Kazuichi Isaka

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.

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.

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.

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.

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.

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).

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.

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.

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.

Microbial community of anammox bacteria immobilized in polyethylene glycol gel carrier

Anaerobic ammonium oxidation (anammox) is a recently discovered microbial pathway in the biological nitrogen cycle and a new cost-effective way to remove ammonium from wastewater. We have so far developed new immobilization technique that anammox bacteria entrapped in polyethylene glycol (PEG) gel carrier. However, fate and behavior of anammox bacteria in a gel carrier is not well understood. In the present study, we focused on the population changes of anammox bacteria in a gel carrier. Three specific primer sets were designed for real-time PCR. For quantification of anammox bacteria in a gel carrier, real-time PCR was performed. The anammox bacteria related to HPT-WU-N03 clone were increased the rate in anammox population, and found to be a major population of anammox bacteria in a gel carrier. Furthermore, from the results of nitrogen removal performance and quantification of anammox bacteria, the correlation coefficient between copy numbers of anammox bacteria and nitrogen conversion rate was calculated as 0.947 in total anammox population. This is the first report that population changes of anammox bacteria immobilized in a gel carrier were evaluated.