetics, and indicate the AHL association with Nitrospira adaptation to various conditions.