A comparitive study of ammonia-oxidizing bacteria in lab-scale industrial wastewater treatment reactors

The diversity and community structure of the b-proteobacterial ammonia oxidising bacteria (AOB) in a range of different lab-scale industrial wastewater treatment reactors were compared. Three of the reactors treat waste from mixed domestic and industrial sources whereas the other reactor treats waste solely of industrial origin. PCR with AOB selective primers was combined with denaturing gradient gel electrophoresis to allow comparative analysis of the dominant AOB populations and the phylogenetic affiliation of the dominant AOB was determined by cloning and sequencing or direct sequencing of bands excised from DGGE gels. Different AOB were found within and between different reactors. All AOB sequences identified were grouped within the genus Nitrosomonas. Within the lab-scale reactors there appeared to be selection for a low diversity of AOB and predominance of a single AOB population. Furthermore, the industrial input in both effluents apparently selected for salt tolerant AOB, most closely related to Nitrosococcus mobilis and Nitrosomonas halophila.

Identification of Bacteria Responsible for Ammonia Oxidation in Freshwater Aquaria

ABSTRACT Culture enrichments and culture-independent molecular methods were employed to identify and confirm the presence of novel ammonia-oxidizing bacteria (AOB) in nitrifying freshwater aquaria. Reactors were seeded with biomass from freshwater nitrifying systems and enriched for AOB under various conditions of ammonia concentration. Surveys of cloned rRNA genes from the enrichments revealed four major strains of AOB which were phylogenetically related to theNitrosomonas marina cluster, theNitrosospira cluster, or the Nitrosomonas europaea-Nitrosococcus mobilis cluster of the β subdivision of the class Proteobacteria. Ammonia concentration in the reactors determined which AOB strain dominated in an enrichment. Oligonucleotide probes and PCR primer sets specific for the four AOB strains were developed and used to confirm the presence of the AOB strains in the enrichments. Enrichments of the AOB strains were added to newly established aquaria to determine their ability to accelerate the establishment of ammonia oxidation. Enrichments containing the Nitrosomonas marina-like AOB strain were most efficient at accelerating ammonia oxidation in newly established aquaria. Furthermore, if the Nitrosomonas marina-like AOB strain was present in the original enrichment, even one with other AOB, only the Nitrosomonas marina-like AOB strain was present in aquaria after nitrification was established.Nitrosomonas marina-like AOB were 2% or less of the cells detected by fluorescence in situ hybridization analysis in aquaria in which nitrification was well established.

Polyclonal Antibodies Recognizing the AmoB Protein of Ammonia Oxidizers of the β-Subclass of the ClassProteobacteria

ABSTRACT A 41-kDa protein of Nitrosomonas eutropha was purified, and the N-terminal amino acid sequence was found to be nearly identical with the sequence of AmoB, a subunit of ammonia monooxygenase. This protein was used to develop polyclonal antibodies, which were highly specific for the detection of the four genera of ammonia oxidizers of the β-subclass of Proteobacteria(Nitrosomonas, including Nitrosococcus mobilis, which belongs phylogenetically to Nitrosomonas;Nitrosospira; Nitrosolobus; andNitrosovibrio). In contrast, the antibodies did not react with ammonia oxidizers affiliated with the γ-subclass ofProteobacteria (Nitrosococcus oceani andNitrosococcus halophilus). Moreover, methane oxidizers (Methylococcus capsulatus, Methylocystis parvus, and Methylomonas methanica) containing the related particulate methane monooxygenase were not detected. Quantitative immunoblot analysis revealed that total cell protein ofN. eutropha consisted of approximately 6% AmoB, when cells were grown using standard conditions (mineral medium containing 10 mM ammonium). This AmoB amount was shown to depend on the ammonium concentration in the medium. About 14% AmoB of total protein was found when N. eutropha was grown with 1 mM ammonium, whereas 4% AmoB was detected when 100 mM ammonium were used. In addition, the cellular amount of AmoB was influenced by the absence of the substrate. Cells starved for more than 2 months contained nearly twice as much AmoB as actively growing cells, although these cells possessed low ammonia-oxidizing activity. AmoB was always present and could even be detected in cells of Nitrosomonas after 1 year of ammonia starvation.

Combined Molecular and Conventional Analyses of Nitrifying Bacterium Diversity in Activated Sludge: Nitrosococcus mobilis and Nitrospira-Like Bacteria as Dominant Populations

ABSTRACT The ammonia-oxidizing and nitrite-oxidizing bacterial populations occurring in the nitrifying activated sludge of an industrial wastewater treatment plant receiving sewage with high ammonia concentrations were studied by use of a polyphasic approach. In situ hybridization with a set of hierarchical 16S rRNA-targeted probes for ammonia-oxidizing bacteria revealed the dominance ofNitrosococcus mobilis-like bacteria. The phylogenetic affiliation suggested by fluorescent in situ hybridization (FISH) was confirmed by isolation of N. mobilis as the numerically dominant ammonia oxidizer and subsequent comparative 16S rRNA gene (rDNA) sequence and DNA-DNA hybridization analyses. For molecular fine-scale analysis of the ammonia-oxidizing population, a partial stretch of the gene encoding the active-site polypeptide of ammonia monooxygenase (amoA) was amplified from total DNA extracted from ammonia oxidizer isolates and from activated sludge. However, comparative sequence analysis of 13 amoA clone sequences from activated sludge demonstrated that these sequences were highly similar to each other and to the corresponding amoA gene fragments ofNitrosomonas europaea Nm50 and the N. mobilisisolate. The unexpected high sequence similarity between theamoA gene fragments of the N. mobilisisolate and N. europaea indicates a possible lateral gene transfer event. Although a Nitrobacter strain was isolated, members of the nitrite-oxidizing genus Nitrobacter were not detectable in the activated sludge by in situ hybridization. Therefore, we used the rRNA approach to investigate the abundance of other well-known nitrite-oxidizing bacterial genera. Three different methods were used for DNA extraction from the activated sludge. For each DNA preparation, almost full-length genes encoding small-subunit rRNA were separately amplified and used to generate three 16S rDNA libraries. By comparative sequence analysis, 2 of 60 randomly selected clones could be assigned to the nitrite-oxidizing bacteria of the genusNitrospira. Based on these clone sequences, a specific 16S rRNA-targeted probe was developed. FISH of the activated sludge with this probe demonstrated that Nitrospira-like bacteria were present in significant numbers (9% of the total bacterial counts) and frequently occurred in coaggregated microcolonies with N. mobilis.