Surface Attachment of Ammonia-Oxidizing Bacteria in Soil

A BSTRACTIndigenous ammonia-oxidizing bacteria (AOB) in a clay loam soil were extremely difficult to release from soil particles compared to most heterotrophic bacteria; less than 1% of indigenous AOB (estimated as potential ammonia oxidation rate) were extractable by the dispersion-density-gradient centrifugation technique. This is at least 10-fold less than the extractability of heterotrophic bacteria. Urea applications to the same soil induced a 5-fold increase in the potential ammonia oxidation rate, and this resulted in a much higher percentage (8%) extractability of AOB. Thus, the newly grown AOB in the urea-treated soil were less strongly attached to the soil particles. The contrast suggests that the strong attachment of indigenous AOB is a gradual process taking place due to a long residence time (infrequent/slow cell division) compared to heterotrophic organisms. However, the contrast could also reflect differences in species composition of the original AOB community and those growing in response to urea inputs. Specific detection of AOB in extinction dilution cultures was done by PCR and sequencing of the products. Considerable diversity was found within the genus Nitrosospira, but severe problems with the specificity of the primers were observed. Two allegedly AOB specific PCR primers pairs were used: one specific for Nitrosospira (SPIRA) and one which should encompass all AOB within the beta- Proteobacteria (GAOB). Only 33% of the cultures that gave PCR products with GAOB also gave products with the SPIRA primer pair, suggesting the presence of AOB other than Nitrosospira. However, the phylogeny based on the sequencing placed all the cultures in various clusters of the Nitrosospira clade, suggesting that the SPIRA primers do not match all members of the Nitrosospira genus. The cultures obtained from the urea-treated soil were different from the others in giving PCR products only with the SPIRA primers and not with the GAOB. Since sequencing also here confirmed the presence of Nitrosospira, these observations suggest that the GAOB primers do not match all AOB species.