Station 1: Flow cytometry (Antonia Ahme)
One device we are using for the community analyses is a so-called flow cytometer. It looks and sounds a lot like R2D2 from Star Wars and helps us to differentiate between populations of single-celled organisms in the Planktotrons. To do this, it sucks up the tiny particles and cells in our sample and sends them to a flow stream, which acts as a little train to carry the cells along. Flowing with this tiny carrier, each single cell is passed by different laser beams. The flow cytometer then measures the size of the shadow that every particle is producing and also the colour and amount of light that is scattered or produced via fluorescence. All this information taken together can tell us which members of the community grow, die or start to appear throughout the experiment (Photo: Sibet Riexinger).
 

Station 2: Single-cell isolation (Antonia Ahme)
The North Sea micro-plankton community is a diverse set of organisms that differ not only in their appearance but also in their capabilities. As we are exposing them to drastic changes in temperatures, some of its members might be able to cope better than others. We would like to get to know these little survivors a bit better and want to understand what makes them stand out from the rest. Therefore, we are isolating them from the original community by picking them with tiny pipettes under the microscope before growing them in the lab. This enables us to examine them comprehensively in further experiments and answer a couple of different questions. For example, are they maybe particularly flexible to match their physiology to new conditions? Or is there a subset of individuals among them that was always better at coping with higher temperatures?

Station 3: Fume hood (Antonia Ahme, Ruben Schulte-Hillen)
Under the fume hood we conduct the sampling procedures that include toxic fixation agents. These are necessary to preserve the status quo of the Planktotron water at specific timepoints for more time-consuming measurements that will be done later on. We perform three different kinds of fixation: one for backup flow cytometry, one for microscopic analyses and one for RNA sequencing. Through the microscope, we can better assess the identity, morphology and abundance of the “bigger” (> 2µm diameter) community members. The RNA helps us to understand what the little critters are doing – it is a messenger molecule translating the genetic information from the DNA into actual cellular processes (Photos: Sibet Riexinger).

Simultaneously to filling up the Planktotrons, the same seawater
including the phytoplankton community was filled into 250 bottles,
each of which was then exposed to different combinations of the
treatment temperatures as well as phosphorus and nitrogen additions.
Just as the planktotrons increase in temperature and then remain
constant from 12°C or 18°C degrees onwards, the incubated bottles
experience the same conditions. However, thanks to the nutrient
addition, we can learn more about the role of different nutrient
concentrations and ratios within the system. This give us the chance to
understand how interactions of potential future temperature and nutrient
conditions shape the phytoplankton community growth, stoichiometry and composition.

Station 5: DNA Filtration (Alexander Sentimenti)
The DNA filtration station is used to sample eukaryotic (little algae and grazers) and prokaryotic (bacteria) DNA from the Planktotrons. It consists of a funnel that gets filled with seawater and a filter underneath that catches the DNA. The water is pumped through the system using a vacuum pump system. To sample the different groups of organisms, two filter sizes are used: 0.8 µm for the eukaryotic DNA and 0.2 µm for the prokaryotic DNA. Once the water from one Planktotron has run through, the filter is carefully removed and placed into a little collection tube with plastic beads and lysis buffer. The beads and lysis buffer aid the DNA extraction that takes place later on. In the meantime, the samples are kept at -80 °C awaiting their extraction. The extracted DNA will then be sequenced and used to track changes in community composition over time (Photos: Sibet Riexinger).

Station 6: Micrograzing (Marco J. Cabrerizo)
One technique we are using to quantify how small herbivorous protists, named micro-zooplankton, predate on tiny photosynthetic organisms i.e. algae, is the dilution method. Through differently diluted water including the natural plankton communities coming from surface waters of the North Sea, we quantify on how much biomass is produced by algae when predators are present and not. Then, we retrieve the little organisms by filtering the seawater samples through special glass-fiber filters placed on a customized filtration system. After that, we extract the pigments retained in such filters using an ethanol-based solution and measure them using a fluorometer, a device that measures the fluorescence signal emitted by these algal pigments, which are also present in all plants all over the world (Photos: Sibet Riexinger).

Station 7: Chlorophyll filtration (Markus Olsson)
Water from the planktotrons/mesocosms are filtered at this station. A filter is placed under these orange cups, a set volume of water is poured into each cup and then a pump creates a vacuum, drawing the water through the filter. The filter has a certain hole size and catches everything that doesn’t go through. At this station we filter for particulate organic carbon, nitrogen & phosphorus, as well as chlorophyll. Measurements of these parameters will tell us more about how the autotrophic (plant-like) part of the community is growing and how biomass is built up differently under the environmental factors we expose them to (Photos: Sibet Riexinger).

Station 8: Daily small sampling (Nancy Kühne)
Daily fluorometric measurements are taken in the planktotrons to determine the in vivo fluorescence. This means that the chlorophyll content in the microalgae community is measured with a fluorometer which helps us to get a first insight into the development of the biomass under the various environmental factors. In addition, the pH, the salinity and the oxygen concentration in the tanks are measured daily with the help of various sensors in order to check whether and how the parameters change due to the different temperature conditions (Photo: Sibet Riexinger).

Station 9: Thermal Performance Curves (Simon Hasselø-Kline)
Where are the optimal temperature and temperature limits of our North Sea community? To investigate this, we have exposed the plankton community to a gradient of 10 different temperatures, with the water samples heated or cooled on heating mats. The fluorescence (indicator of biomass) of the community can be measured with a plate reader. This  exposes the photosynthetic organisms in the water to a certain wavelengths of light, which then emit light at a different wavelength that the plate reader can measure. In this way we can estimate how the growth of the community changes over time at the different temperatures (Photos: Sibet Riexinger).

Life in the ocean is dependent on a variety of different nutrients and elements. Without the cocktail of many nutrients, planktonic life would be unable to grow. Human influence affects the balance of nutrients in the ocean, which can determine which planktonic organisms can grow. We investigate how nutrients may limit or enhance growth, by adding nitrogen, phosphorous and/or silicate to the water samples. The growth of the organisms in the nutrient added water is then measured over time using a plate reader, which allows us to estimate how the concentration of organisms changes over time.