Individual evaluation of nitrite and free nitrous acid inhibition on anammox activity.

The anammox reaction simultaneously utilizes ammonia and nitrite as substrates; however, high nitrite concentrations act as strong inhibitors of the reaction. In this study, inhibition by NO2- and free nitrous acid (FNA) was separately evaluated in continuous feeding tests using different biomass carriers. The influent NO2- concentration was increased under pH 7.6, where FNA is less likely to affect anammox activity. A continuous test using polyethylene glycol (PEG) gel carriers containing immobilized anammox bacteria showed that the inhibition ratio was 13% when the NO2--N concentration in the reactor was 350 mg L-1 (FNA ≤0.06 mg L-1). The relationship between NO2- concentration in the reactor and inhibition ratio increased linearly. Evaluation of the inhibitory effect of FNA by increasing the influent NO2- concentration at pH 6.4, where FNA is easily formed, demonstrated that the relationship between FNA and inhibition ratio could be fitted to a sigmoid curve, and the 50% inhibitory concentration (IC50) of FNA was 0.88 mg L-1. A similar test performed using polyvinyl alcohol carriers containing anammox bacteria on their surface showed the same trend as the PEG gel carriers, with the IC50 for FNA at 0.70 mg L-1. These results indicate that the inhibitory effect of FNA on anammox activity was greater than that of NO2-. The evaluation of these two factors helped identify important operational indicators of the stable application of anammox processes.

Long-Term Limitation Effects of Se(VI), Zn(II), and Ni(II) on Start-Up of the Anammox Process Using Gel Carrier.

Anaerobic ammonia oxidation (anammox) bacteria are inhibited by heavy metals at high concentrations but require trace amounts of some heavy-metal elements for growth and activity maintenance. The present study evaluates the long-term limitation effects of Se(VI), Zn(II), and Ni(II) on the start-up period of an anammox reactor. To strictly limit the levels of heavy metals in the reactor, all tests used ultrapure water as the influent synthetic wastewater and all reactors were installed in a clean booth. The anammox biomass was maintained through the gel entrapment technique. In the absence of Se(VI) and Ni(II), the anammox reactor start-up was 18.9 kg-N (m3-carrier d)-1 (nitrogen conversion rate (NCR) per gel carriers), indicating that Se(VI) and Ni(II) are not required or need not be continuously added to maintain the anammox process. Under Zn(II) limitation, the anammox process failed to start-up and the NCR tended to decrease rapidly. After readdition of 0.005 mg L-1 of Zn(II), the NCR did not decline further and instead partially recovered at a very slow rate. The NCR was completely recovered after adding 0.020 mg L-1 of Zn(II). These results reveal that Zn(II) limitation seriously affects the start-up of the anammox process while Se(VI) and Ni(II) are not required or need not be continuously added to the anammox process.

Effect of inorganic carbon limitation on the nitrogen removal performance of the single-stage reactor containing anammox and nitritation gel carriers.

Herein, the effect of inorganic carbon (IC) limitation on the nitrogen removal performance of the single-stage reactor containing nitritation and anammox gel carriers was investigated. As a result of a continuous feeding test, the effluent ammonium concentration increased as the IC concentration decreased, indicating the deterioration of nitritation activity, not anammox. Furthermore, the sensitivity of IC to anammox and nitritation activity was investigated in anammox and nitritation reactors, respectively. Consequently, the relationship between the effluent IC concentration and nitritation rate was well described using the Michaelis-Menten equation. The apparent Km value of nitritation was calculated as 4.4 mg-C L-1. In anammox reactor, it was calculated as 1.7 mg-C L-1. These results revealed that the affinity of nitritation gel carriers to IC was lower than that of anammox, supporting that nitritation activity was easily deactivated by decrease in the IC concentration rather than anammox. Microbial community analysis revealed that Nitrosomonas europaea and Candidatus Jettenia asiatica were the dominant species of ammonium-oxidizing and anammox bacteria.

Nitrogen and Oxygen Isotope Signatures of Nitrogen Compounds during Anammox in the Laboratory and a Wastewater Treatment Plant.

Isotopic fractionation factors against 15N and 18O during anammox (anaerobic ammonia oxidization by nitrite) are critical for evaluating the importance of this process in natural environments. We performed batch incubation experiments with an anammox-dominated biomass to investigate nitrogen (N) and oxygen (O) isotopic fractionation factors during anammox and also examined apparent isotope fractionation factors during anammox in an actual wastewater treatment plant. We conducted one incubation experiment with high δ18O of water to investigate the effects of water δ18O. The N isotopic fractionation factors estimated from incubation experiments and the wastewater treatment plant were similar to previous values. We also found that the N isotopic effect (15εNXR of -77.8 to -65.9‰ and 15ΔNXR of -31.3 to -30.4‰) and possibly O isotopic effect (18εNXR of -20.6‰) for anaerobic nitrite oxidation to nitrate were inverse. We applied the estimated isotopic fractionation factors to the ordinary differential equation model to clarify whether anammox induces deviations in the δ18O vs δ15N of nitrate from a linear trajectory of 1, similar to heterotrophic denitrification. Although this deviation has been attributed to nitrite oxidation, the O isotopic fractionation factor for anammox is crucial for obtaining a more detailed understanding of the mechanisms controlling this deviation. In our model, anammox induced the trajectory of the δ18O vs δ15N of nitrate during denitrification to less than one, which strongly indicates that this deviation is evidence of nitrite oxidation by anammox under denitrifying conditions.

Quantitative evaluation of inhibitory effect of various substances on anaerobic ammonia oxidation (anammox).

The inhibitory effect of 20 substances of various chemical species on the anaerobic ammonia oxidation (anammox) activity of an enrichment culture, predominated by Candidatus Brocadia, was determined systematically by using a 15N tracer technique. The initial anammox rate was determined during first 25 min with a small-scale anaerobic batch incubation supplemented with possible inhibitors. Although Cu2+ and Mn2+ did not inhibit anammox, the remaining 18 substances [Ni2+, Zn2+, Co2+, [Formula: see text] , Fe2+, 4 amines, ethylenediaminetetraacetic acid (EDTA), ethylenediamine-N,N'-bis (2-hydroxyphenylacetic acid) (EDDHA), citric acid, nitrilotriacetic acid (NTA), N,N-dimethylacetamide (DMA), 1,4-dioxane, dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF) and tetrahydrofuran (THF)] were inhibitory. Inhibitory effect of NTA, EDDHA, THF, DMF, DMA and amines on anammox was first determined in this study. Inhibitory effects of metals were re-evaluated because chelators, which may interfere inhibitory effect, have been used to dissolve metal salts into assay solution. The relative anammox activities as a function of concentration of each substance were described successfully (R2 > 0.91) either with a linear inhibition model or with a Michaelis-Menten-based inhibition model. IC50 values were estimated based on either model, and were compared. The IC50 values of the 4 chelators (0.06-2.7 mM) and 5 metal ions (0.02-1.09 mM) were significantly lower than those of the 4 amines (10.6-29.1 mM) and 5 organic solvents (3.5-82 mM). Although it did not show any inhibition within 25 min, 0.1 mM Cu2+ completely inhibited anammox activity in 240 min, suggesting that the inhibitory effect caused by Cu2+ is time-dependent.

Biological wastewater treatment of 1,4-dioxane using polyethylene glycol gel carriers entrapping Afipia sp. D1.

A biological treatment system for 1,4-dioxane-containing wastewater was developed using the bacterium Afipia sp. D1, which can utilize 1,4-dioxane as the sole carbon source. Strain D1 was entrapped in a polyethylene glycol gel carrier to stably maintain it in a bioreactor, and continuous feeding tests were performed to treat model industrial wastewater containing 1,4-dioxane. 1,4-Dioxane removal activity rapidly increased soon after the start of feeding of influent with 400 mg/L 1,4-dioxane, and the volumetric removal rate reached 0.67 kg dioxane/m(3)/d on day 36 by a stepwise increase in loading. The start-up period of the 1,4-dioxane treatment reactor was approximately 1 month, and stable removal performance was subsequently achieved for more than 1 month. The average 1,4-dioxane effluent concentration and 1,4-dioxane removal efficiency were 3.6 mg/L and 99%, respectively, during stable operation. Further 1,4-dioxane degradation activity of the of the gel carrier was characterized in batch experiments with respect to temperature. The optimum temperature for 1,4-dioxane treatment was 31.7°C, and significant removal was observed at a temperature as low as 6.9°C. The apparent activation energy for 1,4-dioxane degradation was estimated to be 47.3 kJ/mol. This is the first report of the development of a 1,4-dioxane biological treatment system using gel entrapment technology.

Pilot test of biological removal of 1,4-dioxane from a chemical factory wastewater by gel carrier entrapping Afipia sp. strain D1.

A pilot-scale (120 L) bioreactor system using a gel carrier-entrapped pure bacterial strain, Afipia sp. strain D1, capable of degrading 1,4-dioxane as a sole carbon and energy source was constructed and applied to treat real industrial wastewater containing 1,4-dioxane from a chemical factory. Although the wastewater not only contained high concentrations of 1,4-dioxane but also considerable amounts of other organic compounds (73 mg-TOCL(-1) on average), the bioreactor could efficiently remove 1,4-dioxane without significant inhibitory effects. The reactor startup could be completed within approximately 1 month by increasing the 1,4-dioxane loading rate (0.09-0.47 kg-dioxanem(-3)d(-1)) in a stepwise manner. Effective 1,4-dioxane removal was stably maintained for 3 months with an influent 1,4-dioxane of 570-730 mg L(-1), giving an average effluent concentration and removal rate of 3.4 mg L(-1) and 0.46 kg-dioxanem(-3)d(-1), respectively. A 1,4-dioxane loading fluctuation between 0.14 and 0.72 kg-dioxanem(-3)d(-1) did not significantly affect its removal, and more than 99% removal efficiency was constantly maintained. The Monod model could well describe the relationship between the effluent 1,4-dioxane concentration and 1,4-dioxane removal rates of the bioreactors, showing that the half-saturation constant (Ks) was 28 mg L(-1).

Evaluation of inhibitory effects of heavy metals on anaerobic ammonium oxidation (anammox) by continuous feeding tests.

To facilitate the application of anaerobic ammonium oxidation (anammox) to a nitrogen removal process, the effects of heavy metals (Ni, Cu, Co, Zn, and Mo) on anammox bacteria entrapped in gel carriers were examined by conducting continuous feeding tests for each metal. The results show that all anammox activities decreased by more than 10 % when influent concentrations of Ni, Cu, Co, Zn, and Mo were 5, 5, 5, 10, and 0.2 mg/L, respectively. It was observed that the effects of Ni, Cu, Co, and Zn on anammox activity were reversible and that of Mo on anammox activity was irreversible. Anammox activity was not affected when influent containing mixed Ni, Cu, Co, and Zn (0.5 mg/L) was fed into the reactor.

Complete autotrophic denitrification in a single reactor using nitritation and anammox gel carriers.

A novel aerobic denitrification reactor, aerobic denitrification using nitrifying and anoxic ammonium-oxidizing (anammox) bacteria immobilized on gel carriers in a single stage (AIGES), was developed. Two types of gel carriers, a nitritation gel carrier and an anammox gel carrier, were installed in single reactor, and the denitrification performance of simultaneous nitritation and anammox was evaluated. The denitrification performance increased gradually with increased aeration rate, reaching a denitrification rate of 1.4 kg N m(-3) d(-1) 2 weeks after the nitritation and anammox gel carriers were mixed. A high average denitrification efficiency of 82% was confirmed. Stable aerobic denitrification performance was observed for more than half a year. In the startup period of AIGES operation, ammonia-oxidizing bacteria were shown by fluorescence in situ hybridization analysis to grow on the surface layer of anammox gel cubes. These results indicated that anammox gel carriers promptly adapted to an aerobic environment by altering the microbial ecosystem.

Stability of autotrophic nitrogen removal system under four non-steady operations.

Stable nitrogen removal from the digester supernatant for sludge via the nitritation-anammox process under steady operations of ammonium concentration and flow rate has been often reported. In this study, the effects of four non-steady operations, intentional fluctuations of influent concentration from 890 to 650 mg-N/L and hydraulic load of the 10% increase, temporally shutdown for 3-d and maximum capacity of each reactor, were evaluated in the nitritation-anammox process treating digester supernatant for sludge. No serious effects were observed in the anammox reactor because the aeration-control system in the nitritation reactor responded and controlled the nitritation efficiency satisfactorily against intentional fluctuations and temporally shutdown. Finally, the maximum capacity of each reactor was evaluated, and the nitritation rate was found to be 2.3 kg-N/m(3)/d at a DO of 4.0mg/L, and the nitrogen-conversion rate was 9.0 kg-N/m(3)/d.

High-rate denitrification using polyethylene glycol gel carriers entrapping heterotrophic denitrifying bacteria.

This study evaluated the nitrogen removal performance of polyethylene glycol (PEG) gel carriers containing entrapped heterotrophic denitrifying bacteria. A laboratory-scale denitrification reactor was operated for treatment of synthetic nitrate wastewater. The nitrogen removal activity gradually increased in continuous feed experiments, reaching 4.4 kg N m(-3) d(-1) on day 16 (30 °C). A maximum nitrogen removal rate of 5.1 kg N m(-3) d(-1) was observed. A high nitrogen removal efficiency of 92% on average was observed at a high loading rate. In batch experiments, the denitrifying gel carriers were characterized by temperature. Nitrate and total nitrogen removal activities both increased with increasing temperature, reaching a maximum at 37 and 43 °C, respectively. Apparent activation energies for nitrate and nitrite reduction were 52.1 and 71.9 kJ mol(-1), respectively. Clone library analysis performed on the basis of the 16S rRNA gene revealed that Hyphomicrobium was mainly involved in denitrification in the methanol-fed denitrification reactors.

Temperature dependence for anammox bacteria enriched from freshwater sediments.

The anoxic ammonium oxidation (anammox) process has been regarded as an attractive alternative process to treat wastewater containing high ammonium concentrations. By the implementation of anammox process at moderately low temperatures (<25°C), the anammox process will be applied to more various industrial wastewater treatments. In this study, we established enrichment cultures of anammox bacteria from freshwater sediments by using an up-flow column reactor equipped with porous polyester nonwoven fabric at moderately low temperatures. Their nitrogen conversion rates reached 0.07-0.26 kg-N/m³/d. Phylogenetic analysis based on 16S rRNA gene from enrichment cultures revealed the presence of various anammox bacteria affiliated with unknown anammox bacteria as well as known anammox candidates, i.e., Candidatus Kuenenia stuttgartiensis and Candidatus Brocadia fulgida, Candidatus Scalindua wagneri. Anammox bacterial populations were influenced by enrichment conditions, i.e., seed sediments and temperature.

Novel autotrophic nitrogen removal system using gel entrapment technology.

A pilot plant involving a nitritation-anammox process was operated for treating digester supernatant. In the preceding nitritation process, ammonium-oxidizing bacteria were immobilized in gel carriers, and the growth of nitrite-oxidizing bacteria was suppressed by heat-shock treatment. For the following anammox process, in order to maintain the anammox biomass in the reactor, a novel process using anammox bacteria entrapped in gel carriers was also developed. The nitritation performance was stable, and the average nitrogen loading and nitritation rates were 3.0 and 1.7 kg Nm(-3)d(-1), respectively. In the nitritation process, nitrate production was completely suppressed. For the anammox process, the startup time was about two months. Stable nitrogen removal was achieved, and an average nitrogen conversion rate of 5.0 kg Nm(-3)d(-1) was obtained. Since the anammox bacteria were entrapped in gel carriers, stable nitrogen removal performance was attained even at an influent suspended solids concentration of 1500 mg L(-1).

Effects of inorganic carbon limitation on anaerobic ammonium oxidation (anammox) activity.

Anammox bacteria are chemoautotrophic bacteria that oxidize ammonium with nitrite as the electron acceptor and with CO(2) as the main carbon source. The effects of inorganic carbon (IC) limitation on anammox bacteria were investigated using continuous feeding tests. In this study, a gel carrier with entrapped anammox sludge was used. It was clearly shown that the anammox activity deteriorated with a decrease in the influent IC concentration. The relationship between the influent IC concentration and the anammox activity was analyzed using Michaelis-Menten kinetics, and the apparent K(m) was determined to be 1.2mg-C/L. The activity could be recovered by adding IC to the influent. The consumption ratio of IC to ammonium was not constant and mainly depended on the influent ratio of the IC to ammonium concentrations (inf.IC/inf.NH(4)-N). The results indicated that an inf.IC/inf.NH(4)-N ratio of 0.2 in the anammox reactor was ideal for the anammox process using gel cubes.

Effects of nitrite inhibition on anaerobic ammonium oxidation.

In order to assess the stability of nitrogen removal systems utilizing anaerobic ammonium oxidation (anammox), it is necessary to study the toxic effects of nitrite on these biochemical reactions. In this study, the effects of nitrite on anammox bacteria entrapped in gel carriers were investigated using batch and continuous feeding tests. The results showed that the nitrite concentration in a reactor must be less than 274-mg N/L in order to prevent a decrease in the anammox activity, which occurred when the gel carriers were soaked in nitrite solutions with concentrations greater than 274-mg N/L in a batch test. In a continuous feeding test, nitrite inhibition was not observed at low concentrations of nitrite. However, the anammox activity decreased to 10% when the nitrite concentration increased to 750-mg N/L over a 7-day period in the reactor. In addition, it was shown that the effects of nitrogen on the anammox reaction were reversible because the anammox activity completely recovered within 3 days when the influent nitrite concentration was decreased to less than 274-mg N/L.

High-rate nitrogen removal from livestock manure digester liquor by combined partial nitritation-anammox process.

In this study, combination of a partial nitritation reactor, using immobilized polyethylene glycol (PEG) gel carriers, and a continuous stirred granular anammox reactor was investigated for nitrogen removal from livestock manure digester liquor. Successful nitrite accumulation in the partial nitritation reactor was observed as the nitrite production rate reached 2.1 kg-N/m(3)/day under aerobic nitrogen loading rate of 3.8 kg-N/m(3)/day. Simultaneously, relatively high free ammonia concentrations (average 50 mg-NH(3)/l) depressed the activity of nitrite oxidizing bacteria with nitrate concentration never exceeding 3% of TN concentration in the effluent of the partial nitritation reactor (maximum 35.2 mg/l). High nitrogen removal rates were achieved in the granular anammox reactor with the highest removal rate being 3.12 kg-N/m(3)/day under anaerobic nitrogen loading rate of 4.1 kg-N/m(3)/day. Recalcitrant organic compounds in the digester liquor did not impair anammox reaction and the SS accumulation in the granular anammox reactor was minimal. The results of this study demonstrated that partial nitritation-anammox combination has the potential to successfully remove nitrogen from livestock manure digester liquor.

Microbial diversity of anammox bacteria enriched from different types of seed sludge in an anaerobic continuous-feeding cultivation reactor.

Enrichment of anammox bacteria from three types of seed sludge, sewage, digester, and nitrification sludges, was conducted using a nonwoven fabric carrier for immobilizing the anammox bacteria, and the microbial diversity of the enriched anammox culture was investigated. About four months later, simultaneous removals of ammonium and nitrite, and production of a small amount of nitrate, which is unique to the anammox reaction, were observed in all 3 sludge reactors. Results of 16S rRNA gene analysis indicated that anammox bacteria were cultivated and diversified in each sludge type. Moreover, the microbial diversity of anammox bacteria was higher in the enriched culture from sewage sludge compared to the other two types of seed sludge. Bacillus sp. coexisted in the anammox culture cultivated from sewage sludge. These results suggest that differences in the anammox community in the enriched culture were caused by differences in the type of seed sludge.

Innovative treatment system for digester liquor using anammox process.

This study demonstrated that partial nitritation using nitrifying activated sludge entrapped in a polyethylene glycol (PEG) gel carrier, as a pretreatment to anammox process, could be successfully applied to digester liquor of biogas plant at a nitrogen loading rate of 3.0 kg-N/m(3)/d. The nitritation process produced an effluent with a NO(2)-N/NH(4)-N ratio between 1.0 and 1.4, which was found to be suitable for the subsequent anammox process. A high SS concentration (2000-3000 mg/l) in the digester liquor did not affect partial nitritation treatment performances. Effluent from this partial nitritation reactor was successfully treated in the anammox reactor using anammox sludge entrapped in the PEG gel carrier with T-N removal rates of greater than 4.0 kg-N/m(3)/d. Influent BOD and SS contents did not inhibit anammox activity of the anammox gel carrier. The combination of partial nitritation and anammox reactors using PEG entrapped nitrifying and anammox bacteria was shown to be effective for the removal of high concentration ammonium in the digester liquor of a biogas plant.

Anaerobic ammonium oxidation (anammox) irreversibly inhibited by methanol.

Methanol inhibition of anaerobic ammonium oxidation (anammox) activity was characterized. An enrichment culture entrapped in a polyethylene glycol gel carrier was designed for practical uses of wastewater treatment. Batch experiments demonstrated that anammox activity decreased with increases in methanol concentration, and relative activity reached to 29% of the maximum when 5 mM methanol was added. Also, batch experiments were conducted using anammox sludge without immobilization. Anammox activity was evaluated by quantifying (14)N(15)N ((29)N) emission by combined gas chromatography-quadrupole mass spectrometry, and the anammox activity was found to be almost as sensitive to methanol as in the earlier trials in which gel carriers were used. These results indicated that methanol inhibition was less severe than previous studies. When methanol was added in the influent of continuous feeding system, relative activity was decreased to 46% after 80 h. Although the addition was halted, afterwards the anammox activity was not resumed in another 19 days of cultivation, suggesting that methanol inhibition to anammox activity was irreversible. It is notable that methanol inhibition was not observed if anammox activity was quiescent when substrate for anammox was not supplied. These results suggest that methanol itself is not inhibitory and may not directly inhibit the anammox activity.

Nitrogen removal performance using anaerobic ammonium oxidation at low temperatures.

An anaerobic ammonium oxidation (anammox) process for ammonia-rich wastewater treatment has not been reported at temperatures below 15 degrees C. This study used a gel carrier with entrapped anammox bacteria to obtain a stable nitrogen removal performance at low temperatures. In a continuous feeding test, a high nitrogen conversion rate (6.2 kg N m(-3) day(-1)) was confirmed at 32 degrees C. Nitrogen removal activity decreased gradually with decreasing operation temperature; however, it still occurred at 6 degrees C. Nitrogen conversion rates at 22 and 6.3 degrees C were 2.8 and 0.36 kg N m(-3) day(-1), respectively. Moreover, the stability of anammox activity below 20 degrees C was confirmed for more than 130 days. In batch experiments, anammox gel carriers were characterized with respect to temperature. The optimum temperature for anammox bacteria was found to be 37 degrees C. Furthermore, it was clear that the temperature dependence changed at about 28 degrees C. The apparent activation energy in the temperature range from 22 to 28 degrees C was calculated as 93 kJ mol(-1), and that in the range from 28 to 37 degrees C was 33 kJ mol(-1). This value agrees with the result of a continuous feeding test (94 kJ mol(-1), between 6 and 22 degrees C). The nitrogen removal performance demonstrated at the low temperatures used in this study will open the door for the application of anammox processes to many types of industrial wastewater treatment.