Final Report Summary - NITRICOS (Nitrogen removal in coastal sediments: molecular microbial ecology of nitrate reducing bacteria)
Coastal ecosystems such as estuaries, salt marshes and inshore coastal waters are important habitats regulating nutrient exchange between land and sea. However, over the last century increased anthropogenic inputs of nitrogen (N) from sources such as fertiliser run-off, sewage discharges and aquaculture have significantly perturbed these habitats. The increased availability of N stimulates primary production (eutrophication), which may lead eventually to temporary anoxia in the water column, release of toxic sulfide from the sediment and mass mortality of macrofauna and fish stocks (dystrophic crisis). Benthic microbial processes such as denitrification (DN) can in part alleviate the effect of increased N loads naturally removing up to 50 % of the N in many estuaries as dinitrogen gas subsequently lost from the aquatic environment. On the other hand dissimilatory nitrate reduction to ammonium (DNRA) converts nitrate to ammonium that remains in the system and may lead potentially to another cycle of eutrophication. Another important aspect of the nitrate reduction process is that a product of DN is nitrous oxide (N2O), a greenhouse gas 300 times more effective than CO2 that also contributes to stratospheric ozone depletion. In addition, estuaries and shallow coastal waters are considered the most important sources of atmospheric N2O. Therefore, the study of the ecological function of bacterial populations that mediate key processes, such as N removal or release of N2O, and their relation to measured rates in different systems is important to assess the effect of anthropogenic perturbations to the environment. DN and DNRA occur simultaneously in marine sediments and are expected to compete for nitrate and carbon. The balance of activity between these two functional groups and the mechanisms that control competition between the two groups is important to the control of their ecological activity and fate of nitrogen load.
Aim of the project was to study the rate processes involved in N removal (DN, DNRA, Anammox) in marine sediments, (2) to study the spatial changes of the benthic nitrate reducing community based on community nucleic acids, and relate these to the process rate measurements, (3) to determine the effect of environmental factors on the rate processes of nitrate reduction. 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.
During the first period of the current project a detailed study of the variations of the microbenthic community metabolism were followed in an undisturbed intertidal area of the inner part of the Bay of Cadis (Cadis, Spain) on a monthly basis for one and a half year. This included the period of macroalgal bloom occuring during the winter months and resulting in an increased transport of organic matter to the sediment once the bloom collapses. Multivariate multiple regression analysis revealed that the environmental variables explained ca. 40 % of the variation of oxygen flux rates, with the concentration of nutrients and especially silicate having the most explanatory power, suggesting that this nutrient is a limiting nutrient for microbenthic production in the inner Bay of Cadis. The relative importance of each nutrient pool varied throughout the year and showed clear differences between nutrients. Ammonium, phosphate and silicate pools were dominated by the freeze lysable fraction, representing as average: 71, 72 and 56 % of the total pool. Interestingly, exchangeable ammonium pool represented just a 20 % of the total pool, being just two times higher than the PW pool. On the other hand, nitrite and nitrate pools had a higher variability without a clear dominance of any pool, fluctuating throughout the studied period. The dynamics of these pools could have a high impact on the benthic biogeochemistry. The localisation of the main pool in a pore water, inside of the cells or bounded to the sediment is going to condition the fraction being available for biological and chemical processes and, therefore, their use and transformations.
We have attempted to study the diversity and dynamics of the microbial communities using the novel approach of 454?-pyrosequencing. Although this technique as we have applied it so far does not give information on the expression levels of the different organisms or the abundance of functional genes, it provides a much more detailed picture of the community present. So far only a few samples have been analysed and the results already show significant differences in the areas with and without macroalgae within the major groups where denitrification is known to occur (a-, ß-, ?-Proteobacteria). Further analyses of these results are underway.
Following the in situ work a mesocosm experiment to see the effect of increased organic matter input, in the form of macroalgae blooms, on the local seagrass community was conducted. Among other measurements, the production of isotopically labeled nitrogen gas (29N2 and 30N2) was used to calculate rates of denitrification and Anammox, using the differentiation of these processes the production of isotopically labeled nitrous oxide (ratio 46N2O: 45N2O). The results obtained so far show a significant effect of the presence of macroalgae on denitrification rates. No Anammox activity could be detected. To calculate rates DNRA production isotopically labeled ammonium (15N-NH4) is measured and once these samples are analysed we will be able to determine the relative importance of the various processes. In parallel to that, we have analysed the benthic microbial community for the final day of the experiment using again a 454?-pyrosequencing appraoch. A highly diverse and complex ecosystem was found with 23 Phyla, 8 of which represent > 80 % sequences. 15-20 % of the sequences belonged to unclassified bacteria (new groups?), while 2-3 % of the remaining sequences belonged to the rest of the Phyla. It is evident from the results that there is a significant number of species, grouped in microbial groups, especially of Bacteroidetes, that are stimulated by the presence of macroalgae. In contrast, the bacterial groups that re inhibited by the presence of macrolagae is less evident.
In an attempt to answer some interesting questions that have arisen from my previous group in the UK where it was shown that tropical sediments perform primarily dissimilatory nitrate reduction to ammonium and not denitrification, we have performed a study of the benthic metabolism in the Gulf of Nicoya, a tropical estuary of the Pacific coast of Costa Rica. Sediment cores were taken in 5 stations, covering a gradient from the mouth of the Tempisque River to the outer part of the gulf (Isla Caballo). We have measured several variables similar to those for the insitu study and the macroalgal experiment, namely sediment microprofiles were measured in the sediment-water interface using microelectrodes, benthic fluxes using core incubations, nitrogen processes using isotope paring technique, bacterial community composition using molecular techniques and abundance using cytometry etc. The results so far do not show significant differences in the rates along the estuary for the rainy season. Denitrification accounts approximately for 3-5 % of total benthic metabolism. Many more results are underway. This is a fruitful area for further study.
Overall, our results provide further insight in the processes related to nitrogen cycling and its effects on benthic ecosystems and habitats that will allow us to improve management of estuaries and coastal areas with aim the attenuation of the nitrogen load, the effect of macroalgal blooms and the human impact on the environment.
Aim of the project was to study the rate processes involved in N removal (DN, DNRA, Anammox) in marine sediments, (2) to study the spatial changes of the benthic nitrate reducing community based on community nucleic acids, and relate these to the process rate measurements, (3) to determine the effect of environmental factors on the rate processes of nitrate reduction. 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.
During the first period of the current project a detailed study of the variations of the microbenthic community metabolism were followed in an undisturbed intertidal area of the inner part of the Bay of Cadis (Cadis, Spain) on a monthly basis for one and a half year. This included the period of macroalgal bloom occuring during the winter months and resulting in an increased transport of organic matter to the sediment once the bloom collapses. Multivariate multiple regression analysis revealed that the environmental variables explained ca. 40 % of the variation of oxygen flux rates, with the concentration of nutrients and especially silicate having the most explanatory power, suggesting that this nutrient is a limiting nutrient for microbenthic production in the inner Bay of Cadis. The relative importance of each nutrient pool varied throughout the year and showed clear differences between nutrients. Ammonium, phosphate and silicate pools were dominated by the freeze lysable fraction, representing as average: 71, 72 and 56 % of the total pool. Interestingly, exchangeable ammonium pool represented just a 20 % of the total pool, being just two times higher than the PW pool. On the other hand, nitrite and nitrate pools had a higher variability without a clear dominance of any pool, fluctuating throughout the studied period. The dynamics of these pools could have a high impact on the benthic biogeochemistry. The localisation of the main pool in a pore water, inside of the cells or bounded to the sediment is going to condition the fraction being available for biological and chemical processes and, therefore, their use and transformations.
We have attempted to study the diversity and dynamics of the microbial communities using the novel approach of 454?-pyrosequencing. Although this technique as we have applied it so far does not give information on the expression levels of the different organisms or the abundance of functional genes, it provides a much more detailed picture of the community present. So far only a few samples have been analysed and the results already show significant differences in the areas with and without macroalgae within the major groups where denitrification is known to occur (a-, ß-, ?-Proteobacteria). Further analyses of these results are underway.
Following the in situ work a mesocosm experiment to see the effect of increased organic matter input, in the form of macroalgae blooms, on the local seagrass community was conducted. Among other measurements, the production of isotopically labeled nitrogen gas (29N2 and 30N2) was used to calculate rates of denitrification and Anammox, using the differentiation of these processes the production of isotopically labeled nitrous oxide (ratio 46N2O: 45N2O). The results obtained so far show a significant effect of the presence of macroalgae on denitrification rates. No Anammox activity could be detected. To calculate rates DNRA production isotopically labeled ammonium (15N-NH4) is measured and once these samples are analysed we will be able to determine the relative importance of the various processes. In parallel to that, we have analysed the benthic microbial community for the final day of the experiment using again a 454?-pyrosequencing appraoch. A highly diverse and complex ecosystem was found with 23 Phyla, 8 of which represent > 80 % sequences. 15-20 % of the sequences belonged to unclassified bacteria (new groups?), while 2-3 % of the remaining sequences belonged to the rest of the Phyla. It is evident from the results that there is a significant number of species, grouped in microbial groups, especially of Bacteroidetes, that are stimulated by the presence of macroalgae. In contrast, the bacterial groups that re inhibited by the presence of macrolagae is less evident.
In an attempt to answer some interesting questions that have arisen from my previous group in the UK where it was shown that tropical sediments perform primarily dissimilatory nitrate reduction to ammonium and not denitrification, we have performed a study of the benthic metabolism in the Gulf of Nicoya, a tropical estuary of the Pacific coast of Costa Rica. Sediment cores were taken in 5 stations, covering a gradient from the mouth of the Tempisque River to the outer part of the gulf (Isla Caballo). We have measured several variables similar to those for the insitu study and the macroalgal experiment, namely sediment microprofiles were measured in the sediment-water interface using microelectrodes, benthic fluxes using core incubations, nitrogen processes using isotope paring technique, bacterial community composition using molecular techniques and abundance using cytometry etc. The results so far do not show significant differences in the rates along the estuary for the rainy season. Denitrification accounts approximately for 3-5 % of total benthic metabolism. Many more results are underway. This is a fruitful area for further study.
Overall, our results provide further insight in the processes related to nitrogen cycling and its effects on benthic ecosystems and habitats that will allow us to improve management of estuaries and coastal areas with aim the attenuation of the nitrogen load, the effect of macroalgal blooms and the human impact on the environment.