Reason and control strategy for denitrification and anammox sludge flotation in nitrogen removal process: Mechanisms, strategies and perspectives.

Anaerobic biological treatment technology, especially denitrification and anaerobic ammonia oxidation (anammox) technology as mainstream process, played dominant role in the field of biological wastewater treatment. However, the above process was prone to sludge floating during high load operation and thereby affecting the efficient and stable operation of the system. Excessive production of extracellular polymeric substance (EPS) was considered to be the main reason for anaerobic granular sludge flotation, but the summaries in this area were not comprehensive enough. In this review, the potential mechanisms of denitrification and anammox sludge floatation were discussed from the perspective of granular sludge structural characteristics, nutrient transfer, and microbial flora change respectively, and the corresponding control strategies were also summarized. Finally, this paper indicated that future research on sludge flotation should focus on reducing the negative effects of EPS in sludge particles.

Light-Driven Electron Uptake from Nonfermentative Organic Matter to Expedite Nitrogen Dissimilation by Chemolithotrophic Anammox Consortia.

Nonphotosynthetic microorganisms are typically unable to directly utilize light energy, but light might change the metabolic pathway of these bacteria indirectly by forming intermediates such as reactive oxygen species (ROS). This work investigated the role of light on nitrogen conversion by anaerobic ammonium oxidation (anammox) consortia. The results showed that high intensity light (>20000 lx) caused ca. 50% inhibition of anammox activity, and total ROS reached 167% at 60,000 lx. Surprisingly, 200 lx light was found to induce unexpected promotion of the nitrogen conversion rate, and ultraviolet light (<420 nm) was identified as the main contributor. Metagenomic and metatranscriptomic analyses revealed that the gene encoding cytochrome c peroxidase was highly expressed only under 200 lx light. 15N isotope tracing, gene abundance quantification, and external H2O2 addition experiments showed that photoinduced trace H2O2 triggered cytochrome c peroxidase expression to take up electrons from extracellular nonfermentative organics to synthesize NADH and ATP, thereby expediting nitrogen dissimulation of anammox consortia. External supplying reduced humic acid into a low-intensity light exposure system would result in a maximal 1.7-fold increase in the nitrogen conversion rate. These interesting findings may provide insight into the niche differentiation and widespread nature of anammox bacteria in natural ecotopes.

Beyond an Applicable Rate in Low-Strength Wastewater Treatment by Anammox: Motivated Labor at an Extremely Short Hydraulic Retention Time.

The application of anammox technology in low-strength wastewater treatment is still challenging due to unstable nitrite (NO2--N) generation. Partial denitrification (PD) of nitrate (NO3--N) reduction ending with NO2--N provides a promising solution. However, little is known about the feasibility of accelerating nitrogen removal toward the practical application of anammox combined with heterotrophic denitrification. In this work, an ultrafast, highly stable, and impressive nitrogen removal performance was demonstrated in the PD coupling with an anammox (PD/A) system. With a low-strength influent [50 mg/L each of ammonia (NH4+-N) and NO3--N] at a low chemical oxygen demand/NO3--N ratio of 2.2, the hydraulic retention time could be shortened from 16.0 to 1.0 h. Remarkable nitrogen removal rates of 1.28 kg N/(m3 d) and excellent total nitrogen removal efficiency of 94.1% were achieved, far exceeding the applicable capacity for mainstream treatment. Stimulated enzymatic reaction activity of anammox was obtained due to the fast NO2--N jump followed by a famine condition with limited organic carbon utilization. This high-rate PD/A system exhibited efficient renewal of bacteria with a short sludge retention time. The 16S rRNA sequencing unraveled the rapid growth of the genus Thauera, possibly responsible for the incomplete reduction of NO3--N to NO2--N and a decreasing abundance of anammox bacteria. This provides new insights into the practical application of the PD/A process in the energy-efficient treatment of low-strength wastewater with less land occupancy and desirable effluent quality.

Anammox sludge preservation: Preservative agents, temperature and substrate.

The difficulties of enrichment and preservation of anaerobic ammonium oxidation bacteria (AnAOB) greatly limit their application in practice. Herein, traditional and emerging preservative agents (e.g., EPS + N2H4, betaine, glycerol and trehalose) were evaluated for their preservation of AnAOB-dominant sludge at different temperatures (e.g., 4 °C and room temperature). In addition, the effects of substrates on preservation were also considered. The results showed that adding betaine or glycerol at 4 °C was the optimal strategy for preserving anammox granular sludge. The relative anammox activities (rAA) increased by 145.26% and 158.30% at the recovery phase, respectively. Moreover, the absolute abundances of functional gene hzsA increased by 339% and 46%, respectively. Although the granular properties and microbial community structures changed during the preservation, the general performance of anammox granules could effectively restored. Collectively, this study provides the optimal strategies for anammox sludge preservation at low temperatures.

Molecular Insight into the Binding Property and Mechanism of Sulfamethoxazole to Extracellular Proteins of Anammox Sludge.

Antibiotics are widely found in nitrogen-containing wastewater, which may affect the operation stability of anaerobic ammonium oxidation (anammox)-based biological treatment systems. Extracellular polymeric substances (EPSs) of anammox sludge play a pivotal role in combining with antibiotics; however, the exact role and how the structure of the leading component of EPSs (i.e., extracellular proteins) changes under antibiotic stress remain to be elucidated. Here, the interaction between sulfamethoxazole and the extracellular proteins of anammox sludge was investigated via multiple spectra and molecular simulation. Results showed that sulfamethoxazole statically quenched the fluorescent components of EPSs, and the quenching constant of the aromatic proteins was the largest, with a value of 1.73 × 104 M-1. The overall binding was an enthalpy-driven process, with ΔH = -75.15 kJ mol-1, ΔS = -0.175 kJ mol-1 K-1, and ΔG = -21.10 kJ mol-1 at 35 °C. The O-P-O and C═O groups responded first under the disturbance of sulfamethoxazole. Excessive sulfamethoxazole (20 mg L-1) would decrease the ratio of α-helix/(ß-sheet + random coil) of extracellular proteins, resulting in a loose structure. Molecular docking and dynamic simulation revealed that extracellular proteins would provide abundant sites to bind with sulfamethoxazole, through hydrogen bond and Pi-Akyl hydrophobic interaction forces. Once sulfamethoxazole penetrates into the cell surface and combines with the transmembrane ammonium transport domain, it may inhibit the NH4+ transport. Our findings enhance the understanding on the interaction of extracellular proteins and sulfamethoxazole, which may be valuable for deciphering the response property of anammox sludge under the antibiotic stress.

How anammox responds to the emerging contaminants: Status and mechanisms.

Numerous researches have been carried out to study the effects of emerging contaminants in wastewater, such as antibiotics, nanomaterials, heavy metals, and microplastics, on the anammox process. However, they are fragmented and difficult to provide a comprehensive understanding of their effects on reactor performance and the metabolic mechanisms in anammox bacteria. Therefore, this paper overviews the effects on anammox processes by the introduced emerging contaminants in the past years to fulfill such knowledge gaps that affect our perception of the inhibitory mechanisms and limit the optimization of the anammox process. In detail, their effects on anammox processes from the aspects of reactor performance, microbial community, antibiotic resistance genes (ARGs), and functional genes related to anammox and nitrogen transformation in anammox consortia are summarized. Furthermore, the metabolic mechanisms causing the cell death of anammox bacteria, such as induction of reactive oxygen species, limitation of substrates diffusion, and membrane binding are proposed. By offering this review, the remaining research gaps are identified, and the potential metabolic mechanisms in anammox consortia are highlighted.

Response of anammox granules to the simultaneous exposure to macrolide and aminoglycoside antibiotics: Linking performance to mechanism.

Antibiotic pollution is becoming increasingly severe due to its extensive use. The potential application of the anaerobic ammonium oxidation (anammox) process in the treatment of wastewater containing antibiotics has attracted much attention. As common antibiotics, spiramycin (SPM) and streptomycin (STM) are widely used to treat human and animal diseases. However, their combined effects on the anammox process remain unknown. Therefore, this study systematically evaluated the response of the anammox process to both antibiotics. The half maximal inhibitory concentrations of SPM and STM were determined. The continuous-flow anammox system could adapt to SPM and STM at low concentrations, while antibiotics at high concentrations exhibited inhibitory effects. When the concentrations reached 5 mg L-1 SPM and 50 mg L-1 STM, the nitrogen removal efficiency dramatically decreased and then rapidly recovered within 8 days. Correspondingly, the abundances of dominant bacteria and genes also changed with antibiotic concentrations. In general, the anammox process showed a stable performance and a high resistance to SPM and STM, suggesting that acclimatization by elevating the concentrations was beneficial for the anammox process to obtain resistance to different antibiotics with high concentrations. This study provides guidance for the stable operation of anammox-based biological treatment of antibiotics containing wastewater.

Anammox Granules Acclimatized to Mainstream Conditions Can Achieve a Volumetric Nitrogen Removal Rate Comparable to Sidestream Systems.

The implementation of mainstream anammox has gained increasing attention. In this study, the feasibility of using sidestream anammox granules to start up mainstream reactors was investigated by comparing two switching strategies. A maximum nitrogen removal potential of 3.6 ± 0.2 kg N m-3 d-1 was obtained for the reactor after direct switching to mainstream conditions (70 mg TN L-1, 15 °C). Nevertheless, the reactor preacclimatized to 25 °C (Ma) exhibited a higher nitrogen removal potential of 7.0 ± 0.3 kg N m-3 d-1 at 15 °C, which is the highest volumetric nitrogen removal rate of mainstream anammox reactors to date. Candidatus Kuenenia stuttgartiensis was identified as the dominant anammox bacterium, and its relative abundance in two reactors remained stable throughout the whole operation (200 days). Moreover, with the aid of acclimatization, the activation energy was reduced and the specific growth rate became higher. These results indicated that the physiological evolution of the dominant anammox bacterium instead of interspecies selection was the main reason for the high potential during the switch to mainstream conditions. Therefore, using sidestream anammox granules as seed sludge to start up mainstream reactors was demonstrated to be feasible, and a switching strategy of acclimatization at 25 °C was recommended.

Performance, kinetics characteristics and enhancement mechanisms in anammox process under Fe(II) enhanced conditions.

In order to explore the performance, kinetics characteristics and enhancement mechanisms in anammox process under ferrous iron enhanced conditions, a laboratory-scale UASB anammox reactor has been built up and operated for 534 days. Experimental results showed that the Anammox process was successfully started up in a short operation period and the TNRE reached 83.34 ± 2.96% with a maximum total nitrogen removal rate of 14.4 kg m-3 d-1 after long-term operated under influent Fe(II) concentration of 5.3 mg L-1. Simulation results using different kinetic models showed that the Stover-Kincannon model and the Grau second-order model were useful for describing the anammox performance under Fe(II) enhanced conditions. Extracellular polymeric substance (EPS) act a pivotal part in the granulation of Anammox sludge and the improvement of anammox activity. Iron improved the hydrophobicity of the sludge by reducing the PN/PS ratios, and also increased the Anammox granular diameter. The granular diameter of higher than 2.00 accounted for 58.3% of the total sludge. At the same time, the presence of iron decreased EPS levels, and also decreased the iron adsorption ability to sludge. More iron was transported into Anammox, which improved the nitrogen removal ability in the Anammox reactor.

Deciphering the toxic effects of antibiotics on denitrification: Process performance, microbial community and antibiotic resistance genes.

The extensive application of antibiotics, and the occurrence and spread of antibiotic resistance genes (ARGs) shade health risks to human and animal. The long-term effects of sulfamethoxazole (SMX) and tetracycline (TC) on denitrification process were evaluated in this study, with the focus on nitrogen removal performance, microbial community and ARGs. Results showed that low-concentration SMX and TC (<0.2 mg L-1) initially caused a deterioration in nitrogen removal performance, while higher concentrations (0.4-20 mg L-1) of both antibiotics had no further inhibitory influences. The abundances of ARGs in both systems generally increased during the whole period, and most of them had significant correlations with intI1, especially efflux-pump genes. Castellaniella, which was the dominant genus under antibiotic pressure, might be potential resistant bacteria. These findings provide an insight into the toxic effects of different antibiotics on denitrification process, and guides future efforts to control antibiotics pollution in ecosystems.

The occurrence, maintenance, and proliferation of antibiotic resistance genes (ARGs) in the environment: influencing factors, mechanisms, and elimination strategies.

Here, we review the possible reasons responsible for the occurrence, maintenance and proliferation of antibiotic resistance genes (ARGs) in the environment, as well as the corresponding mechanisms of their development, diffusion and transfer. Additionally, elimination strategies are also discussed. The factors that influence the development of ARGs are selection pressure, including that from antibiotics, metal and multiple other factors, co-resistance and cross-resistance, microbial consortium structure, nutrients in the environment and oxidative stress responses. Process parameters, transport pathways, and elimination strategies to reduce the health risk caused by ARGs are also reviewed in detail. Moreover, knowledge gaps and future opportunities of ARGs are addressed.

Short-term effects of nanoscale Zero-Valent Iron (nZVI) and hydraulic shock during high-rate anammox wastewater treatment.

The stability and resilience of an anaerobic ammonium oxidation (anammox) system under transient nanoscale Zero-Valent Iron (nZVI) (50, 75 and 100 mg L-1), hydraulic shock (2-fold increase in flow rate) and their combination were studied in an up-flow anaerobic sludge blanket reactor. The response to the shock loads can be divided into three phases i.e. shock, inertial and recovery periods. The effects of the shock loads were directly proportional to the shock intensity. The effluent quality was gradually deteriorated after exposure to high nZVI level (100 mg L-1) for 2 h. The higher effluent sensitivity index and response caused by unit intensity of shock was observed under hydraulic and combined shocks. Notably, the specific anammox activity and the content of heme c were considerably reduced during the shock phase and the maximum loss rates were about 30.5% and 24.8%, respectively. Nevertheless, the extracellular polymeric substance amount in the shock phase was enhanced in varying degrees and variation tendency was disparate at all the tested shock loads. These results suggested that robustness of the anammox system was dependent on the magnitude shocks applied and the reactor resistance can be improved by reducing hydraulic retention time with the increase of nZVI concentration under these circumstances.

Summary of the preservation techniques and the evolution of the anammox bacteria characteristics during preservation.

The anaerobic ammonium oxidation (anammox) process is a promising wastewater treatment method for biological nitrogen removal. A sufficient amount of active anammox sludge as a seed is crucial to the fast establishment and stability of the anammox process. Anammox bacteria is a kind of microorganism which is sensitive to the environmental conditions, e.g., oxygen, temperature. The optimum temperature and pH for the growth of the anammox bacteria are 30-40 °C and 6.7-8.3. A proper preservation technique allows fast start-up of the anammox process, overcoming the long doubling time of anammox biomass. The preservation of the anammox sludge is influenced by various factors, e.g., preservation techniques, duration, temperature, substrates, and protective agents. During preservation, the characteristics of the anammox biomass, including the bioactivity, heme c content, extracellular polymeric substances (EPS), and sludge morphology, change with time. The optimum preservation technique is not invariable and it depends on the purpose of preservation (precedence of bioactivity or quantity), the bacterial community, and other parameters. It is important for the preserved anammox biomass to achieve reactivation so that stable anammox reactors can be established as soon as possible. However, because the preservation process is complicated, the knowledge regarding preservation is far from complete, and much future work will be required to increase the understanding of preservation.

Long-term effects of heavy metals and antibiotics on granule-based anammox process: granule property and performance evolution.

The feasibility of the anaerobic ammonium oxidation (anammox) process to treat synthetic swine wastewater containing antibiotics and heavy metals was studied in this work. Nitrogen removal performance and granule characteristics were tracked by continuous-flow monitoring to evaluate the long-term joint effects of Cu and Zn and of Cu and oxytetracycline (OTC). Cu and Zn with a joint loading rate (JLR) of 0.04 kg m(-3) day(-1) did not affect the performance, while a JLR of 0.12 kg m(-3) day(-1) caused a rapid collapse in performance. Cu and OTC addition with a JLR of 0.04 kg m(-3) day(-1) for approximately 2 weeks induced significant nitrite accumulation. Granule characteristic analysis elucidated the disparate inhibition mechanisms of heavy metals and antibiotics: the internalization of heavy metals caused metabolic disorders, whereas OTC functioned as a growth retarder. However, anammox reactors could adapt to a JLR of 0.04 kg m(-3) day(-1) via self-regulation during the acclimatization to subinhibitory concentrations, which had a stable nitrogen removal rate (>8.5 kg m(-3) day(-1)) and removal rate efficiency (>75 %) for reactors with Cu-OTC addition. Therefore, this study supports the great potential of using anammox granules to treat swine wastewater.

Influence of preservation temperature on the characteristics of anaerobic ammonium oxidation (anammox) granular sludge.

Preserving active anaerobic ammonium oxidation (anammox) biomass is a potential method for securing sufficient seeding biomass for the rapid start-up of full-scale anammox processes. In this study, anammox granules were cultured in an upflow anaerobic sludge blanket (UASB) reactor (R0), and then the enriched anammox granules were preserved at 35, 20, 4, and -30 °C. The subsequent reactivation characteristics of the granules were evaluated in four UASB reactors (denoted R1, R2, R3, and R4, respectively) to investigate the effect of preservation temperature on the characteristics of anammox granules and their reactivation performance. The results demonstrated that 4 °C was the optimal preservation temperature for maintaining the biomass, activity, settleability, and integrity of the anammox granules and their cellular structures. During the preservation period, a first-order exponential decay model may be used to simulate the decay of anammox biomass and activity. The protein-to-polysaccharide ratio in the extracellular polymeric substances and the heme c content could not effectively indicate the changes in settleability and activity of the anammox granules, respectively, and a loss of bioactivity was positively associated with the degree of anaerobic ammonium-oxidizing bacteria cell lysis. After 42 days of storage, the anammox granules preserved at 4 °C (R3) exhibited a better recovery performance than those preserved at 20 °C (R2), -30 °C (R4), and 35 °C (R1). The comprehensive comparison indicated that 4 °C is the optimal storage temperature for anammox granular sludge because it promotes improved maintenance and recovery performance properties.

Analyzing the revolution of anaerobic ammonium oxidation (anammox) performance and sludge characteristics under zinc inhibition.

In the present study, the short- and long-term effects of Zn(II) on the anaerobic ammonium oxidation (anammox) performance and sludge characteristics were evaluated. The anammox activity decreased with increasing Zn(II) concentration and pre-exposure time in short-term tests. The half maximal inhibitory concentration (IC50) of Zn(II) was found to be 25.0 mg L(-1). The 24 and 48-h pre-exposure time was a restricted factor impacting the anammox activity, and washing the inhibited sludge with buffer solution only worked under 0 and 24-h pre-exposure time. The anammox sludge could tolerate 5 mg L(-1) Zn(II) but was suppressed at 8 mg L(-1). The inhibited performance could be remitted, as the combination strategies were applied, and after the short term of recovery period, the inhibited sludge characteristics were remitted to the normal.

Start-up of granule-based denitrifying reactors with multiple magnesium supplementation strategies.

In the present work, the effect of Mg(2+) supplementation on the start-up of a denitrification process and the granulation of denitrifying sludge was investigated in three upflow anaerobic sludge blanket (UASB) reactors. The reactors R1 and R2 were continuously and intermittently, respectively, supplied with 50 mg L(-1) Mg(2+), whereas R0 was used as the control. The nitrogen loading rate (NLR) and organic loading rate (OLR) gradually increased, and extremely high values were obtained (36.0 kgN m(-3) d(-1) and 216.0 kgCOD m(-3) d(-1), respectively). Granulation occurred in R1 first, but the reactor capacities were comparable. Suffering from starvation, the R0-R2 performances were comparable. At the end of the experiment, the average diameter of the granules in R0, R1, and R2 were 1.67, 1.72 and 1.68 mm, respectively, and the settling velocities of the granules in R1 and R2 were 1.14-fold the speed of R0. The specific denitrifying activity (SDA) of the sludge from the reactors supplied with Mg(2+) was greater than the reactor without Mg(2+). Intermittent Mg(2+) supplementation was identified as the best choice to be utilized to cultivate denitrifying granules, which was consistent with kinetic analysis.

Evidence for the cooccurrence of nitrite-dependent anaerobic ammonium and methane oxidation processes in a flooded paddy field.

Anaerobic ammonium oxidation (anammox) and nitrite-dependent anaerobic methane oxidation (n-damo) are two of the most recent discoveries in the microbial nitrogen cycle. In the present study, we provide direct evidence for the cooccurrence of the anammox and n-damo processes in a flooded paddy field in southeastern China. Stable isotope experiments showed that the potential anammox rates ranged from 5.6 to 22.7 nmol N2 g(-1) (dry weight) day(-1) and the potential n-damo rates varied from 0.2 to 2.1 nmol CO2 g(-1) (dry weight) day(-1) in different layers of soil cores. Quantitative PCR showed that the abundance of anammox bacteria ranged from 1.0 × 10(5) to 2.0 × 10(6) copies g(-1) (dry weight) in different layers of soil cores and the abundance of n-damo bacteria varied from 3.8 × 10(5) to 6.1 × 10(6) copies g(-1) (dry weight). Phylogenetic analyses of the recovered 16S rRNA gene sequences showed that anammox bacteria affiliated with "Candidatus Brocadia" and "Candidatus Kuenenia" and n-damo bacteria related to "Candidatus Methylomirabilis oxyfera" were present in the soil cores. It is estimated that a total loss of 50.7 g N m(-2) per year could be linked to the anammox process, which is at intermediate levels for the nitrogen flux ranges of aerobic ammonium oxidation and denitrification reported in wetland soils. In addition, it is estimated that a total of 0.14 g CH4 m(-2) per year could be oxidized via the n-damo process, while this rate is at the lower end of the aerobic methane oxidation rates reported in wetland soils.

Transient and long-term effects of bicarbonate on the ANAMMOX process.

In this study, the effects of both transient and long-term inorganic carbon (IC) addition on the anaerobic ammonium oxidation (ANAMMOX) process under pseudo-steady-state and substrate inhibitions were analyzed using reactor performance and measures of sludge activity. Compared with the nitrogen removal rate (NRR) of 3.42 kg N m(-3) day(-1) in the control bioreactor (ICDR) without IC, the peak NRR reached 21.0 kg N m(-3) day(-1) in the reactor (ICAR) with sufficient IC added. It was revealed that the long-term addition of bicarbonate significantly enhanced the performance of the ANAMMOX reactor. The optimum HCO3 (-)/TN ratio was considered to be 1.20, which is lower than that in normal conditions. The IC concentration affected biomass activity, and the transient addition or removal of IC to differing sludge media caused a significant loss of activity. Sufficient addition of IC alleviated the inhibition of excess substrates, while the inhibition was aggravated by the IC limitation. The half-maximal (50 %) inhibitory concentrations of substrate for the sludge were 295 mg L(-1) NO2 (-)-N and 361 mg L(-1) NH4 (+)-N with 120 mg L(-1) of fixed HCO3 (-) and 346 mg L(-1) NO2 (-)-N and 456 mg L(-1) NH4 (+)-N with unlimited IC, respectively. Changing the HCO3 (-)/TN (in milligrams per milligram) ratio resulted in the variation of ANAMMOX stoichiometric ratios. Sludge characterization parameters in the ICDR, including biomass, extracellular polymeric substances, heme C, and so on, were lower than those in ICAR. Filamentous bacteria and spherical bacteria were also observed in the reactor with limited IC.

Mechanistic insight into microbial interaction and metabolic pattern of anammox consortia on surface-modified biofilm carrier with extracellular polymeric substances.

The extremely slow growth rate of anaerobic ammonia oxidation (anammox) bacteria limits full-scale application of anammox process worldwide. In this study, extracellular polymeric substances (EPS)-coated polypropylene (PP) carriers were prepared for biofilm formation. The biomass adhesion rate of EPS-PP carrier was 12 times that of PP carrier, and EPS-PP achieved significant enrichment of E. coli BY63. The 120-day continuous flow experiment showed that the EPS-PP carrier accelerated the formation of anammox biofilm, and the nitrogen removal efficiency increased by 10.5 %. In addition, the abundance of Candidatus Kuenenia in EPS-PP biofilm was 27.1%. Simultaneously, amino acids with high synthesis cost and the metabolites of glycerophospholipids related to biofilm formation on EPS-PP biofilm were significantly up-regulated. Therefore, EPS-PP carriers facilitated the rapid formation of anammox biofilm and promoted the metabolic activity of functional bacteria, which further contributed to the environmental and economic sustainability of anammox process.