Effect of nitrite concentration on the distribution and competition of nitrite-oxidizing bacteria in nitratation reactor systems and their kinetic characteristics
Introduction
Recent interest in novel nitrogen removal technologies via nitrite pathway, such as SHARON and Anammox process, attract researchers’ interest because of their economic advantages in saving costs of aeration and organic carbons (Schmidt et al., 2003). The above processes rely on the competition, elimination or inhibition of nitrite-oxidizing bacteria (NOB) so that the oxidation of nitrite to nitrate is blocked. Nitrite concentration in the conventional nitrogen removal wastewater treatment remains very low by the simultaneous transformation of nitrite to nitrate. Several mechanisms are suggested for the inhibition of nitrite oxidation (Anthonisen et al., 1976; Hellinga et al., 1998; Kuai and Verstraete, 1998; Philips et al., 2002), but they are not always equally effective and it is believed that the nitrite oxidation rate and degree of inhibition are different for each NOB species.
Former studies on NOB identification in wastewater treatment plants showed complicated and ambiguous results. Nitrobacter had been known as the key NOB in wastewater treatment plants (Grady and Lim, 1980). However, the opinion was dramatically challenged when no Nitrobacter-related organisms were detected in nitrifying activated sludge samples by fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes specific for the genus Nitrobacter (Wagner et al., 1996). Further investigations with 16S rDNA sequencing and in situ hybridization using oligonucleotide probe revealed that genus Nitrospira was the responsible NOB and Nitrobacter species were rarely found in nitrification systems (Burrell et al., 1998; Juretschko et al., 1998; Schramm et al., 1998, Schramm et al., 1999) indicating that Nitrospira species, not Nitrobacter, were numerically dominant and carried out nitrite oxidation in the nitrifying bioreactors. In sewage treatment plants Nitrospira occurs predominantly embedded in cell aggregates such as flocs or biofilms (Juretschko et al., 1998; Koops and Pommerening-Roser, 2001). Unfortunately, all attempts to isolate Nitrospira from activated sludge and to grow them in pure culture have been unsuccessful and, consequently, the knowledge of the microbiology of Nitrospira species is still limited.
On the other hand, Daims et al. (2001a) reported that Nitrobacter and Nitrospira-like bacteria co-existed in a nitrifying sequencing batch reactor (SBR) where nitrite concentration varies significantly during an operation cycle. They suggested that Nitrobacter could take advantage of the temporarily elevated nitrite concentration and compete successfully with Nitrospira in the SBR. The co-existence of Nitrospira and Nitrobacter was also observed in a full scale activated sludge plant (Coskuner and Curtis, 2002) and a leachate treatment plant where nitrite concentration was highly maintained (Kim et al., 2006). Recently, Kelly et al. (2005) also reported that membrane hybridization format and terminal restriction fragment length polymorphism (T-RFLP) fingerprinting revealed that both Nitrospira and Nitrobacter were contributing populations in a full-scale activated sludge treatment plant.
Physiological study on NOB showed that under aerobic conditions Nitrospira species only took up inorganic carbon and pyruvate but not acetate, butyrate, and propionate while many species of Nitrobacter were able to grow mixotrophically with the above organic compounds (Prosser, 1989). In addition, no uptake by Nitrospira of any of the carbon sources was observed under anoxic or anaerobic conditions (Daims et al., 2001a) while Nitrobacter utilized pyruvate in an anoxic condition (Bock et al., 1988). Therefore, Nitrobacter species might have competitiveness over Nitrospira because they can utilize organic components in an aerobic or anoxic condition. The above result also supports the possibility of the co-existence of Nitrobacter and Nitrospira in a nitrogen removal activated sludge plant by nitrification and denitrification.
With respect to kinetic characteristics of NOB, Schramm et al. (1999) suggested that Nitrospira represent K strategists adapted to low nitrite and oxygen concentrations, while Nitrobacter, as an r strategist, thrives if nitrite and oxygen are present in higher concentrations. The K/r hypothesis was also supported by the fact that Nitrospira can be enriched from activated sludge to a certain degree when nitrite concentration in the growth medium is low, while higher nitrite concentrations select for Nitrobacter (Bartosch et al., 2002). Nevertheless, no quantitative data from continuous reactor systems have been reported to confirm the hypothesis in engineered systems except Wagner et al. (2002) who compared the dominance of NOB after addition of Nitrobacter to the chemostats. Some pure culture experiments with Nitrobacter species showed that the specific nitrite-oxidizing activities were 5.1–42 fmol/cell h (Prosser, 1989). Ehrich et al. (1995) reported the specific nitrite-oxidizing activity of Nitrospira moscoviensis, which is isolated from a corroded iron pipe of a heating system in Moscow, to be 6.3 fmol/cell h. It is hard to tell that Nitrobacter is an r-strategist from the above specific activities, moreover, considering the potential inaccuracy of the most probable number (MPN) technique used for enumerating NOB.
In situ analysis with microelectrodes estimated the nitrite-oxidizing activity of Nitrospira as 0.02 fmol/cell h in a nitrifying biofilm system where ammonia-oxidizing bacteria (AOB) compete with NOB for oxygen (Schramm et al., 1999). The AOB might have out-competed NOB for oxygen because AOB are thought to possess lower Ko values for oxygen than NOB (Focht and Verstraete, 1977; Prosser, 1989). Nitrite-oxidizing activity may also be limited by the activity of AOB which provide nitrite to NOB. For the above reasons, the estimated in situ activity may be different from the physiological potential of NOB. No in situ activities of Nitrobacter in wastewater treatment systems are available yet. Therefore, it is meaningful to investigate the distribution and specific activities of Nitrospira and Nitrobacter in mixed culture systems.
The objectives of this study were to investigate the effects of nitrite concentration, nitrite load, and starvation conditions as a selection pressure on the distribution of Nitrobacter and Nitrospira in two different nitratation reactor systems, and possibly to measure specific nitrite-oxidizing activities of Nitrobacter and Nitrospira, and finally to examine the validity of the K/r hypothesis. For the purpose, a continuous biofilm airlift reactor (CBAR) running under nitrite limiting conditions and an SBR with high nitrite conditions were operated for 90 days. Nitrite was supplied to the reactors as the only energy source in order to eliminate the growth of ammonia oxidizers, and subsequently to provide more realistic nitrite-oxidation activity.
The distribution of NOB in the biofilm and sludge flocs was determined by FISH with confocal laser scanning microscopy (CLSM). The specific nitrite oxidation activities of each NOB were also estimated from the results of nitrite oxidation kinetics and NOB concentration measurements in the biofilm samples by quantitative FISH image analyses.
Section snippets
Bioreactors for nitrite oxidation
Both CBAR and SBR used the same size of airlift-type reactors which have a concentric tube inside equipped with a three-phase separator and a total reactor volume of 5 L. Height, riser diameter and down comer diameter of the reactor were 77, 4 and 7 cm, respectively. The reactors were operated in parallel at pH 7.2 by the addition of 1 M HCl and 1 M NaHCO3, and the temperature was maintained at 22–26 °C.
Basalt with a mean diameter of 200 μm was used as a carrier material for biofilm development in
Nitrite oxidation in CBAR and SBR
The CBAR was continuously operated for 90 days with nitrite as the sole energy source and Fig. 1 shows the operation results. In phase A, the inlet nitrite concentration and load were kept at 200 mg N/L and 0.29 kg N/m3 d, respectively. In phase B, the load was increased to 0.73 kg N/m3 d by increasing the inlet nitrite concentration to 500 mg N/L. In both phases, nitrite was completely oxidized to nitrate and nitrite concentration was maintained at nearly zero. Dissolved oxygen was maintained at 4–7 mg/L
Conclusions
In the continuous biofilm airlift reactor where nitrite concentration was maintained very low, Nitrospira (59%) was the dominant NOB while Nitrobacter occupied about 5% of total bacteria. In the sequencing batch reactor where nitrite concentration was relatively high, Nitrobacter (64%) was the dominant NOB while Nitrospira occupied only 3% of total bacteria. In situ analysis showed that the specific activity of Nitrobacter (93.8 mg/g NOB h) is higher than that of Nitrospira (10.5 mg/g NOB h). The
Acknowledgments
This research was supported by the research grant from Hallym University, Korea. Support from Korea Research Foundation Grant (KRF-2004-041-D00401) is also acknowledged.
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