Metagenomics reveals the response of desert steppe microbial communities and carbon-nitrogen cycling functional genes to nitrogen deposition.

Introduction: Nitrogen (N) deposition seriously affects the function of carbon (C) and N cycling in terrestrial ecosystems by altering soil microbial communities, especially in desert steppe ecosystems. However, there is a need for a comprehensive understanding of how microorganisms involved in each C and N cycle process respond to N deposition. Methods: In this study, shotgun metagenome sequencing was used to investigate variations in soil C and N cycling-related genes in the desert steppe in northern China after 6 years of the following N deposition: N0 (control); N30 (N addition 30 kg ha-1 year-1): N50 (N addition 50 kg ha-1 year-1). Results: N deposition significantly increased the relative abundance of Actinobacteria (P < 0.05) while significantly decreased the relative abundances of Proteobacteria and Acidobacteria (P < 0.05). This significantly impacted the microbial community composition in desert steppe soils. The annual addition or deposition of 50 kg ha-1 year-1 for up to 6 years did not affect the C cycle gene abundance but changed the C cycle-related microorganism community structure. The process of the N cycle in the desert steppe was affected by N deposition (50 kg ha-1 year-1), which increased the abundance of the pmoA-amoA gene related to nitrification and the nirB gene associated with assimilation nitrite reductase. There may be a niche overlap between microorganisms involved in the same C and N cycling processes. Discussion: This study provides new insights into the effects of N deposition on soil microbial communities and functions in desert steppe and a better understanding of the ecological consequences of anthropogenic N addition.

Identification of bacteria and fungi responsible for litter decomposition in desert steppes via combined DNA stable isotope probing.

Introduction: Soil microorganisms play crucial roles in determining the fate of litter in desert steppes because their activities constitute a major component of the global carbon (C) cycle. Human activities lead to increased ecosystem nitrogen (N) deposition, which has unpredictable impacts on soil microorganism diversity and functions. Nowadays, it is necessary to further study the succession of these microorganisms in the process of litter decomposition in desert steppe, and explore the effect of N deposition on this process. This issue is particularly important to resolve because it contributes to the broader understanding of nutrient cycling processes in desert steppes. Methods: In this study, DNA stable isotope probing (DNA-SIP) was used to study changes in soil bacterial and fungal community composition and function during 8 weeks of culture of 13C-labeled litter in desert steppes. Results: The results were as follows: (1) Actinomycetota, Pseudomonadota, and Ascomycota are the main microorganisms involved in litter decomposition in desert steppes; (2) N deposition (50 kg ha-1 year-1) significantly increased the relative abundance of some microorganisms involved in the decomposition process; and (3) N deposition likely promotes litter decomposition in desert steppes by increasing the abundances of N cycles bacteria (usually carrying GH family functional genes). Discussion: These findings contribute to a deeper understanding of the C assimilation mechanisms associated with litter residue production, emphasizing the importance of extensive C utilization.

Effective degradation of quinoline by catalytic ozonation with MnCexOy catalysts: performance and mechanism.

Quinoline inevitably remains in the effluent of coking wastewater treatment plants due to its bio-refractory nature, which might cause unfavorable effects on human and ecological environments. In this study, MnCexOy was consciously synthesized by α-MnO2 doped with Ce3+ (Ce:Mn = 1:10) and employed as the ozonation catalyst for quinoline degradation. After that, the removal efficiency and mechanism of quinoline were systematically analyzed by characterizing the physicochemical properties of MnCexOy, investigating free radicals and monitoring the solution pH. Results indicated that the removal rate of quinoline was greatly improved by the prepared MnCexOy catalyst. Specifically, the removal efficiencies of quinoline could be 93.73, 62.57 and 43.76%, corresponding to MnCexOy, α-MnO2 and single ozonation systems, respectively. The radical scavenging tests demonstrated that •OH and •O2- were the dominant reactive oxygen species in the MnCexOy ozonation system. Meanwhile, the contribution levels of •OH and •O2- to quinoline degradation were about 42 and 35%, respectively. The abundant surface hydroxyl groups and oxygen vacancies of the MnCexOy catalyst were two important factors for decomposing molecular O3 into more •OH and •O2-. This study could provide scientific support for the application of the MnCexOy/O3 system in degrading quinoline in bio-treated coking wastewater.

Application of biological soil crusts for efficient cadmium removal from acidic mine wastewater.

Utilizing an acid-resistant biological soil crust (BSC) species that we discovered, we developed a device capable of efficiently removing cadmium (Cd) from mine wastewater with varying levels of acidity. Our research has demonstrated that this particular BSC species adapts to acidic environments by regulating the balance of fatty acids and acid-resistant enzymes. At a Cd concentration of 5 mg/L, the BSC grew well. When the initial Cd concentration was 2 mg/L, and the flow rate was set at 1 mL/min (at pH levels of 3, 4, and 5), BSC had a high removal rate of Cd, and the removal rate increased with the increase of pH (from 90% to 97%). Chemisorption is the primary removal mechanism in the initial stage, where the functional groups and minerals on the surface of the BSC play a significant role. In addition, BSC also adapts to Cd stress by changing bacterial community structure. It was discovered through infrared spectroscopy and two-dimensional correlation analysis that hydrophilic groups, specifically phosphate and carboxyl groups, exhibited the highest reactivity during the Cd binding process. Protein secondary structure analysis confirmed that as the pH increased, the adsorption capacity of the BSC increased; making biofilm formation easier. This study presents a novel approach for the treatment of acidic wastewater.

Evaluation of metal pollution characteristics using water and moss in the Luanchuan molybdenum mining area, China.

Luanchuan is rich in molybdenum resources, and mining activities are frequent, but over-mining can cause serious metal pollution to the local environment. To explore the degree of metal pollution caused by mining activities, the content characteristics and spatial distribution of metals in mining areas were studied by measuring the concentrations of Fe, Mn, Zn, Ba, Mo, Cu, Cr, Co, V, and W in surface water and mosses of mining areas. In addition, the metal pollution index (HPI), contamination factor (CF), and pollution load index (PLI) were used to evaluate metal pollution, and factor analysis was used to analyze the sources of metals. The results of the analysis of surface water at the mine site indicate the most abundant element in surface water, with a maximum concentration of 3713.8 µg/L, and its content far exceeds the water quality standard of Class III of the Environmental Quality Standard for Surface Water. The results of the HPI analysis showed that nearly 90% of the surface water was moderately contaminated (HPI ≥ 15). The results of the analysis of atmospheric deposition at the mine site confirm that the metal elements with a high threat to the atmospheric environment are Mo and W. The results of PLI indicate that the level of atmospheric deposition pollution in the study area is severe (PLI > 4). Factor analysis indicated that rock weathering and mining activities were the main sources of metals. This study provides a theoretical basis for the investigation and control of metal pollution in similar metal mining areas.

[Remediation of Petroleum-contaminated Soil by Highly Efficient Oil-degrading Bacteria and Analysis of Its Enhancement Mechanism].

A 120-day in situ remediation of oil-contaminated soil was carried out by using highly efficient oil-degrading bacteria. The effects of bio-enhanced remediation and changes in soil physicochemical properties and enzyme activities were investigated. Combined with metagenomic sequencing and bioinformatics analysis, the strengthening mechanism was revealed. The results showed that compared with the blank control group (Ctrl), the degradation rate of total petroleum hydrocarbons in the bioremediation group (Exp-BT) was significantly increased, reaching 81.23%. During enhanced bioremediation by highly efficient oil-degrading bacteria, the pH of the soil was stable, the oxidation capacity of the system was improved, and the electrical conductivity was in the range suitable for agricultural activities. Lipase and dehydrogenase maintained high activity during repair. In addition, the analysis of the initial contaminated soil (B0), the highly efficient oil-degrading bacteria obtained from domestication (GZ), and the soil samples after bioremediation (BT) in the obtained samples showed that, at the phylum level, the total proportion of Proteobacteria and Actinobacteria increased by 17.1%. At the genus level, the abundance of Nocardioides, Achromobacter, Gordonia, and Rhodococcus increased significantly. The species and function contribution analysis of COG and KEGG proved that the above bacterial genera had important contributions to the degradation of petroleum hydrocarbons. A high abundance of petroleum hydrocarbon-related metabolic enzymes and five petroleum hydrocarbon-related degradation genes was found in the soil after remediation:alkM, tamA, rubB, ladA, and alkB. The analysis showed that the introduction of the exogenous petroleum hydrocarbon-degrading bacteria group enhanced the metabolic activity of microorganism-related enzymes and the expression of corresponding functional genes.

Coupled systems of pre-denitrification and partial nitritation/anammox improved functional microbial structure and nitrogen removal in treating swine manure digestate.

This study evaluated the functional activity and microbial structure of a pre-denitrification and single-stage partial nitritation/anammox process (DB-SNAP) coupled system for effectively treating swine manure digestate (SMD). At influent ammonium concentrations of (1000 to 1500) mg/L, the pre-denitrification reactor increased the nitrogen removal efficiency (NRE) by 5%, resulting in an average NRE of 96%. The DB-SNAP and nitrogen-limited strategy facilitated the rapid adoption of anammox bacteria (AnAOB) in the SMD, maintaining a high specific rate of 0.3gN/gVSS/d. A high secretion of tightly bound extracellular polymeric substances (76 mg/gVSS to 102 mg/gVSS) promoted micro-granule aggregation and stability. Moreover, Ca. Kuenenia, an AnAOB genus, was highly enriched from 21% to (27 to 30) %, whereas Nitrospira, a nitrite-oxidizing bacteria, was significantly suppressed to (0 to 0.05) %. These findings will provide valuable guidance in implementing the anammox process in swine wastewater treatment.

Highly efficient and reusable Mg-Fe layered double hydroxides anchored in attapulgite for uranium uptake from wastewater.

Micro/nano interface adsorption is an effective strategy for separating uranium from aqueous solutions. However, their undesirable capture efficiency and poor cycling stability limit their practical application. In this study, we developed a clay-based micro-adsorbent constructed using attapulgite (ATP) and Mg-Fe layered double hydroxides (Mg-Fe LDHs) for uranium uptake from wastewater. The surface charge affinity between ATP and Mg-Fe LDHs contributed to the robust heterostructure of the ATP@Mg-Fe LDHs adsorbent, thereby enabling a uniform distribution of Mg-Fe LDHs on the ATP surface. Thus, the aggregation behavior of Mg-Fe LDHs was significantly reduced and stellated with an improved dispersion performance of this ATP@Mg-Fe LDHs micro-composite in an aqueous solution. The uranium adsorption capacity was 670.21 mg/g, which is the maximum among previously reported clay-based adsorbents. Notably, a satisfying performance was achieved for the adsorbent stability; the uranium adsorption efficiency remained as high as 97% after eight cycles of adsorption-desorption. The ATP@Mg-Fe LDHs adsorbent for separating UO22+ from water is a promising system that combines efficiency, capacity, selectivity, and reusability, and has potential for scaled-up applications.

Efficient Removal of Heavy Metals from Contaminated Sunflower Straw by an Acid-Assisted Hydrothermal Process.

The removal of heavy metals is crucial to the utilization of contaminated biomass resources. In this study, we report an efficient process of hydrothermal conversion (HTC) of sunflower straw (Helianthus annuus L.) to remove heavy metals. The effect of different HTC temperatures and concentrations of HCl additives on heavy metal removal efficiency was investigated. The results revealed that increasing the temperature or concentration of HCl promoted the transfer of heavy metals from hydrochar to liquid products during HTC. The heavy metals removed to the liquid products included up to 99% of Zn and Cd, 94% of Cu, and 87% of Pb after hydrothermal conversion with a temperature of 200 °C and HCl 2%. The species of heavy metals in hydrochars converted from unstable to stable with an increase in temperature from 160 °C to 280 °C. The stable fractions of heavy metals in the acidic condition decreased as the acid concentration increased. This aligns well with the high transfer efficiency of heavy metals from the solid phase to the liquid phase under acidic conditions. The FTIR indicated that the carboxy and hydroxy groups decreased significantly as the temperature increased and the concentration of HCl increased, which promoted the degradation of sunflower straw. A scan electron microscope showed that the deepening of the destruction of the initial microstructure promotes the transfer of heavy metals from hydrochars to liquid phase products. This acid-assisted hydrothermal process is an efficient method to treat biomass containing heavy metals.

The effects of salinity changes on anammox performance: The response rule and tolerance mechanism.

Some wastewaters contain high concentrations of ammonia coexisting with large amounts of salt, which might negatively affect the anaerobic ammonium oxidation (anammox) process. In this study, the performance of the anammox process under different saline conditions was investigated using an upflow anaerobic sludge bed-anammox system. After long-term operating for 275 days, the results indicated that the nitrogen removal efficiency remained high under the 0-40 g NaCl/L, and low salinity (15 g NaCl/L) substantially promoted specific anammox activity. Affected by the saline environment, the appearance, color, and shape of sludge notably changed, and the amount of extracellular polymeric substances gradually increased with increasing salinity, which might be one of the reasons for the strong salt tolerance of the system. Chloroflexi and Planctomycetes were the dominant strains under long-term salinity, and Brocadiaceae_g_ unclassified exhibited halophilic characteristics. The redundancy analysis results showed that the concentration of influent NH4 + -N and salinity were the main environmental factors affecting the microbial community of the system. PRACTITIONER POINTS: Provides data to support the maximum value for salinity wastewater treatment with anammox processes' tolerance of 40 g NaCl/L. EPS changes may be responsible for the response to salinity challenges and provide direction for high salinity wastewater treatment. Brocadiaceae_g_ unclassified exhibited a halophilic quality. And it can be focused on to improve treatment efficiency.

The Aggregation and Dissolution of Citrate-Coated AgNPs in High Ammonia Nitrogen Wastewater and Sludge from UASB-Anammox Reactor.

Silver nanoparticles (AgNPs) are released into the sewage pipes and ultimately wastewater treatment plants during manufacturing, use, and end-life disposal. AgNPs in wastewater treatment plants aggregate or dissolve, and may affect the microbial community and subsequent pollutant removal efficiency. This study aims to quantitatively investigate the fate of AgNPs in synthetic high ammonia nitrogen wastewater (SW) and sludge from an up-flow anaerobic sludge blanket (UASB) anammox reactor using a nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), transmission electron microscope (TEM), and atomic absorption spectroscopy (AAS). Results showed that 18.1 mM NH4+, 2.11 mM Mg2+ in SW caused less negative zeta potential (ζ-potential, -18.4 vs. -37.4 mV), aggregation (388.8 vs. 21.5 nm), and settlement (80%) of citrate-coated AgNPs (cit-AgNPs) in 220 min. The presence of 18.5 mM Cl- in SW formed AgCl2-, AgCl(aq) and eventually promoted the dissolution (9.3%) of cit-AgNPs. Further exposure of SW-diluted AgNPs to sludge (42 mg L-1 humic acid) and induced a more negative ζ-potential (-22.2 vs. -18.4 mV) and smaller aggregates (313.4 vs. 388.8 nm) due to the steric and hindrance effect. The promoted Ag dissolution (34.4% vs. 9.3%) was also observed after the addition of sludge and the possible reason may be the production of Ag(NH3)2+ by the coexistence of HA from sludge and NH4+ from SW. These findings on the fate of AgNPs can be used to explain why AgNPs had limited effects on the sludge-retained bacteria which are responsible for the anammox process.

Preparation and Characterization of Red Mud/Fly Ash Composite Material (RFCM) for Phosphate Removal.

In this study, a new red mud/fly ash composite material (RFCM) for phosphate removal was prepared by granulation and activation methods, using bauxite residue (red mud, RM) as the main raw material, adding with some fly ash and a few adhesives. The effects of different types of RM and adhesives on RFCM for phosphate removal were discussed. It was found that RFCM prepared from sintering red mud and cement waste performed better on phosphate removal than that prepared from Bayer red mud and common industrial adhesives. After calcination activated at appropriate temperature around 800-900℃, the specific surface area of RFCM increased, and new substances with hydroxyl (-OH) appeared on the surface of RFCM, which were the functional groups for phosphate removal. Mechanism of RFCM for phosphate removal was speculated as a combination of physical adsorption, chemical adsorption and chemical precipitation, which mainly depended on ligand exchange and chemical reaction. This research will provide a potential application of bauxite residue in environmental remediation.

Impacts of salt shocking and the selection of a suitable reversal agent on anammox.

In this study, an anaerobic ammonium oxidation (anammox) reactor, which was inhibited by a salinity of 50 g NaCl L-1 during a long-term experiment, was rapidly restarted by decreasing the salinity to 20 g NaCl L-1 and adding biomass. The effects of exposure time and shock concentrations on the anammox reactor indicate that anammox granular sludge has a high tolerance to salinity and strong ability for self-recovery. The nitrogen removal efficiency was higher than 50% after exposure to 50 g NaCl L-1 for 66 h. To shorten the time taken for effluent nitrogen concentrations to attain national standards (GB18918-2002) after the anammox reactor was shocked with NaCl, reactor performance (i.e., recovery) after the addition of K+, glycine betaine, Fe2+, and hydroxylamine were compared after the reactor was inhibited by 80 g NaCl L-1. The results indicate that hydroxylamine was the best reversal agent. The recovery time of the anammox reactor could be shortened by 50% following the addition of hydroxylamine. The most favorable NH2OH-N/NO2--N concentration ratio for improving nitrogen removal of anammox was 1:11. The abundances of Planctomycetes and its genera Candidatus Kuenenia and Brocadiaceae_g_unclassified increased after repeated salinity shock-recovery phases, indicating that Candidatus Kuenenia and Brocadiaceae_g_unclassified are able to adapt to NaCl shocking and recovery.

Effect of ultrasound on oil recovery from crude oil containing sludge.

To recover oil from crude oil containing sludge is still a research hot topic from the view of sustainability, in which ultrasonic has been proven to be an efficient and environment friendly technique. However, the effect of sludge characteristic on ultrasonic-assisted oil recovery efficiency is little known. In this study, the analysis of variance (ANOVA) was conducted based on six types of crude oily sludge with hydrophilicity and lipophilicity separately and five different ultrasonic operation factors (ultrasonic power (A), frequency (B), time (C), initial temperature (D) and pH (E)). The results showed that the oil recovery efficiency was mainly affected by the ultrasonic power and hydrophilicity of sludge (the highest 92% of oil recovery rate was achieved with the ultrasonic power of 240 W and hydrophilic sludge). Moreover, the wettability, decreased average particle size and increased specific surface area of sludge were found after ultrasonic treatment. Besides, changes in the oil component, such as the decrease of asphaltenes along with an increase of saturates, were also further observed. Therefore, the findings in this study can provide technical support for the practical application of ultrasonic technology in different kinds of oily sludge treatment.

Nutrients removal performance of a denitrifying phosphorus removal process in alternate anaerobic/anoxic-aerobic double membrane bioreactors (A2N-DMBR).

An alternate anaerobic/anoxic-aerobic double membrane bioreactors process (A2N-DMBR) was proposed to improve denitrifying phosphorus removal efficiency. The system was operated for 70 d under different nitrogen/phosphorus (N/P) ratios with synthetic wastewater to present the performance evaluation of nutrients removal and microbial community structure in the A2N-DMBR process. The results showed that when the influent total phosphorus (TP) was 6.4 mg/L, the corresponding N/P ratio of 8.8, the high removal capacity of nitrogen and phosphorus could be achieved with the average effluent TP and total nitrogen (TN) concentration of 0.8 mg/L and 12.0 mg/L, respectively. Periodical test showed that pH and oxidation-reduction potential (ORP) could be used as control parameters for anaerobic phosphate release, and ORP was also closely related with the phosphate uptake in anoxic phase. The high-throughput sequencing analysis revealed that the Proteobacteria and Xanthomonadales-nobank related to biological nitrogen and phosphorus removal was domination bacteria at phylum and genus level in A2N-DMBR system, with the proportion of 42.5% and 39.1%, respectively. Furthermore, Dechloromonas, which was further detected as putative denitrifying phosphorus accumulating organisms (DPAOs), was enriched (9.9%) in the system.

Performance and microbial community of anammox in presence of micro-molecule carbon source.

Because ammonium (NH4+-N) coexists with organic matter in some wastewaters, the possible adverse influences of organic matter become a major concern in the applications of anaerobic ammonium oxidation (anammox). In this study, the effects of acetate, as a representative of micro-molecule organic matter, on anammox were investigated. Efficient nitrogen removal was realized because denitrifying bacteria and anammox bacteria (AnAOB) had a better synergistic effect under the condition of chemical oxygen demand (COD) concentrations lower than 251 ±â€¯7 mg L-1. Furthermore, the nitrogen removal efficiency (NRE) decreased to 82.02 ±â€¯3.14% when COD was increased to 730 ±â€¯9 mg L-1, and effluent free ammonia (FA) reached 21.93 ±â€¯4.71 mg L-1 might be one of factors leading to inhibition. However, the nitrogen-removal contribution rate of anammox remained steady at 61.97 ±â€¯2.84% at COD of 730 ±â€¯9 mg L-1, which indicated that anammox was still dominant in the system. AnAOB, such as Ca. Kuenenia and Ca. Jettenia, and denitrifying bacteria, such as Denitratisoma and Thauera, were found to coexist in the reactor. Interestingly, Ca. Kuenenia presented in the trend of first decreased then increased with the increasing of organic matter concentration, which might be one of reasons that anammox played an important role in nitrogen removal at high COD concentration.

Effects of temperature on anammox performance and community structure.

A lab-scale anammox up-flow anaerobic sludge blanket (UASB) reactor was run to investigate the influence of temperature on anammox performance and community structure. The anammox system had a higher substrate tolerance at 13 °C than at 18 °C. The adverse effects caused by the use of a lower temperature (8 °C) could be restored. The nitrogen removal rate (NRR) decreased with decreasing in situ specific anammox activity (SAA). Interestingly, the ex situ SAA acclimated at 23 °C, when exposed to ex situ temperatures of 33 and 28 °C, was higher than for those acclimated at 33 and 28 °C. No shift was observed in the optimum temperature for ex situ SAA in the whole lowering process of anammox UASB. More extracellular polymeric substances were produced in response to cooler conditions (18 °C and 13 °C). Ca. Kuenenia became much more abundant (55.18% of the microbial community) and had a competitive advantage over other anammox bacteria (AnAOB) at 13 °C.

Nitrogen removal performance and microbial community structure in the start-up and substrate inhibition stages of an anammox reactor.

In this study, the nitrogen removal performance and microbial community structure were investigated during the start-up, instability, and recovery stages of an anaerobic ammonium oxidation (anammox) reactor loaded with compound carriers (shale ceramsite and suspended ball carrier). The results indicated that the anammox reactor successfully started up on 116th d when the nitrogen loading rate (NLR) reached 0.72 ± 0.05 kg N m-3 d-1. The anammox reactor ran well with free ammonia (FA) at 13.65 ± 2.69 mg/L and free nitrous acid (FNA) at 39.49 ± 10.95 µg/L, indicating that its tolerance for FA and FNA was higher than that of granular sludge anammox reactors. The anammox system was inhibited when FA and FNA reached 29.65 mg/L and 77.02 µg/L, respectively. The tolerance of anammox bacteria towards FA and FNA decreased after this inhibition. The nitrogen removal performance could be efficiently recovered by decreasing the influent substrate concentration and increasing the hydraulic retention time (HRT). Candidatus Brocadia and Candidatus Jettenia, two genus-level anammox bacteria, were detected in this reactor using a high-throughput sequencing technique. After high substrate shock, the abundance of Candidatus Brocadia decreased while that of Candidatus Jettenia increased, which might be due to the competition between Candidatus Jettenia and Candidatus Brocadia. The relationships between anammox communities and operational factors were investigated via redundancy analysis (RDA), which showed that FA was the principal factor affecting the microbial community structure during the operation stage.

Substrate inhibition and concentration control in an UASB-Anammox process.

An UASB-Anammox reactor was operated for more than one year to study the process performance variations respond to the nitrogen loading rate (NLR) and substrate concentration. The IC10 (451.1mg/L), IC50 (725.3mg/L) and the prospected threshold of influent total nitrogen (TN) concentration were simulated. A stable TN removal efficiency was obtained when the TN influent was controlled. The disequilibrium distribution of the substrate following the plug flow with the height of the reactor resulted in significant variations in specific Anammox activity from the bottom to the top of the reactor (348→3mgN/gVSS/d). With long term acclimation, the nitrogen removal capacity of Anammox sludge varied significantly, with the most activated sludge obtained in the bottom part a 100 times capacity greater than that of the top. A stable performance with high removal efficiency in the constructed UASB-Anammox reactor was obtained when the influent TN concentration was below 451.1mg/L.

Long-term operation performance and variation of substrate tolerance ability in an anammox attached film expanded bed (AAFEB) reactor.

An anammox attached film expanded bed (AAFEB) reactor was operated to study the long-term performance and the variation of substrate tolerance ability. The results indicated that the nitrogen loading potential (NLP) was significantly enhanced from 13.56gN·(L·d)(-)(1) to 20.95gN·(L·d)(-)(1) during the stable operation period. The inhibitory concentration of 10% (IC10) for free ammonia (FA), free nitrous acid (FNA) and SNinf (diluted substrate concentration) increased from 18mg/L, 12µgL(-1) and 370mgNL(-)(1) to 31mg/L, 19µgL(-1) and 670mgNL(-)(1), respectively. However, the substrate shock of 2500mgNL(-)(1) for 24h terribly weakened the treatment performance and substrate tolerance ability of the reactor. The results of batch tests indicated that the existence of lag phase made the AAFEB reactor more vulnerable to substrate variation. The SNinf was accurate to be used to monitor the reactor performance and should be maintained below 320mgNL(-)(1) to ensure the absolute stable operation.