Final Activity Report Summary - DEFUNIREG (Diversity and ecological function of benthic nitrate reducing populations along an estuarine nitrate gradient) Over the last century estuarine and coastal ecosystems have been subject to perturbation due to increased anthropogenic inputs of nitrogen from diverse sources such as fertiliser run-off, sewage discharges and aquaculture. The increased availability of nitrogen stimulates production of marine algae (eutrophication), with adverse effects to a wide range of ecosystem services as it may lead eventually to temporary anoxia in the water column, release of toxic sulfide from the sediment and mass mortality of benthic organisms and fish stocks. Natural processes in coastal sediment involving microbes influence nitrogen load and its effect on the environment. Of particular importance is the process of denitrification (dissimilatory nitrate reduction, DN) which can remove up to 50% of the load. Denitrifying bacteria respire nitrate or nitrite from the environment to gain their energy, in the same way as humans use oxygen, and reduce it to dinitrogen gas that escapes in the atmosphere and is subsequently lost from the aquatic environment. However, in estuarine sediments, another process, that of nitrate ammonification (dissimilatory nitrate reduction to ammonium, DNRA), competes with denitrification for nitrate. Nitrate ammonifying bacteria gain heir energy by reducing nitrate to ammonium which then remains in the system in a form readily available to marine algae and may lead to further eutrophication events. Therefore, understanding the benthic processes involved in the removal of nitrogen is important for the management and reduction of nutrient load to coastal waters.The first step in both processes (DN and DNRA) is identical (nitrate reduction to nitrite) and is catalysed by one of the two types of nitrate reductase enzyme, nar and nap, that have different roles depending on the bacterial group involved and environmental conditions. Following this first step, different enzymes are involved in each process, nirS for DN and nrfA for DNRA.The river Colne estuary (east coast of UK), where our study was conducted, is a typical example of a macrotidal, hypernutrified, muddy estuary where strong gradients of nitrate and ammonium are found due to inputs from the river and a sewage treatment plant at the head of the estuary. Higher denitrification rates have been observed at the head decreasing towards the mouth of the estuary following the overlying water concentrations of nitrate. The characteristics of the estuary and the considerable data on denitrification available for the sediments of this estuary constitute it a model system for the study of diversity and functional ecology of nitrate reducing communities.Aim of the project was to study the spatial distribution of genes coding for key functional enzymes in the benthic nitrate reducing community along an estuarine nitrate concentration and relate these to process rates. This allowed us to test existing hypotheses concerning the factors that determine the distribution and competition outcome of these two functional groups of bacteria, DN and DNRA bacteria, involved in the reduction of nitrate and nitrogen cycling. To accomplish this, we used biogeochemical and modern molecular techniques that have only recently started being applied in environmental studies.The results clearly showed that the nitrogen removal capacity of the sediments decreases from the head towards the mouth of the estuary, as well as with increasing depth in the sediment. This corresponds with a decrease in water column nitrate concentrations and indicates that the microbial communities in the surface sediments respond to ambient nitrate concentrations. In addition, the contribution of each enzyme (nar vs nap) confirmed previous laboratory models as the greater proportion
