In Situ Characterization of Nitrospira -Like Nitrite-Oxidizing Bacteria Active in Wastewater Treatment Plants

Abstract

Uncultivated Nitrospira-like bacteria in different biofilm and activated-sludge samples were investigated by cultivation-independent molecular approaches. Initially, the phylogenetic affiliation of Nitrospira-like bacteria in a nitrifying biofilm was determined by 16S rRNA gene sequence analysis. Subsequently, a phylogenetic consensus tree of the Nitrospira phylum including all publicly available sequences was constructed. This analysis revealed that the genus Nitrospira consists of at least four distinct sublineages. Based on these data, two 16S rRNA-directed oligonucleotide probes specific for the phylum and genus Nitrospira, respectively, were developed and evaluated for suitability for fluorescence in situ hybridization (FISH). The probes were used to investigate the in situ architecture of cell aggregates of Nitrospira-like nitrite oxidizers in wastewater treatment plants by FISH, confocal laser scanning microscopy, and computer-aided three-dimensional visualization. Cavities and a network of cell-free channels inside the Nitrospira microcolonies were detected that were water permeable, as demonstrated by fluorescein staining. The uptake of different carbon sources by Nitrospira-like bacteria within their natural habitat under different incubation conditions was studied by combined FISH and microautoradiography. Under aerobic conditions, the Nitrospira-like bacteria in bioreactor samples took up inorganic carbon (as HCO 3 ؊ or as CO 2 ) and pyruvate but not acetate, butyrate, and propionate, suggesting that these bacteria can grow mixotrophically in the presence of pyruvate. In contrast, no uptake by the Nitrospira-like bacteria of any of the carbon sources tested was observed under anoxic or anaerobic conditions. Nitrification, the oxidation of ammonia to nitrate catalyzed by bacteria, is a key part of global nitrogen cycling (37). In the first step of nitrification, chemolithoautotrophic ammonia oxidizers transform ammonia to nitrite, which is subsequently oxidized to nitrate by the nitrite-oxidizing bacteria (8). All isolated chemolithoautotrophic, nitrite-oxidizing bacteria belong to one of four different genera (7) Nitrobacter (alpha subclass of Proteobacteria), Nitrococcus (gamma subclass of Proteobacteria), Nitrospina (delta subclass of Proteobacteria), and Nitrospira (phylum Nitrospira). While species of the genus Nitrobacter have been isolated from a variety of environments, including soil and fresh water, it was long assumed that the other three genera were confined to marine environments (7). In recent studies, however, bacteria related to the genus Nitrospira were also found to occur in different nonmarine habitats. While the first described species of this genus, Nitrospira marina, was isolated from ocean water (49), the second isolated species, N. moscoviensis, was cultured from an iron pipe of a heating system in Moscow, Russia (16). These two species are the only cultivated representatives of the genus Nitrospira, but numerous related bacteria have recently been detected by comparative analy...

An innovative respirometric method to assess the autotrophic active fraction: Application to an alternate oxic–anoxic MBR pilot plant

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Structure and function of the microbial community in a full-scale enhanced biological phosphorus removal plant

“…Some filamentous bacteria, such as Chloroflexi (Bjornsson et al, 2002;Kragelund et al, 2007) and 'Candidatus Microthrix parvicella' (Blackall et al, 1996), may occasionally be present and cause bulking problems. Micro-organisms involved in N removal (nitrification/denitrification) may be present, and include betaproteobacterial ammoniaoxidizing bacteria (Mobarry et al, 1996) and nitrite oxidizers belonging to the phylum Nitrospira (Daims et al, 2001). Potential denitrifiers may be RPAO, Azoarcus, Thauera and Aquaspirillum (Thomsen et al, 2004(Thomsen et al, , 2007.…”

Expanded metabolic versatility of ubiquitous nitrite-oxidizing bacteria from the genus Nitrospira

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