교육데이터 활용•지원 서비스

Experimental Design A six-level, dose-response ocean acidification experiment was run on a natural microbial community from nearshore Antarctica, between 19th November and 7th December 2014. Seawater was collected from approximately 1 km offshore of Davis Station, Antarctica (68◦ 35’ S, 77◦ 58’ E), pre-filtered (200 μm), and transferred into six 650 L tanks (minicosms) located in a temperature-controlled shipping container. Six CO2 levels were achieved by altering the fugacity of carbon dioxide (ƒCO2) within each minicosms. The ƒCO2 was adjusted stepwise to the target concentrations for each minicosm (343, 506, 634, 953, 1140, 1641 μatm) over a five-day period using 0.2 μm filtered seawater enriched with CO2. This acclimation to CO2 was conducted at low light (0.9 ± 0.2 μmol m−2 s−1) so there was low growth of the phytoplankton. Light levels were then increased over a further two days to 90.52 ± 21.45 μmol m−2 on a 19:5 light/dark non-limiting light cycle. After this acclimation period, the microbial community was allowed to grow for 10 days (days 8-18), during which the ƒCO2 levels within each minicosm was adjusted daily to maintain the target ƒCO2 level for each minicosm, and light levels were kept constant. No nutrients were added during the experiment. For a more detailed description of minicosm set-up, lighting and carbonate chemistry see; Davidson, A. T., McKinlay, J., Westwood, K., Thomson, P. G., van den Enden, R., de Salas, M., Wright, S., Johnson, R., and Berry, K.:Enhanced CO2 concentrations change the structure of Antarctic marine microbial communities, Mar. Ecol. Prog. Ser., 552, 93-113, 2016. Deppeler, S. L., Petrou, K., Westwood, K., Pearce, I., Pascoe, P., Schulz, K. G., and Davidson, A. T. Ocean acidification effects on productivity in a coastal Antarctic marine microbial community, Biogeosciences, 15(1), 2018. Sample Collection Samples of 40-400 L were collected and sequentially size-fractionated filtered onto 293 mm biomass filters with 3.0 and 0.1 μm pore-sized polyethersulfone membrane filters (Pall XE20206 Disc 3.0 μm Versapor 293 mm and 656552 Disc 0.1 μm Supor 293 mm) using the design of the Global Ocean Sampling expedition (Rusch et al., 2007). Samples were collected on days 0 (immediately after seawater collection), 12 (mid-exponential growth) and 18 (end of experiment). On day 0, 400 L of seawater was collected from the reservoir tank (pre-filtered 200 μm), from which all the minicosms were filled, to allow characterisation of the initial community. This sample was collected from the reservoir, and not the minicosms, due to the large volume needed to collect sufficient microbial biomass on the filters. On day 12 and 18, 40 L was collected from each minicosm for filtration. The later samples were of a smaller volume due to the increase in biomass in the minicosms during the experiment, meaning less volume of water was required to gain sufficient material on the filters to perform molecular analysis. The filter membranes containing the concentrated microbial biomass were stored in 15 mL of storage buffer, flash frozen in liquid nitrogen and stored at - 80◦C. The storage buffer was freshly prepared on each sampling day with a mixture of 2.5 mM EGTA, 2.5 mM EDTA, 0.1 mM Tris-EDTA, RNA Later (0.5x house prepared), 1 mM PMSF and Protease Inhibitor Cocktail VI (Ng et al., 2010). Between samples the filtration apparatus was sequentially washed with 2 x 25 L 0.1 M NaOH, 2 x 25 L 0.07% Ca(OCl)2 and 2 x 25 L fresh water. All samples were stored and transported at -80◦C to the Australian Antarctic Division, Hobart, Australia for DNA extraction. DNA Extraction and Sequencing The DNA was extracted from half of each filter (3.0 and 0.1 μatm per sample) via the method described in Rusch et al. (2007). In short, the filters were cut into small pieces and agitated in a lysozyme and sucrose buffer for 60 minutes and underwent three freeze/thaw cycles in a Proteinase K solution. This was followed by a gentler agitation