Grazing rate of microzooplankton measured experimentally in the North Atlantic during the Maria S. Merian cruise MSM26, spring 2013

The microzooplankton grazing dilution experiments were conducted at stations 126, 127, 131 and 133-137, following Landry & Hassett (1982). Seawater samples (whole seawater - WSW) were taken via Niskin bottles mounted on to a CTD Rosette out of the chlorophyll maximum at each station. Four different dilution levels were prepared with WSW and GF/F filtered seawater - 100% WSW, 75% WSW, 50% WSW and 25% WSW. The diluted WSW was filled in 2.4 L polycarbonate bottles (two replicates for every dilution level). Three subsamples (250 - 500 mL depending on in situ chlorophyll) of the 100% WSW were filtered on to GF/F filters (25 mm diameter) and chlorophyll was extracted in 5 mL 96% ethanol for 12-24 hours. Afterwards it was measured fluorometrically before and after the addition of HCl with a Turner fluorometer according to Jespersen and Christoffersen (1987) on board of the ship. In addition, one 250 mL subsample of the 100% WSW was fixed in 2% Lugol (final concentration), to determine the microzooplankton community when back at the Institute for Hydrobiology and Fisheries Science in Hamburg. Also, one 50 mL subsample of the 100% WSW was fixed in 1 mL glutaraldehyde, to quantify bacteria abundance. The 2.4 L bottles were put in black mesh-bags, which reduced incoming radiation to approximately 50% (to minimize chlorophyll bleaching). The bottles were incubated for 24 hours in a tank on deck with flow-through water, to maintain in situ temperature. An additional experiment was carried out to test the effect of temperature on microzooplankton grazing in darkness. Therefore, 100% WSW was incubated in the deck tank and in two temperature control rooms of 5 and 15°C in darkness (two bottles each). The same was done with bottles where copepods were added (five copepods of Calanus finmarchicus in each bottle; males and females were randomly picked and divided onto the bottles). In addition, two 100% WSW bottles with five copepods each were incubated at in situ temperature at 100% light level (without mesh-bags). All experiments were incubated for 24 hours and afterwards two subsamples of each bottle were filtered on to GF/F filters (25 mm diameter); 500 - 1000 mL depending on in situ chlorophyll. One 250 mL subsample of one of the two replicates of each dilution level and each additional experiment (temperature and temperature/copepods) was fixed in 5 mL lugol for microzooplankton determination. One 50 mL subsample of one of the two 100% WSW bottles as well as of one of the additional experiments without copepods was fixed in 1 mL glutaraldehyde for bacteria determination later on. Copepods were fixed in 4% formaldehyde for length measurements and sex determination.

Oxygen contents, sulfate concentrations, and changes of IPLs under different storage conditions of samples from ODP Leg 204 and IODP Expedition 311

We have studied the effects of slow infiltration of oxygen on microbial communities in refrigerated legacy samples from ocean drilling expeditions. Storage was in heat-sealed, laminated foil bags with a N2 headspace for geomicrobiological studies. Analysis of microbial lipids suggests that Bacteria were barely detectable in situ but increased remarkably during storage. Detailed molecular examination of a methane-rich sediment horizon showed that refrigeration triggered selective growth of ANME-2 archaea and a drastic change in the bacterial community. Subsequent enrichment targeting methanogens yielded exclusively methylotrophs, which were probably selected for by high sulfate levels caused by oxidation of reduced sulfur species. We provide recommendations for sample storage in future ocean drilling expeditions.

Biogeochemistry measurements in a mesocosm experiment in the Bay of Hopavagen, Norway, in August 2012

The goal of this work has been to examine the influence of upper ocean food web structure and functioning on both the natural and artificially enhanced sequestration of carbon within the ocean. Data obtained in the mesocosm experiment run in the Bay of Hopavågen in August 2012 are used to assess the extent to which organic matter produced within four different food webs is retained in the upper ocean food web versus remineralized back to carbon dioxide and inorganic nutrients (ammonium, dissolved silicon, phosphate) versus exported from the system in the form of rapidly sinking particles. The experiment was carried out in a set of 12 mesocosms covering, in triplicate, 2 different phytoplankton communities (diatom versus non-diatom) exposed to 2 different zooplankton communities (-copepod and +copepod). These starting conditions were established by first filling the bags, roughly simultaneously, with seawater from the Bay of Hopavågen. Mesozooplankton were then removed to the most complete extent possible immediately removed from half of the mesocosms through repeated vertical hauls of a plankton net (200 µm mesh). Nitrate and phosphate was added to half mesocosms daily to promote the growth of non-siliceous phytoplankton (e.g. dinoflagellates or coccolithophores). To the other half of the mesocosms, nitrate, phosphate, and silicate were added to promote the growth of diatoms. Material was allowed to settle and the two distinct phytoplankton populations were allowed to develop for 4 days, after which copepods collected from the Bay of Hopavågen were added back to the half of the N+P mesocosms and to the half of the N+P+Si mesocosms from which mesozooplankton had not been removed at the beginning. This yielded a set of four initial starting conditions (N+P-copepods, N+P+copepods, N+P+Si-copepods, and N+P+Si+copepods). In the primary mesocosms, samples for a set of core parameters were taken every time the mesocosms were sampled. Samples for particulates (PIC, BSi, POC, PON) were collected on GF/F or 0.4 µm polycarbonate.

Mesocosm experiment Cape Verde 2012: Data

Two 7-day mesocosm experiments were conducted in October 2012 at the Instituto Nacional de Desenvolvimento das Pescas (INDP), Mindelo, Cape Verde. Surface water was collected at night before the start of the respective experiment with RV Islândia south of São Vicente (16°44.4'N, 25°09.4'W) and transported to shore using four 600L food safe intermediate bulk containers. Sixteen mesocosm bags were distributed in four flow-through water baths and shaded with blue, transparent lids to approximately 20% of surface irradiation. Mesocosm bags were filled from the containers by gravity, using a submerged hose to minimize bubbles. The accurate volume inside the individual bags was calculated after addition of 1.5 mmol silicate and measuring the resulting silicate concentration. The volume ranged from 105.5 to 145 L. The experimental manipulation comprised addition of different amounts of inorganic N and P. In the first experiment, the P supply was changed at constant N supply in thirteen of the sixteen units, while in the second experiment the N supply was changed at constant P supply in twelve of the sixteen units. In addition to this, "cornerpoints" were chosen that were repeated during both experiments. Four cornerpoints should have been repeated, but setting the nutrient levels in one mesocosm was not succesfull and therefore this mesocosm also was set at the center point conditions. Experimental treatments were evenly distributed between the four water baths. Initial sampling of the mesocosms on day 1 of each run was conducted between 9:45 and 11:30. After nutrient manipulation, sampling was conducted on a daily basis between 09:00 and 10:30 for days 2 to 8.

Mesocosm experiment Cape Verde 2012: Setup

Two 7-day mesocosm experiments were conducted in October 2012 at the Instituto Nacional de Desenvolvimento das Pescas (INDP), Mindelo, Cape Verde. Surface water was collected at night before the start of the respective experiment with RV Islândia south of São Vicente (16°44.4'N, 25°09.4'W) and transported to shore using four 600L food safe intermediate bulk containers. Sixteen mesocosm bags were distributed in four flow-through water baths and shaded with blue, transparent lids to approximately 20% of surface irradiation. Mesocosm bags were filled from the containers by gravity, using a submerged hose to minimize bubbles. The accurate volume inside the individual bags was calculated after addition of 1.5 mmol silicate and measuring the resulting silicate concentration. The volume ranged from 105.5 to 145 L. The experimental manipulation comprised addition of different amounts of inorganic N and P. In the first experiment, the P supply was changed at constant N supply in thirteen of the sixteen units, while in the second experiment the N supply was changed at constant P supply in twelve of the sixteen units. In addition to this, "cornerpoints" were chosen that were repeated during both experiments. Four cornerpoints should have been repeated, but setting the nutrient levels in one mesocosm was not succesfull and therefore this mesocosm also was set at the center point conditions. Experimental treatments were evenly distributed between the four water baths. Initial sampling of the mesocosms on day 1 of each run was conducted between 9:45 and 11:30. After nutrient manipulation, sampling was conducted on a daily basis between 09:00 and 10:30 for days 2 to 8.

Mesocosm experiment Cape Verde 2012: Environmental conditions (PAR, cloud cover, pump)

Two 7-day mesocosm experiments were conducted in October 2012 at the Instituto Nacional de Desenvolvimento das Pescas (INDP), Mindelo, Cape Verde. Surface water was collected at night before the start of the respective experiment with RV Islândia south of São Vicente (16°44.4'N, 25°09.4'W) and transported to shore using four 600L food safe intermediate bulk containers. Sixteen mesocosm bags were distributed in four flow-through water baths and shaded with blue, transparent lids to approximately 20% of surface irradiation. Mesocosm bags were filled from the containers by gravity, using a submerged hose to minimize bubbles. The accurate volume inside the individual bags was calculated after addition of 1.5 mmol silicate and measuring the resulting silicate concentration. The volume ranged from 105.5 to 145 L. The experimental manipulation comprised addition of different amounts of inorganic N and P. In the first experiment, the P supply was changed at constant N supply in thirteen of the sixteen units, while in the second experiment the N supply was changed at constant P supply in twelve of the sixteen units. In addition to this, "cornerpoints" were chosen that were repeated during both experiments. Four cornerpoints should have been repeated, but setting the nutrient levels in one mesocosm was not succesfull and therefore this mesocosm also was set at the center point conditions. Experimental treatments were evenly distributed between the four water baths. Initial sampling of the mesocosms on day 1 of each run was conducted between 9:45 and 11:30. After nutrient manipulation, sampling was conducted on a daily basis between 09:00 and 10:30 for days 2 to 8.

Benthic anammox and dentrification rates measured in a slurry incubation experiments at four different stations in the Arabian Sea off Pakistan during the METEOR cruise M74/2 in 2007

Vertical distributions of benthic denitrification and anammox rates within the sediment were estimated from slurry incubation experiments. Rates were used to calculate the contribution of anammox and denitrification to the total N-loss. Briefly, MUC sediment cores were sliced in 2 cm intervals and the sediment was diluted and incubated with degassed bottom water in a gas tight bag. After pre-incubating the bags for 2 h, 15N-labeled substrates were injected into the bags and the slurries were thoroughly mixed. Incubations were performed in the dark at in situ temperatures. The N2 isotope ratio (28N2, 29N2, and 30N2) was determined by gas chromatography-isotopic ratio mass spectrometry (VG Optima, Micromass) and calculated according to Kuypers et al. (2005) and Holtappels et al. (2011), respectively.Furthermore, total organic carbon and nitrogen concentrations were measured of core sediment layers corresponding to those used for rate measurements. Concentrations of organic carbon and nitrogen were determined by combustion/gas chromatography (Carlo Erba NA-1500 CNS analyzer) of dried sediment samples after acidification. The same sediment layer were also used to extract nucleic acids. The concentrations of the DNA in the samples were measured spectrophotometrically with a NanoDrop instrument (Thermo Fisher Scientific Inc.). The biomarker functional gene nirS, encoding the cd1-containing nitrite reductase, for both denitrifiers and marine anammox bacteria were quantified with real-time PCR, using the primers cd3aF/R3cd (5'-GTSAACGTSAAGGARACSGG-3' (Michotey et al., 2000)/5'-GASTTCGGRTGSGTCTTGA-3'; Throback et al., 2004) and Scnir372F/Scnir845R (5'-TGTAGCCAGCATTGTAGCGT-3'/5'-TCAAGCCAGACCCATTTGCT-3'; Lam et al., 2009).