Nitrospiralike Bacteria In Wastewater Treatment Plants

Aerobic nitrification is a two-stage process driven by chemolithoautotrophic bacteria: ammonia oxidizers like Nitrosomonas species convert ammonia to nitrite, and nitrite oxidizers like Nitrobacter species oxidize nitrite further to nitrate. According to textbook knowledge, Nitrosomonas and Nitrobacter species catalyze aerobic nitrification in wastewater treatment plants.90,91 The widespread opinion that Nitrobacter species are the predominant nitrite oxidizers in wastewater treatment arose because these bacteria can be isolated from most nitrifying activated sludge samples. However, this concept was challenged when molecular techniques were applied on nitrite-oxidizing bacteria in nitrifying bioreactors. Quantitative dot blot or FISH with rRNA-targeted probes failed to detect Nitrobacter in nitrifying aquarium Alters92 or in activated sludge,93 respectively. These findings launched a hunt for the real nitrite oxidizers in such habitats. Two years later, bacteria related to the genus Nitrospira were detected by 16S rRNA sequence analysis in a nitrite-oxidizing, laboratory-scale bioreactor.94 At approximately the same time, Hovanec et al.95 found 16S rRNA sequences of Nitrospira--like bacteria in biofilms from freshwater aquaria and confirmed, by quantitative dot blot with Nitrospira-specific oligonuc-leotide probes, that these organisms were highly abundant in the biofilms. The genus Nitrospira contains only two cultured species, Nitrospira marina,96 and Nitrospira moscoviensis91 which are chemolithoautotrophic nitrite oxidizers. These organisms belong to a deep-branching bacterial lineage (the phylum Nitrospirae), which is not related to the genus Nitrobacter.91 The detection of uncultured Nitrospira-like bacteria in nitrifying bioreactors was a surprise because the two cultured Nitros-pira species had never been found in similar habitats. However, it was still unclear whether the Nitrospira-like bacteria, which had been detected in laboratory-scale reactors and in aquaria, were also key nitrite oxidizers in full-scale wastewater treatment plants. The importance of these organisms for wastewater treatment was confirmed when FISH with a Nitrospira-specific, rRNA-targeted probe revealed great abundance of Nitrospira-like bacteria in a large industrial wastewater treatment plant.9 All attempts to isolate and culture these Nitrospira-like bacteria were unsuccessful. Interestingly, a Nitrobacter strain could be isolated from the same activated sludge although FISH failed to detect Nitrobacter cells in situ, indicating that the cell density of Nitrobacter in the sludge was much lower than the density of Nitrospira-like bacteria. This discovery made clear that the apparent importance of Nitrobacter for wastewater treatment was merely an artifact of cultivation-dependent methods to detect bacteria in environmental samples. The key role of Nitrospira-like bacteria for nitrite oxidation in wastewater treatment was confirmed by later studies, which used FISH and microelectrodes in order to correlate local abundance of Nitrospira-like bacteria with zones of active nitrite oxidation in biofilms.32,98,99

The kinetics of nitrification was studied decades ago with pure cultures of Nitrosomonas and Nitrobacter species. The results have influenced the design and operation of nitrifying bioreactors in all industrialized countries. In particular, industrial wastewater treatment plants often suffer from unpredictable irregularities or even total breakdowns of nitrification performance. Many of these problems may be caused by yet unknown biological differences between Nitrobacter, the model organism for bioreactor design, and Nitrospira-like bacteria, which are the real nitrite oxidizers in the bioreactors. The physiology of these Nitrospira-like bacteria is difficult to investigate because pure cultures are not available and the only cultured Nitrospira species live in habitats that are markedly different from wastewater treatment plants. However, molecular methods have provided first insight into the biology of Nitrospira-like bacteria. A set of new rRNA-targeted probes, which covers the genus Nitrospira and all other lineages of the phylum Nitrospirae, was used to detect and quantify Nitrospira-like bacteria in nitrifying bioreactors9,31,32 (Figure 15.4). These probes were also applied in FISH-MAR experiments, which revealed that the Nitrospira-like bacteria found in wastewater

FIGURE 15.4 (Color Figure 15.4 appears following page236.) Detection ofNitrospira-like bacteria in a nitrifying biofilmby FISH with probe S-G-Ntspa-662-a- A-1831 and the EUB probe mix, which targets most Bacteria5 Nitrospira-like bacteria (eightgray) are simultaneously stained by probes S-G-Ntspa-662-a-A-18 and EUB338-I.

FIGURE 15.4 (Color Figure 15.4 appears following page236.) Detection ofNitrospira-like bacteria in a nitrifying biofilmby FISH with probe S-G-Ntspa-662-a- A-1831 and the EUB probe mix, which targets most Bacteria5 Nitrospira-like bacteria (eightgray) are simultaneously stained by probes S-G-Ntspa-662-a-A-18 and EUB338-I.

treatment plants fix CO2 under aerobic conditions, but can also use pyruvate as additional carbon source.31 Based on results obtained with FISH and microelec-trodes, Schramm et al.98 proposed that Nitrospira-like bacteria could be K-strategists with a high affinity for nitrite and lower growth rates, while Nitrobacter species could be r-strategists with a lower affinity for nitrite and higher growth rates. This means that Nitrospira-like bacteria could reach high cell densities when the nitrite concentration is particularly low, and that Nitrobacter species could not grow under such conditions. This hypothesis could explain why Nitrospira-like bacteria outcompete Nitrobacter in wastewater treatment plants, where nitrite is continuously available in low concentrations. We have hitherto detected coexistence of Nitrobacter and Nitrospira-like bacteria in only one pilot-scale sequencing batch biofilm reactor receiving high loads of ammonia (Figure 15.5). The activity of ammonia-oxidizing bacteria caused a pronounced nitrite peak during each operational cycle of the reactor.100 The temporal shifts of the nitrite concentration created ecological niches for Nitrospira-like bacteria and for Nitrobacter in the biofilm.31,101

Measurements with microelectrodes have revealed that Nitrospira-like bacteria living in biofilms can cope with relatively low partial pressures of oxygen.102 Interestingly, the cultured species N. moscoviensis is able to transfer electrons from H2 to nitrate in the absence of oxygen.97 The uncultured Nitrospira-like bacteria may have similar capabilities, which would confer high physiological and ecological flexibility.

FIGURE 15.5 (Color Figure 15.5 appears following page 236.) Simultaneous detection of ammonia-oxidizing bacteria (Nitrosomonas sp.) and two different populations of nitrite-oxidizing bacteria (Nitrospira sp. and Nitrobacter sp.) in a nitrifying sequencing batch biofilm reactor. Nitrosomonas cells were stained by FISH with probe S-*-Nsm-0651-a-A-18,30 Nitrospira cells with probe S-G-Ntspa-662-a-A-18,31 and Nitrobacter cells with probe S-G-Nbac-1035-a-A-18.93

FIGURE 15.5 (Color Figure 15.5 appears following page 236.) Simultaneous detection of ammonia-oxidizing bacteria (Nitrosomonas sp.) and two different populations of nitrite-oxidizing bacteria (Nitrospira sp. and Nitrobacter sp.) in a nitrifying sequencing batch biofilm reactor. Nitrosomonas cells were stained by FISH with probe S-*-Nsm-0651-a-A-18,30 Nitrospira cells with probe S-G-Ntspa-662-a-A-18,31 and Nitrobacter cells with probe S-G-Nbac-1035-a-A-18.93

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