We planned to do three experiments, one for each key process (nitrogen fixation, nitrification, and denitrification). In each experiment, we enclosed our organisms of interest in a container so that no water or air is exchanged with the environment. We will refer to this approach as a chamber incubation.
The incubations were carried out at Schiacchetiello beach in the Gulf of Naples. Here you can find a curious pattern in the growth form of the sponge. Commonly you will see them growing on rocky or calcareous surfaces, but here, they can be found growing like a ring surrounding the lower part of the leaves. Then, other larger sponges seem to either fuse together or grow from a shoot to the neighbors.
We collected P. oceanica and C. nucula growing alone and in association and incubated them in transparent incubation chambers fixed on crates. We incubated the sponge alone, the plant alone, both together, and both absent (which means using only seawater) to see how each of the key processes is affected by each organism. For the nitrogen fixation experiment, we added 15N2 gas as isotopic tracer; for the nitrification experiment 15NH4+ and for denitrification we added 15NO3–. These tracers are molecules that contain a slightly different version of nitrogen. It has one additional neutron in its atom and is in nature less abundant than the version you can see in the periodic table. Because we have the tools to detect this heavier version of the nitrogen, we can add a known quantity to our chambers and track where it is going.