WOBEC SOP Autonomous Sampling

Weddell Sea Observatory of Biodiversity and Ecosystem Change – WOBEC Standard Operating Procedures Technical Documentation December 2025 To be cited as Wietz, Matthias, Nils Van den Steen, and Anton Van de Putte. ‘WOBEC SOP Autonomous Sampling’. Zenodo, 12 December 2025. https://doi.org/10.5281/zenodo.17912847. Co-funded by the European Union (c) The authors This report is licensed under the Creative Commons Attribution International license Versions Date Comment Responsible Approved First draft M. Wietz 12/12/2025 Formatting N. Van den Steen Table of Contents 1 Description of sampling gear .......................................................................................................... 3 2 Sampling gear deployment .............................................................................................................. 4 2.1 Deployment ............................................................................................................................................. 4 2.2 Calibration ............................................................................................................................................... 4 2.3 Post-Deployment ................................................................................................................................... 5 2.4 Common Issues ...................................................................................................................................... 5 3 Sample sorting-collection-preservation procedure ............................................................... 5 4 Sample analysis method ................................................................................................................... 5 5 References .............................................................................................................................................. 6 Method responsible: Matthias Wietz 1 Description of sampling gear This SOP applies to the autonomous collection of seawater and sinking particles over entire annual cycles via moored, automated devices (Figure 1). This work will establish yearround inventories of the Biological Carbon Pump in the WOBEC area. Per sampling event, Remote Access Samplers (RAS gen3, McLane USA; see manual) automatically collect 500 mL of seawater from the mixed layer (approx. 40-50m depth) in pre-programmed intervals into Kynar® bags (catalog number KYN-500) containing 700 µL of saturated mercuric chloride solution (7.5%). The RAS tubing is acid-rinsed after each sampling event. Sediment traps (K/MT 234, KUM Germany; see manual) collect sinking particles over preprogrammed intervals (15-30 days depending on the season) in mesopelagic water layers (1000-3000 m) into vials containing 400 mL artifical seawater containing 8 mL of saturated mercuric chloride solution. For both RAS and traps, the chemical fixation preserves the sample until eDNA analyses after recovery. Autonomous sampling operations have been well established over multiple years in the Arctic FRAM Observatory [1–3], ensuring consistent results under safe handling and negligible risks. Moorings are furthermore equipped with sensor packages for continuous measurements of temperature, salinity, density, chlorophyll fluorescence, and nitrate concentrations (Table 1). Figure 1: Autonomous Remote Access Sampler (left) and sediment trap (middle). Devices are deployed on year-round moorings together with sensor packages attached below (right). Secondly, permanently installed in the bow of Polarstern, is the AUTOFIM autonomous underway filtration system. In intervals of 100 nautical miles, this system will automatically pump seawater from the ship’s intake (~10m depth) and capture microbes on polycarbonate filters. 2 Sampling gear deployment 2.1 Deployment Mooring operations are done on the main working deck using the large aft crane. Work will be done under strict safety rules (wearing helmets, no walking under running wires and heavy weights). A dedicated mooring technician is always onboard, and works closely with the experienced crew. RAS, traps and sensors have been deployed in early 2025 within secondary user proposal “SeaCaT” (GPF 24-1/029) at 66° 28,910' S 0° 02,884' E as well as 65° 43,100' S 36° 37,749' W. On the WOBEC expedition PS152, these will be recovered. In case of future WOBEC missions, new deployments are envisioned at similar locations under a deployment / recovery cycle every 2-3 years, in alignment and cooperation with the AWI-HAFOS program and the FRAM Observatory. The average ship time per mooring deployment and recovery are 7h and 9h, respectively (including reference CTD casts). All parameters are listed in Table 1. Table 1: Instrument parameters Instrument Sample / data type Sampling interval / volume Remote Access Sampler Water & eDNA Weekly / 500mL Sediment trap Particles & eDNA Biweekly to monthly / 200mL CTD sensor Temperature / salinity / density Continuously ECO Triplet sensor Chlorophyll Continuously SUNA / ISUS sensor Nitrate Continuously AUTOFIM Water & eDNA Every 100nm / 4L 2.2 Calibration Sensors on moorings are calibrated in collaboration with AWI sections Physical Oceanography and Biogeochemistry prior to the expedition; all equipment is brought to the ship in ready form. RAS and traps are carefully maintained and prepared in the home lab. 2.3 Post-Deployment Recovered instruments are rinsed with seawater, and samples removed. Mercuric chloridepoisoned samples are carefully handled using laboratory safety measures (gloves, safety goggles). Samples are immediately placed at 4°C, minimizing any exposure. We require approximately 10m3 of container space to store the equipment, until unloading in the home port. 2.4 Common Issues The team has ample experience in the handling of moored instruments, founding on years of autonomous sampling in the Arctic Ocean. 3 Sample sorting-collection-preservation procedure Samples are permanently preserved (i.e. killing all biological activity) by the mixing with mercuric chloride. The sampling containers are closed after recovery and stored as-is at 4°C. This minimizes any potential exposure to mercuric chloride, yet does not affect subsequent molecular and biogeochemical analyses. AUTOFIM samples are collected on polycarbonate filters, and stored at -20C. 4 Sample analysis method All molecular work and bioinformatic analyses occur in the home institution, within approx. 6 months after recovery. Preserved samples from RAS and traps are processed using established procedures, as outlined in [1-2]. Briefly, RAS water samples are filtered onto 0.22 Sterivex cartridges and DNA extracted using the PowerWater kit. Prior, a 50 mL aliquot has been withdrawn for quantification of nitrate, phosphate and silicate. Trap particle samples are split into several subsamples for biogeochemical measurements (e.g. POC, biogenic silica) as well as DNA extraction using the PowerWater kit. All DNA extracts are amplicon-sequenced using primers targeting prokaryotes, microeukaryotes, and metazoans – resulting in microbial biodiversity inventories in seasonal and environmental dimensions (relative abundances). Details about sample handling and processing are found in [4]. The workflow for microbial biodiversity assessments is described here. The resulting biomolecular sequence data will be submitted in raw form to the European Nucleotide Archive. In addition, the data will be stored on the AWI Server / Tape Archive, and sample IDs shared within WOBEC along with instructions for access. Sensor data will be submitted to PANGAEA. All data is made available within the WOBEC consortium immediately. Public access will follow after a maximum of two years after sampling, enabling FAIR science. All bioinformatic code will be deposited at Github and Zenodo for full reproducibility. 5 References 1. von Appen W-J, Waite AM, Bergmann M, Bienhold C, Boebel O, Bracher A, et al. Sea-ice derived meltwater stratification slows the biological carbon pump: results from continuous observations. Nat Commun 2021; 12: 7309. 2. Wietz M, Metfies K, Bienhold C, Wolf C, Janssen F, Salter I, et al. Impact of preservation method and storage period on ribosomal metabarcoding of marine microbes: Implications for remote automated samplings. Frontiers in Microbiology 2022; 13: 999925. 3. Priest T, Oldenburg E, Popa O, Dede B, Metfies K, von Appen W-J, et al. Seasonal recurrence and modular assembly of an Arctic pelagic marine microbiome. Nat Commun 2025; 16: 1326. 4. Wietz M, Bienhold C, Metfies K, Torres-Valdés S, von Appen W-J, Salter I, et al. The polar night shift: Seasonal dynamics and drivers of Arctic Ocean microbiomes revealed by autonomous sampling. ISME Commun 1:76.