Microbial nitrous oxide production and nitrogen cycling associated with aquatic invertebrates

Abstract

Nitrogen cycling is intimately linked to the activity of microorganisms that mediate the diverse nitrogen transformations and play a fundamental role in regulating the fate of nitrogen in the Earth s terrestrial and aquatic ecosystems. Microbial activity is influenced by physical, chemical, and biological factors that can be profoundly shaped by macrofaunal organisms, especially in benthic aquatic systems. This thesis therefore aimed at investigating the interactions between microorganisms and benthic aquatic invertebrates and their role in biogeochemical nitrogen cycling, especially regarding the production of nitrous oxide (N2O). This intermediate and by-product of microbial nitrogen cycling processes (mainly nitrification and denitrification) is of great importance as a greenhouse gas and ozone-depleting substance in the atmosphere. To date, the biogenic N2O sources remain poorly quantified in the global N2O budget. Natural N2O production mainly takes place in soils, sediments, and water bodies, but also occurs in the anoxic gut of earthworms and freshwater invertebrates. This thesis investigated for the first time the N2O emission potential of marine invertebrates that densely colonize coastal benthic ecosystems. An initial screening effort in the German Wadden Sea and Aarhus Bay, Denmark, revealed a large variety of marine invertebrate species as N2O emitters (Chapter 2). Statistical analysis showed that the N2O emission potential is not restricted to a certain taxonomic group or feeding guild, but rather correlates with body weight, habitat, and the presence of microbial biofilms on the shell or exoskeleton of the animals. This suggests that N2O emission from marine invertebrates is not necessarily due to denitrification in the gut, but may also result from microbial activity on the external surfaces of the animal. The novel pathway of N2O production in shell biofilms was investigated in detail by a combination of short-term and long-term incubation experiments, stable isotope experiments, microsensor measurements, and molecular analysis (Chapters 3 and 4). Investigations on three marine (Mytilus edulis, Littorina littorea, Hinia reticulata) and one freshwater mollusc species (Dreissena polymorpha) revealed that shell biofilms significantly contribute to the total animal-associated N2O production via both denitrification and nitrification. Ammonium excretion by the molluscs was sufficient to sustain nitrification-derived N2O production in the shell biofilms and thus potentially decouples invertebrate-associated N2O production from environmental nitrogen concentrations. This was demonstrated in detail for the snail H. reticulata, which promotes growth and N2O production of its shell biofilm by enriching its immediate surroundings with dissolved inorganic nitrogen. The shrimp Litopenaeus vannamei, the most important crustacean species in aquaculture worldwide, was found to emit N2O at the highest rate recorded for any marine invertebrate so far (Chapter 5). The shrimp gut represents a transient anoxic habitat in which ingested bacteria produce N2O due to incomplete denitrification. At high stocking densities, L. vannamei may significantly contribute to the N2O supersaturation observed in the rearing tank of the shrimp aquaculture. In an additional study, the fate of nitrogen was investigated in an animal-bacteria-microalgae interaction occurring in intertidal flats (Chapter 6). Diatoms were found to store more nitrate intracellularly when the polychaete Hediste diversicolor stimulated the activity of nitrifying bacteria by excretion of ammonium and oxygenation of the sediment. This intricate interplay alters the forms and availability of the important nutrient nitrogen in marine sediments. Conceptually, benthic invertebrates represent hotspots of microbial nitrogen cycling that add specific features to the general marine nitrogen cycle, such as the noticeable N2O production and the partial decoupling of microbial activity from ambient nutrient supply. In particular, this thesis revealed that invertebrate-associated N2O production constitutes an important link between reactive nitrogen in aquatic environments and atmospheric N2O and is controlled by environmental, autecological, and physiological factors.