(Appendix A) Pigment content, microbial biomass and activity, and grain size characteristics in caged and control sites of the Arctic long-term observatory HAUSGARTEN

Present theories of deep-sea community organization recognize the importance of small-scale biological disturbances, originated partly from the activities of epibenthic megafaunal organisms, in maintaining high benthic biodiversity in the deep sea. However, due to technical difficulties, in situ experimental studies to test hypotheses in the deep sea are lacking. The objective of the present study was to evaluate the potential of cages as tools for studying the importance of epibenthic megafauna for deep-sea benthic communities. Using the deep-diving Remotely Operated Vehicle (ROV) "VICTOR 6000", six experimental cages were deployed at the sea floor at 2500 m water depth and sampled after 2 years (2y) and 4 years (4y) for a variety of sediment parameters in order to test for caging artefacts. Photo and video footage from both experiments showed that the cages were efficient at excluding the targeted fauna. The cage also proved to be appropriate to deep-sea studies considering the fact that there was no fouling on the cages and no evidence of any organism establishing residence on or adjacent to it. Environmental changes inside the cages were dependent on the experimental period analysed. In the 4y experiment, chlorophyll a concentrations were higher in the uppermost centimeter of sediment inside cages whereas in the 2y experiment, it did not differ between inside and outside. Although the cages caused some changes to the sedimentary regime, they are relatively minor compared to similar studies in shallow water. The only parameter that was significantly higher under cages at both experiments was the concentration of phaeopigments. Since the epibenthic megafauna at our study site can potentially affect phytodetritus distribution and availability at the seafloor (e.g. via consumption, disaggregation and burial), we suggest that their exclusion was, at least in part, responsible for the increases in pigment concentrations. Cages might be suitable tools to study the long-term effects of disturbances caused by megafaunal organisms on the diversity and community structure of smaller-sized organisms in the deep sea, although further work employing partial cage controls, greater replication, and evaluating faunal components will be essential to unequivocally establish their utility.

Dissolved lithium and silicon through time in microbial dissolution experiments on ODP Leg 209 peridotite

Using peridotite drilled during Ocean Drilling Program Leg 209, a series of enrichment cultures were initiated on board the ship to stimulate microbially enhanced dissolution of olivine. Dissolution was estimated by measured changes in dissolved Li and Si in the media through time (up to 709 days). The results suggest that there was no significant difference between the amounts of dissolved Li and Si in most of the inoculated microbial cultures compared to the control cultures. Alternative explanations for this are that 1. No microbes are living in the culture tubes that can affect the dissolution rates of olivine, 2. The control cultures have microbes effecting the dissolution of olivine as well as the inoculated cultures, 3. Not enough time has passed to build up a large enough microbial population to effect the dissolution of the olivine in the culture tubes, 4. Microbes act to suppress dissolution of olivine instead of enhancing dissolution, and 5. Abiotic dissolution overshadows microbially enhanced dissolution. Further work is required to test these alternatives.

Microbial community composition in fresh water at different stations

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes was used to investigate the phylogenetic composition of bacterioplankton communities in several freshwater and marine samples. An average of about 50% of the cells were detected by probes for the domains Bacteria and Archaea. Cells were concentrated from water samples (1 to 100 ml) on white polycarbonate filters (diameter, 47 mm; pore size, 0.2 mm; type GTTP 4700 [Millipore, Eschborn, Germany]) by applying a vacuum of <25 kPa. They were subsequently fixed by covering the filter with 3 ml of a freshly prepared, phosphate-buffered saline (pH 7.2)-4% paraformaldehyde (Sigma, Deisenhofen, Germany) solution for 30 min at room temperature. Airdried filters are ready for hybridization and can be stored at 220°C or room temperature for several months without showing apparent changes. Probes BET42a, GAM42a, and PLA886 were used with competitor oligonucleotides as described previously amongst others in Manz et al., (1992; doi:10.1016/S0723-2020(11)80121-9). The filters were transferred to a vial containing 50 ml of prewarmed (48°C) washing solution (70 mM NaCl, 20 mM Tris-HCl [pH 7.4], 5 mM EDTA, 0.01% sodium dodecyl sulfate) and incubated freely floating without shaking at 48°C for 15 min. The filter sections were dried on Whatman 3M paper (Whatman Ltd., Maidstone, United Kingdom) and covered with 50 ml of DAPI solution (1 mg/ml in distilled water filtered through at 0.2-mm filter) for 5 min at room temperature in the dark. For each sample and probe, more than 500 cells were enumerated; for the DAPI examination, more than 1,500 cells were counted per sample. All probe-specific cell counts are presented as the percentage of cells visualized by DAPI. The mean abundances and standard deviations were calculated from the counts of 10 to 20 randomly chosen fields on each filter section. All counts were corrected by subtracting the counts obtained with the negative control NON338. Mean and standard deviation were calculated from the counts of 10 to 20 randomly chosen fields on each filter section.

Microbial community composition and bacterioplankton at time series station Helgoland Roads, North Sea

A process of global importance in carbon cycling is the remineralization of algae biomass by heterotrophic bacteria, most notably during massive marine algae blooms. Such blooms can trigger secondary blooms of planktonic bacteria that consist of swift successions of distinct bacterial clades, most prominently members of the Flavobacteriia, Gammaproteobacteria and the alphaproteobacterial Roseobacter clade. This study explores such successions during spring phytoplankton blooms in the southern North Sea (German Bight) for four consecutive years. The surface water samples were taken at Helgoland Island about 40 km offshore in the southeastern North Sea in the German Bight at the station 'Kabeltonne' (54° 11.3' N, 7° 54.0' E) between the main island and the minor island, Düne (German for 'dune') using small research vessels (http://www.awi.de/en/expedition/ships/more-ships.html). Water depths at this site fluctuate from 6 to 10 m over the tidal cycle. Samples were processed as described previously (Teeling et al., 2012; doi:10.7554/eLife.11888.001) in the laboratory of the Biological Station Helgoland within less than two hours after sampling. Assessment of absolute cell numbers and bacterioplankton community composition was carried out as described previously (Thiele et al., 2011; doi:10.1016/B978-0-444-53199-5.00056-7). To obtain total cell numbers, DNA of formaldehyde fixed cells filtered on 0.2 mm pore sized filters was stained with 4',6-diamidino-2-phenylindole (DAPI). Fluorescently labeled cells were subsequently counted on filter sections using an epifluores-cence microscope. Likewise, bacterioplankton community composition was assessed by catalyzedreporter deposition fluorescence in situ hybridization (CARD-FISH) of formaldehyde fixed cells on 0.2 mm pore sized filters.