Community analysis of β-subgroup ammonia in sewage sludge amended soil
European legislation has increased pressure on the use of land to represent the major disposal option for sewage sludge. Owing to their importance in regulating soil fertility, much research has been conducted into the effects of sewage sludge application on soil microorganisms. However, little knowledge is known about its effects on community dynamics of the beta-subgroup ammonia oxidising bacteria. This is despite the fact that nitrification activity of these bacteria plays an important role in soil nitrogen cycling. This investigation aimed to examine community dynamics of the beta-subgroup ammonia oxidising bacteria in sewage sludge amended soil by employing recently developed molecular based techniques. Firstly, a soil DNA extraction protocol was identified that allowed routine nested PCR amplification of 16S rDNA using beta-subgroup ammonia oxidiser directed primers. Reproducibility observed in denaturant gradient gel electrophoresis (DGGE) profiles suggested that 0.5 g samples used for DNA extraction allowed consistent detection of dominant beta-subgroup ammonia oxidiser community members. The effects of applying primary treatment sewage sludge to a variety of contrasting soils on (3-subgroup ammonia oxidiser community structure was unclear. This was partially hampered by lack of specificity of PCR primers for non ammonia oxidiser 16S rDNA sequences. Further, through measurement of net nitrification, there was no indication that sewage sludge addition stimulated the activity of nitrifier populations. Nevertheless, this study indicated the usefulness of DGGE for screening multiple environmental samples. A set of hierarchical oligonucleotide probes exhibiting specificity at the group, genus and cluster level were optimised using a non-radioactive system. These probes were used to assess the effects of application of aerobically digested sewage sludge to soil for 4 y on beta-subgroup ammonia oxidiser community structure. Despite significant changes in measurable soil parameters including net nitrification activity no changes were observed in beta-subgroup ammonia oxidiser community structure. This indicated resilience of these communities to change. A final study was conducted examining the effects of application of anaerobically digested sewage sludge to soil on beta-subgroup ammonia oxidiser community structure. Inhibition of nitrification by acetylene indicated the presence of a viable population of ammonia oxidising bacteria in continuously shaken samples of sludge. DGGE and oligonucleotide probing analysis provided evidence that sewage populations had the potential to outcompete indigenous soil populations of beta-subgroup ammonia oxidising bacteria. This was despite the fact that MPN enumeration suggested that soil populations of these bacteria were in some cases 10-fold greater than sludge populations. Evidence was also provided that suggested net nitrification to be an unreliable indicator of ammonia oxidiser activity in soil. It is possible to conclude that community structure of the beta-subgroup ammonia oxidising bacteria may be altered by application of sewage sludge to soil. The effects on community structure are likely to be influenced by both the type and level of sludge applied to soil.