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  • General description: The associated file contains sediment pigment data from the antFOCE project 4127. Units: all pigment data in ug/g, 0 = below detection limit of HPLC. Sample collection details: At the start and end of the antFOCE experiment, four sediment core samples were taken from inside and outside each chamber or open plot by divers. The top 1 cm of the cores was then removed and placed in the dark, first at -20ºC for 2 hours, then at -80ºC until analysis at the Australian Antarctic division. Pigment analysis Frozen samples were transported under liquid N2 to a freeze drier (Dynavac, model FD-5), in pre-chilled flasks with a small amount of liquid N2 added. Custom made plumbing fitted to the freeze drier enabled samples to be purged with N2 to prevent photo-oxidation up until solvent extraction. Prior to pigment extraction five 2 g stainless steel ball bearings were added to homogenise the freeze dried sediment. The samples were bead beaten for 1 minute (Biospec products). Subsamples (~0.05 g) were immediately transferred to cryotubes with 700 µl of dimethylformamide (DMF) for two hours. Samples were kept at -80ºC and under a safe light (IFORD 902) at all times. All pigment concentrations are standardised to sediment weight. Pigments were extracted with dimethylformamide (DMF 700 µl) over a two hour period at -20ºC. Zirconia beads, and 100 µl of Apo 8 and an internal standard were added to each sub-sample. After a two hour extraction, sub-samples were bead beaten for 20 seconds and then placed in a centrifuge with filter cartridge inserts for 14 minutes at 2500 rpm at -9ºC to separate the solvent from the sediment. The supernatant was transferred into to a vial and placed in a precooled rpHPLC autosampler. The rpHPLC system used is described in Hodgson et al. (1997). Pigment detection was at 435, 470 and 665 nm for all chlorophylls and carotenoids, with spectra from 300–700 nm being collected every 0.2 seconds. Pigment identification was carried out using a combination of rpHPLC and normal phase HPLC retention times, light absorbance spectra and reference standards (see Hodgson et al., 1997). These techniques assisted in the accurate identification of pigments and their derivatives to a molecular level and enabled several pigment derivatives to be analysed. The HPLC was previously calibrated with authentic standards and protocols outlined in SCOR (1988). Data set headers: (A)Treatment: Example code 4127_SOP7_6-1-15_PlotB_R1, = prodject code_Standard Operating Procedure(SOP) used to collect samples(see antFOCE parent file)_ Date_Chamber/plot(A,B,C,D)_replicate core within Chamber/plot(1,2,3) (B) BB carot= BB caroten, type of pigment detected by HPLC. See Wright, S.W., Jeffrey, S.W. and Mantoura, R.F.C. eds., 2005. Phytoplankton pigments in oceanography: guidelines to modern methods. Unesco Pub for more details. (C) Chl c1 = Chlorophyll derivatives see Wright, S.W., Jeffrey, S.W. and Mantoura, R.F.C. eds., 2005. Phytoplankton pigments in oceanography: guidelines to modern methods. Unesco Pub for more information. (D) Chl c2 = Chlorophyll derivatives see Wright, S.W., Jeffrey, S.W. and Mantoura, R.F.C. eds., 2005. Phytoplankton pigments in oceanography: guidelines to modern methods. Unesco Pub for more information. (E) Chl c3 = Chlorophyll derivative see Wright, S.W., Jeffrey, S.W. and Mantoura, R.F.C. eds., 2005. Phytoplankton pigments in oceanography: guidelines to modern methods. Unesco Pub for more information. (F) Chla = Chlorophyll a see Wright, S.W., Jeffrey, S.W. and Mantoura, R.F.C. eds., 2005. Phytoplankton pigments in oceanography: guidelines to modern methods. Unesco Pub for more information. (G) Ddx =Diadinoxanthin see Wright, S.W., Jeffrey, S.W. and Mantoura, R.F.C. eds., 2005. Phytoplankton pigments in oceanography: guidelines to modern methods. Unesco Pub for more information (H) dtx = Diatoxanthin pigment. see Wright, S.W., Jeffrey, S.W. and Mantoura, R.F.C. eds., 2005. Phytoplankton pigments in oceanography: guidelines to modern methods. Unesco Pub for more information (I) epi = Chlorophyll epimer pigment. see Wright, S.W., Jeffrey, S.W. and Mantoura, R.F.C. eds., 2005. Phytoplankton pigments in oceanography: guidelines to modern methods. Unesco Pub for more information. (j) Fuc = Fucoxanthin pigment. see Wright, S.W., Jeffrey, S.W. and Mantoura, R.F.C. eds., 2005. Phytoplankton pigments in oceanography: guidelines to modern methods. Unesco Pub for more information. (k) Gyro2 = Gyroxanthin pigment. see Wright, S.W., Jeffrey, S.W. and Mantoura, R.F.C. eds., 2005. Phytoplankton pigments in oceanography: guidelines to modern methods. Unesco Pub for more information. (L) Pras = Prasanthin pigment. see Wright, S.W., Jeffrey, S.W. and Mantoura, R.F.C. eds., 2005. Phytoplankton pigments in oceanography: guidelines to modern methods. Unesco Pub for more information. (m) Zea = Zeaxanthin pigment. see Wright, S.W., Jeffrey, S.W. and Mantoura, R.F.C. eds., 2005. Phytoplankton pigments in oceanography: guidelines to modern methods. Unesco Pub for more information. (n) Date = Samples taken at the start of antFOCE experiment or at the end (o) chamber = The antFOCE chamber (A,B,C,D) (p) Treatment = The associated pH level in chambers (Acidified ~7.8, Control ~8.2) (Q) Position = Samples were taken within chambers and outside chambers (outside, inside) (r) rep= Subsamples were taken within each chamber/position (R1=replicate one, R1-R4) Spatial coordinates: 66.311500 S, 110.514216 E Dates: between 1/12/2014 and 1/3/2015 Timezone:UTC+11

  • Long-term experiment on increased CO2 level on krill physiology. Krill were exposed to a range of CO2 conditions 400-4000ppm over a year, and their growth, mortality, and physiology were monitored. -List of files- Ericson Krill Ocean Acidification Study Raw Data_for data centre.xlsx: This file contains data on krill growth, mortality, physiology, and biochemistry, as well as information on water chemistry throughout 1 year period of the experiment. Ericson et al. Adult krill OA MS final submission.pdf: Unpublished manuscript of the experiment including all methods of the experiment.

  • Chlorophyll data was used to measure growth rates of sea ice algae in CO2 incubations. Sea ice brine microalgae was collected from sackholes. Replicate samples were incubated in ambient air (~0.04% CO2), 0.1% CO2, 1.0% CO2 and 2.0% CO2 concentrations. AT the end of the incubations the 50 ml samples were filtered through a 25 mm GF/F filter using vacuum filtration. The filters were placed in 15 ml plastic falcon tubes containing 10 ml of methanol, covered in aluminium foil and kept in the dark at 4 degrees C for 12 hours. Chl a concentration was measured using a 10AU Turner fluorometer following the acidification method of Strickland and Parsons (1972). Data in spread sheet shows the extracted chl + phaeophytin, phaeophytin and chlorophyll concentrations (micro grams l-1) for each of the three experiments. Data were collected at SIPEX Ice Stations 1-8 and SIPEX CTD stations 2-5

  • The data reports the pigment concentrations and results of CHEMTAX analysis for 2 summer seasons in Antarctic. In 2008/09 three experiments in which 6 x 650 l minicosms (polythene tanks) were used to incubate natural microbial communities (less than 200 um diameter) at a range of CO2 concentrations while maintained at constant light, temperature and mixing. The communities were pumped from ice-free water ~60 m offshore on 30/12/08, 20/01/09 and 09/02/09. These experiments received no acclimation to CO2 treatment. A further experiment was performed in 2014/15 using water helicoptered from ~ 1 km offshore amongst decomposing fast ice on 19/11/14. This experiment included a 5 day period during which the community was exposed top low light and the CO2 was gradually raised to the target value for each tank, followed by a two day period when the light was raised to an irradiance that was saturating but not inhibitory for photosynthesis. A range of coincident measurements were performed to quantify the structure and function of the microbial community (see Davidson et al. 2016 Mar Ecol Prog Ser 552: 93–113, doi: 10.3354/meps11742 and Thomson et al 2016 Mar Ecol Prog Ser 554: 51–69, 2016, doi: 10.3354/meps11803). The data provides a matrix of samples against component pigment concentration and the output from CHEMTAX that best explained the phytoplankton composition of the community based on the ratios of the component pigments. For the 2008/09 experiments, samples were obtained every 2 days for 10, 12 and 10 days in experiments 1, 2 and 3 respectively. In 2014/15 samples were obtained from each incubation tank on days 1,3, 5, and 8 during th acclimation period and every 2 days until day 18 thereafter. For each sample a measured volume was filtered through 13 mm Whatman GF/F filters for 20 mins. Filters were folded in half, blotted dry, and immediately frozen in liquid nitrogen for analysis in Australia. Pigments were extracted, analysed by HPLC, and quantified following the methods of Wright et al. (2010). Pigments (including Chl a) were extracted from filters with 300 micro l dimethylformamide plus 50 micro l methanol, containing 140 ng apo-8'-carotenal (Fluka) internal standard, followed by bead beating and centrifugation to separate the extract from particulate matter. Extracts (125 micro l) were diluted to 80% with water and analysed on a Waters HPLC using a Waters Symmetry C8 column and a Waters 996 photodiode array detector. Pigments were identified by comparing retention times and spectra to a mixed standard sample from known cultures (Jeffrey and Wright, 1997), run daily before samples. Peak integrations were performed using Waters Empower software, checked manually for corrections, and quantified using the internal standard method (Mantoura and Repeta, 1997).

  • Parent node for data sets for the Antarctic Free Ocean Carbon Dioxide Enrichment experiment (antFOCE), AAS project 4127. Project Summary: Currently, a quarter of the CO2 we emit is absorbed by the ocean. CO2 absorption in seawater changes its chemistry – reducing ocean pH (raising its acidity) – which has significant impacts on biological processes and serious implications for the resilience of marine ecosystems. As CO2 is more soluble in cold water we expect polar ecosystems to bear the heaviest burden of this 'ocean acidification'. We will perform the first in situ polar CO2 enrichment experiment to determine the likely impacts of ocean acidification on Southern Ocean sea-floor communities under increasing CO2 emissions.

  • This metadata record contains an Excel spreadsheet with Operational Taxonomic Units (OTUs) gained from Eukaryotic 18S rDNA PCR amplification and high-throughput sequencing of samples from Biofilm slides deployed as part of the antFOCE experiment in the austral summer of 2014/15 at Casey station, East Antarctica. Refer to antFOCE report section 4.5.3 for deployment, sampling and analysis details. https://data.aad.gov.au/metadata/records/AAS_4127_antFOCE_Project4127 Sampling design 2 trays of 8 horizontal standard glass microscope slides (72 x 25 mm) per chamber. Four of the glass slides were scored with a diamond pencil approximately 18 mm from the right hand end of the slide and deployed scored side up. The remaining four slides were unmodified. Slides were sampled at: - Tmid - one tray per chamber / open plot. The sampled try was repopulated with fresh slides and redeployed - Tend – 2 slides trays per chamber / open plot. Sampling procedure After 31 days deployment, 1 slide tray per chamber / open plot was sampled. At Tend both trays in each chamber / open plot were sampled. To minimize disturbance while being raised to the surface, each tray was removed from the tray holder by divers and placed in a seawater filled container with a lid. On the surface, slides were removed from the tray using ethanol sterilized forceps. The four unscoured slides per chamber / open plot were placed in a plastic microscope slide holder with a sealable lid. The scoured slides were placed individually in 70 ml plastic sample jars. Lab procedure - Casey The slide holder (4 unscoured slides) from each chamber / open plot was frozen at -20C immediately upon return to the lab. The scoured slides were preserved in sea water containing 1% final concentration glutaraldehyde in separate jars. Preservation Issue: Scoured slides were not refrigerated, either at Casey, during RTA or in Kingston before the 26th Nov 2015, when they were transferred to the 4C Cold Store. antFOCE Background The antFOCE experimental system was deployed in O’Brien Bay, approximately 5 kilometres south of Casey station, East Antarctica, in the austral summer of 2014/15. Surface and sub-surface (in water below the sea ice) infrastructure allowed controlled manipulation of seawater pH levels (reduced by 0.4 pH units below ambient) in 2 chambers placed on the sea floor over natural benthic communities. Two control chambers (no pH manipulation) and two open plots (no chambers, no pH manipulation) were also sampled to compare to the pH manipulated (acidified) treatment chambers. Details of the antFOCE experiment can be found in the report – "antFOCE 2014/15 – Experimental System, Deployment, Sampling and Analysis". This report and a diagram indicating how the various antFOCE data sets relate to each other are available at: https://data.aad.gov.au/metadata/records/AAS_4127_antFOCE_Project4127 AntFOCE biofilm DNA methods Laurence Clarke, Shane Powell, Bruce Deagle DNA extraction The biofilm was removed from the top of each slide with a cotton swab and DNA extracted directly from the swab using the MoBio PowerBiofilm DNA isolation kit following the manufacturer’s protocol. Extraction blanks were extracted in parallel to detect contamination. Eukaryotic 18S rDNA PCR amplification and high-throughput sequencing DNA extracts were PCR-amplified in triplicate with primers designed to amplify 140-170 bp of eukaryotic 18S ribosomal DNA (Jarman et al. 2013). The forward primer was modified to improve amplification of protists. Table 1. First and second round primers, including MID tags (Xs). ILF_ProSSU3'F_X TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG XXXXXX CACCGCCCGTCGCWMCTACCG ILR_SSU3'R_Y GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG XXXXXX GGTTCACCTACGGAAACCTTGTTACG msqFX AATGATACGGCGACCACCGAGATCTACAC XXXXXXXXXX TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG msqRY CAAGCAGAAGACGGCATACGAGAT XXXXXXXXXX GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG PCR amplifications were performed in two rounds, the first to amplify the 18S region and add sample-specific multiplex-identifier (MID) tags and Illumina sequencing primers, the second to add the P5 and P7 sequencing adapters and additional MIDs. Each reaction mix for the first PCR contained 0.1 µM each of forward and reverse primer, 0.2 µg/µL BSA, 0.2 U Phusion DNA polymerase in 1 x Phusion Master Mix (New England Biolabs, Ipswich, MA, USA) and 1 micro L DNA extract in a total reaction volume of 10 micro L. PCR thermal cycling conditions were initial denaturation at 98 degrees C for 30 secs, followed by 25 cycles of 98 degrees C for 5 secs, 67 degrees C for 20 secs and 72 degrees C for 20 secs, with a final extension at 72 degrees C for 5 min. Replicate PCR products were pooled then diluted 1:10 and Illumina sequencing adapters added in a second round of PCR using the same reaction mix and thermal cycling conditions as the first round, except the concentration of BSA was halved (0.1 micro g/micro L), and the number of cycles was reduced to 10 with an annealing temperature of 55 degrees C. Products from each round of PCR were visualized on 2% agarose gels. Second round PCR products were pooled in equimolar ratios based on band intensity. The pooled products were purified using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA) and the concentration of the library measured using the Qubit dsDNA HS assay on a QUBIT 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA). The pool was diluted to 2 nM and paired-end reads generated on a MiSeq (Illumina, San Diego, CA, USA) with MiSeq Reagent Nano kit vs (300-cycles). Bacterial 16S rDNA PCR amplification and high-throughput sequencing Bioinformatics Reads were sorted by sample-specific MIDs added in the second round PCR using the MiSeq Reporter software. Fastq reads were merged using the -fastq_mergepairs command in USEARCH v8.0.1623 (Edgar 2010). Merged reads were sorted by "internal" 6 bp MID tags, and locus-specific primers trimmed with custom R scripts using the ShortRead package (Morgan et al. 2009), with only reads containing perfect matches to the expected MIDs and primers retained. Reads for all samples were dereplicated and global singletons discarded (-derep_fulllength -minuniquesize 2), and clustered into OTUs with the UPARSE algorithm (Edgar 2013) using the '-cluster_otus' command. Potentially chimeric reads were also discarded during this step. Reads for each sample were then assigned to OTUs (-usearch_global -id .97), and an OTU table generated using a custom R script. Taxonomy was assigned to each OTU using MEGAN version 5.10.5 (Huson et al. 2011) based on 50 hits per OTU generated by BLASTN searches against the NCBI 'nt' database (downloaded August 2015). Default LCA parameters were used, except Min support = 1, Min score = 100, Top percent = 10. Alpha and beta-diversity analyses were performed based on a rarefied OTU table with QIIME v1.8.0 (alpha_rarefaction.py, beta_diversity_through_plots.py, Caporaso et al. 2010). References Caporaso JG, Kuczynski J, Stombaugh J, et al. (2010) QIIME allows analysis of high-throughput community sequencing data. Nature Methods 7, 335-336. Huson DH, Mitra S, Ruscheweyh HJ, Weber N, Schuster SC (2011) Integrative analysis of environmental sequences using MEGAN4. Genome Research 21, 1552-1560. Jarman SN, McInnes JC, Faux C, et al. (2013) Adelie penguin population diet monitoring by analysis of food DNA in scats. PLoS One 8, e82227.

  • Metadata record AAS_4127_antFOCE_EnvironmentalData contains seafloor Ambient Light and ambient Seawater Temperature data sets collected at the antFOCE site during the experiment. Ambient Light data was collected using Photosynthetically Active Radiation sensors (Odyssey Dataflow 392 photo diode light meters) distributed around the antFOCE site as well as several inside the experimental chambers and open plots. Seawater Temperature data were collected using Onset Hoboware Tidbit v2 (UTBI-001) temperature loggers attached to the outside of various pieces of the underwater experimental infrastructure across the antFOCE site. Refer to antFOCE report section 2.3 for deployment, sampling and on-station analysis details. https://data.aad.gov.au/metadata/records/AAS_4127_antFOCE_Project4127 Background The antFOCE experimental system was deployed in O'Brien Bay, approximately 5 kilometres south of Casey station, East Antarctica, in the austral summer of 2014/15. Surface and sub-surface (in water below the sea ice) infrastructure allowed controlled manipulation of seawater pH levels (reduced by 0.4 pH units below ambient) in 2 chambers placed on the sea floor over natural benthic communities. Two control chambers (no pH manipulation) and two open plots (no chambers, no pH manipulation) were also sampled to compare to the pH manipulated (acidified) treatment chambers. Details of the antFOCE experiment can be found in the report – "antFOCE 2014/15 – Experimental System, Deployment, Sampling and Analysis". This report and a diagram indicating how the various antFOCE data sets relate to each other are available at: https://data.aad.gov.au/metadata/records/AAS_4127_antFOCE_Project4127

  • Carbonate chemistry data sets for the Antarctic Free Ocean Carbon Dioxide Enrichment experiment, Casey Station, East Antarctica, 2014/15. Project Summary: Currently, a quarter of the CO2 we emit is absorbed by the ocean. CO2 absorption in seawater changes its chemistry – reducing ocean pH (raising its acidity) – which has significant impacts on biological processes and serious implications for the resilience of marine ecosystems. As CO2 is more soluble in cold water we expect polar ecosystems to bear the heaviest burden of this 'ocean acidification'. We will perform the first in situ polar CO2 enrichment experiment to determine the likely impacts of ocean acidification on Southern Ocean sea-floor communities under increasing CO2 emissions.

  • Refer to antFOCE report section 4.5.2 for deployment, sampling and analysis details. https://data.aad.gov.au/metadata/records/AAS_4127_antFOCE_Project4127 The download file contains an Excel workbook with one data spreadsheet and one of notes relevant to the data. The data are the total number of each organism collected from artificial substrate units (plastic pot scourers) deployed in chambers or open plots during the antFOCE experiment (Data = Number of Individuals). Analysis methods are detailed in the Notes spreadsheet. Background The antFOCE experimental system was deployed in O’Brien Bay, approximately 5 kilometres south of Casey station, East Antarctica, in the austral summer of 2014/15. Surface and sub-surface (in water below the sea ice) infrastructure allowed controlled manipulation of seawater pH levels (reduced by 0.4 pH units below ambient) in 2 chambers placed on the sea floor over natural benthic communities. Two control chambers (no pH manipulation) and two open plots (no chambers, no pH manipulation) were also sampled to compare to the pH manipulated (acidified) treatment chambers. Details of the antFOCE experiment can be found in the report – “antFOCE 2014/15 – Experimental System, Deployment, Sampling and Analysis”. This report and a diagram indicating how the various antFOCE data sets relate to each other are available at: https://data.aad.gov.au/metadata/records/AAS_4127_antFOCE_Project4127

  • Refer to antFOCE report section 4.4.5 for deployment, sampling and analysis details. https://data.aad.gov.au/metadata/records/AAS_4127_antFOCE_Project4127 One camera and flash unit was mounted on the top middle section of each chamber to take one photo of the sediment every 30 minutes. The download file contains two folders with the photos taken from the 28th of January to the 23rd of February 2015 – one for Chamber A and one for Chamber C. A video time-lapse compilation of the Chamber A images is also included. Malfunctioning cameras deployed on Chamber A and C and on B and D during this same period and at other times, meant that no useful images were obtained. Background The antFOCE experimental system was deployed in O'Brien Bay, approximately 5 kilometres south of Casey station, East Antarctica, in the austral summer of 2014/15. Surface and sub-surface (in water below the sea ice) infrastructure allowed controlled manipulation of seawater pH levels (reduced by 0.4 pH units below ambient) in 2 chambers placed on the sea floor over natural benthic communities. Two control chambers (no pH manipulation) and two open plots (no chambers, no pH manipulation) were also sampled to compare to the pH manipulated (acidified) treatment chambers. Details of the antFOCE experiment can be found in the report – "antFOCE 2014/15 – Experimental System, Deployment, Sampling and Analysis". This report and a diagram indicating how the various antFOCE data sets relate to each other are available at: https://data.aad.gov.au/metadata/records/AAS_4127_antFOCE_Project4127