Are bacteria or archaea the major players in nitrogen fertiliser loss in agricultural soils?

Which microorganisms are responsible for nitrogen fertiliser loss? Nitrogen is the major fertiliser required for crop production. In the recent past, nitrogen fertiliser has been added to crops in an inorganic form, as an ammonium salt. Ammonia is converted to nitrate by two groups of soil microorganisms in a process termed nitrification: ammonia oxidisers convert ammonia to nitrite, which is converted to nitrite by nitrite oxidisers. Ammonium is retained within the soil but nitrate is readily leached and can accumulate to high, polluting levels in groundwater used to supply drinking water. In addition, ammonia oxidation is accompanied by production of nitrous oxide, a potent greenhouse gas, which is also produced by reduction of nitrate. Nitrification therefore leads to pollution and to significant losses of applied nitrogen fertiliser. Nitrogen fertiliser loss can be reduced by application of nitrification inhibitors with inorganic fertilisers or by use of fertilisers which release ammonia slowly, such as composted manure. Until recently it was believed that the most important soil ammonia oxidisers were bacteria. However, this view was overturned by the recent discovery of organisms belonging to another microbial domain, the archaea, which can also oxidise ammonia. These organisms belong to a subgroup of archaea, the crenarchaea, which are present in all soils but which have never been isolated in the laboratory. We therefore require cultivation-independent, molecular techniques to assess their presence and importance. These techniques show that ammonia oxidising crenarchaea are usually more abundant than ammonia oxidising bacteria and also appear to have greater activity. There is also evidence that they may prefer lower ammonia concentrations than bacteria. Inorganic nitrogen fertilisers generate high concentrations and crenarchaeal ammonia oxidisers may therefore have a bigger role in nitrification in sustainable systems, using organic fertiliser. To determine the importance of crenarchaeal ammonia oxidisers, we will use a combination of field studies, microcosms and physiological experiments to address four objectives. 1. Determination of the relative responses of bacterial and crenarchaeal ammonia oxidisers to ammonia concentration. 2. Determination of whether different fertilisers select for different ammonia oxidiser communities. 3. Assessment of conversion of different fertilisers by bacteria and crenarchaea. 4. Determination of contributions of crenarchaea and bacterial to ammonia oxidation in subsoils. 5. Determination of relative sensitivities of crenarchaea and bacteria to nitrification inhibitors. We will determine the abundances and activities of ammonia oxidising bacteria and crenarchaea in two field sites, with contrasting soils, that have been treated for many years with either inorganic N fertiliser or composted manure. Molecular techniques will be used to determine the population sizes of the two microbial groups. Activity will be investigated by quantification of the levels of expression of amoA, a key gene in ammonia oxidation that encodes part of the protein ammonia monooxygenase. The influence of different fertilisers and of nitrification inhibitors will be investigated using cells extracted from the different soils and in small-scale soil microcosms. We will also use molecular techniques to determine whether particular subgroups within ammonia oxidising bacteria or archaea are influenced differently by ammonia concentration or by nitrification inhibitors. The findings will benefit those in agriculture, environmental agencies and industry by increasing understanding of the roles of microorganisms in nitrogen fertiliser loss and the impacts of different microbial groups on nitrification in traditional and sustainable systems and in subsoils. The findings will also be important to researchers and environmentalists interested in the links between biodiversity and ecosystem function.

Nitrification leads to significant losses of ammonia-based fertilisers which can be reduced by sustainable strategies, including use of slow-release fertilisers or inhibitors. Our understanding of factors determining losses and benefits of alternative fertilisation strategies is based on the assumption that bacteria are responsible for nitrification in agricultural soils and that cultivated bacterial ammonia oxidisers represent natural communities. Molecular techniques have demonstrated much greater diversity than laboratory cultures and led to the discovery that non-thermophilic crenarchaea can oxidise ammonia and may be more important in controlling nitrification and fertiliser loss. There is a belief, and some evidence, that crenarchaeal ammonia oxidisers prefer lower ammonia concentrations. Their contribution to nitrification may, therefore, be particularly important in sustainable systems, using organic fertilisers, and in subsoils, where ammonia concentrations will be lower. The sensitivity of crenarchaeal ammonia oxidisers to nitrification inhibitors may differ from that of bacteria. This project will test hypotheses regarding the responses of crenarchaeal and bacterial ammonia oxidisers to ammonia concentration and the consequences for their contributions to soil nitrification. The influence of ammonia concentration and inhibitors will be investigated in cells extracted from soil and, in situ, by assessment of transcriptional activity. Long-term selection will be studied in field sites fertilised with inorganic or organic N, which will also be used to determine differences in communities with soil depth. Soil microcosms will be used to determine the influence of different fertiliser strategies on ammonia oxidiser activity, the contribution of different groups and the influence of nitrification inhibitors. The hypotheses will be linked by utilisation of quantitative data from different objectives to predict community structure and nitrification rates.