Ammonia-Oxidising Bacteria in Soil : Studies of diversity and abundance using 16S rRNA gene analysis

Abstract: Nitrification is the process whereby ammonia is oxidised to nitrate. The first step of this process is carried out by the chemoautotrophic, ammonia-oxidising bacteria (AOB), which convert ammonia to nitrite. Nitrification makes soil nitrogen more mobile thereby increasing its availability to plants and microorganisms, but at the same time, this nitrogen pool is more susceptible to nitrogen losses. Nitrate is mobile and easily lost through leaching, which can lead to serious environmental problems, such as eutrophication. Increased levels of nitrification can also contribute to atmospheric pollution, through the production of the greenhouse gasses, NO and N20. Since nitrification is fundamentally linked to both crop productivity and environmental pollution, studies that increase our understanding of the process and the organisms involved are important. This study had two main aims: to establish molecular biological methods for examining AOB in soil; and to use these methods in investigations into the diversity and abundance of AOB in arable and forest soils. Throughout the investigations, the 16S rRNA molecule in the AOB was used as a target gene.A new method for the quantification of AOB in soil was established - 'real-time PCR'. The method is a PCR technique, based on the continuous measurement of DNA concentrations during amplification. Populations of AOB were quantified using samples of arable soil (unfertilised and fertilised with nitrogen) and coniferous forest soil (limed and unlimed). The frequency of AOB was three times higher in the fertilised compared with the unfertilised arable soil, i.e. 6 x 107 and 2 x 107 cells g-1 of soil (dw), respectively. The frequency of AOB in the forest soil samples was in the same range as in the arable soils, i.e. -107-108 cells g-1 of soil (dw). Thus, AOB density appears to have an upper limit of ∼108 cells g-1 of soil (dw). This limit is independent of the soil environment investigated.Bead-beating was used to extract DNA from soil, and the effects of varying the duration and the attachment of AOB to soil particles during cells lysis were assessed. Free-living AOB and those associated with soil particles were separated by density gradient centrifugation. The PCR products of partial 16S rRNA gene sequences, which had been amplified from DNA extracted from arable soil, were either separated on denaturing gradient gel electrophoresis (DGGE) or quantified using real-time PCR. The results suggested that the beadbeating should last about 100 s in order to lyse as many AOB as possible and that attachment of AOB to surfaces does not affect the number of different AOB-like sequences obtained during the time-course. The composition and abundance of each AOB fraction was compared. The composition of 16S rDNA sequences was different in the two bacterial fractions, both in the fertilised and the unfertilised arable soils. Thus, direct DNA extraction is preferable, since neither of the bacterial fractions represents the total AOB diversity in the soil. The particle-associated fraction contained at least 70% of the total AOB populations in both soils.The effects of liming on the diversity of soil AOB in acid coniferous forests were investigated, using electrophoretic separation of PCR products by SSCP (single-strand conformation polymorphism) and DGGE. Liming had a clear effect on the composition of the AOB population in the upper soil horizons. Sequences similar to those of known AOB were only detected in the limed plots. In the unlimed control plot, the most frequently detected sequences were from a group that, according to the phylogenetic analysis, was not closely related to other bacterial groups. These sequences may represent acid-tolerant ammonia-oxidisers. This difference in the composition of the AOB population may reflect the increase in nitrification caused by liming. Furthermore, the effect of liming on AOB numbers, demonstrated using real-time PCR, was most pronounced early in the growing season. The AOB populations in the acidic, unlimed, control soils reached the same density as those in the limed soils during the autumn.