Nitrite-oxidizing bacteria, Nitrospira, distribution in the outer layer of the biofilm from filter materials of a recirculating water system for the goldfish Carassius auratus

We used a clone library method to investigate changes in the biofilm microflora associated with the conditioning of filter materials in a recirculating water system for the culture of goldfish Carassius auratus. The microbial density was higher in the outer layer of biofilm from filter materials (1.7 × 1010–3.0 × 1010 cells/g) than in the inner layer (1.5 × 109–2.4 × 109 cells/g) throughout the experimental period. The clone library method using bacterial 16S rRNA genes collected from the outer layer of filter material yielded sequences from four (day 8), nine (day 15), twelve (day 22) and nine (day 64) taxonomic categories of bacteria including Acidobacteria, Actinobacteria, Bacilli, Bacteroidetes, Flavobacteria, Fusobacteria, Nitrospira, α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, Sphingobacteria, Verrucomicrobia and unclassified bacteria. The inner layer yielded sequences from six (day 8), eight (day 15), five (day 22) and five (day 64) taxonomic categories of bacteria including Acidobacteria, Actinobacteria, Bacilli, Flavobacteria, α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, Sphingobacteria, Verrucomicrobia and unclassified bacteria. Bacteria in the outer layer of biofilm belonged predominantly to the genera Acinetobacter, Cetobacterium, Comamonas, Flectobacillus, Flavobacterium, Ideonella and Pseudomonas, whereas those in the inner layer were predominantly members of the genera Flavobacterium, Flexibacter, Ideonella, Janthinobacterium, Pedobacter and Pseudomonas. As the filter materials became conditioned, the population of nitrite-oxidizing bacteria related to Nitrospira was restricted to the outer layer of the biofilm. In addition, phylogenetic analysis indicated the presence of both an indigenous Nitrospira and a distinct type of Nitrospira that appeared after conditioning had begun.