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Diatom frustule cleaning protocol
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Previously published methods for determining diatom bound 15N values required
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adaptation due to the high organic carbon concentrations in Guaymas Basin sediments [Robinson
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et al., 2004; Robinson and Sigman, 2008]. A more universal cleaning method for use with all
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sediment, culture and water column samples was developed to allow more direct data
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comparisons. Specific details for cleaning culture and water column diatoms differ slightly from
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sediments and will be reported elsewhere [Horn et al., 2011].The most significant change
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incorporated here was the use of potassium permanganate oxidation in place of hydrogen
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peroxide to reduce variability of 15N values of culture and water column samples [Horn et al.,
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2011].
Several adaptations to the persulfate-denitrifier method were tested to determine how best
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to remove excess organic carbon without affecting the 15N value of nitrogen contained within
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diatom frustules. We added an initial potassium permanganate oxidation prior to any cleaning
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methods described by Robsinson et al. [2004] and Robinson and Sigman [2008], replaced the
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hydrogen peroxide (H2O2) oxidation with a potassium persulfate oxidation, and added additional
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perchloric oxidations prior to dissolution of the frustules. Described below is a detailed
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description of the adapted cleaning procedures.
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An initial oxidation was necessary to prevent particle aggregating during the physical
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separation of more dense aluminosilicates and metal oxides from biogenic silica. The use of
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H2O2 and potassium permanganate as oxidizing agents was compared. The permanganate
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oxidation followed the procedure described by Hasle and Fryxell [1970]. Briefly, 50%
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concentration H2SO4 was added to the samples and then 3 mL of saturated potassium
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permanganate was added in 0.5 mL increments. In order to reduce the excess potassium
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permanganate, saturated oxalic acid was added slowly until the solution was clear. The H2O2
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oxidation involved the addition of 10 mL of cold 10% H2O2 to the dry sample with brief
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sonication. After 20 minutes at room temperature, 10 mL of 2 M HCl was added to the sediment-
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H2O2 mixture, and sonication repeated. The samples reacted for an additional 30 minutes before
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rinsing. After this initial oxidation, the physical separation and reductive cleaning of the opal
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fraction, as described in Robinson et al. [2004] were used for all samples. In the published
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method, the oxidation after the reductive cleaning used H2O2. Here, we replaced the H2O2
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oxidation with a second potassium permanganate oxidation [Hasle and Fryxell, 1970] after
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which the sediments were further oxidized using perchloric acid at 100˚C [Robinson and Sigman,
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2008]. The samples that were initially oxidized using potassium permanganate removed more
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organic nitrogen than the H2O2 oxidation with far less variability, 16.8 ± 0.8 µmol N per gram of
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opal compared to 26.7 ± 8.0 µmol N per gram of opal (Figure S2). For the diatom bound  15N
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method to be effective, the  15N value should measure the 15N value of the nitrogen pool
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contained within the frustule, not nitrogen from organic matter outside of or adhering to the
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frustule. The lower standard deviation of the 15N values associated with the potassium
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permanganate oxidation suggests that this method consistently removed similar quantities of
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external organic matter.
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Perchloric acid was previously used to oxidize external organic matter from the frustules
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[Robinson and Sigman, 2008]. Data from cleaning diatoms grown in culture indicate that
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additional perchloric acid oxidation steps remove more organic matter and improve the precision
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of the 15N measurement (Horn and Morales, unpublished data). A comparison of results where
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the samples were subjected to 4, 5, or 6 perchloric acid treatments showed that the precision of
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the 15N values was the best when 4 perchloric acid treatments were used. The standard
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deviation of 6 samples with 4 perchloric acid oxidations was 0.35‰, 5 oxidations was 0.63‰
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and 6 perchloric acid oxidations was 0.57‰ (Figure S3). We interpret the higher standard
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deviation associated with 5 or more perchloric acid oxidation steps as resulting from possible
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alteration of the diatom-bound organic N. Each additional cleaning subjects the samples to
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additional heating thus a higher likelihood for opal dissolution. For future work using
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sedimentary diatom bound 15N measurements, it is suggested that the number of perchloric acid
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oxidations be determined for each specific sedimentary environment.
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References
Hasle, G. R., and G. A. Fryxell (1970), Diatoms: cleaning and mounting for light and electron
microscopy, Transactions of the American Microscopical Society, 89, 469-474.
Horn, M., et al. (2011), Nitrogen isotopic relationship between diatom-bound and bulk organic
matter of cultured polar diatoms, Paleoceanography, doi:10.1029/2010PA002080, in press.
Robinson, R. S., et al. (2004), Revisiting nutrient utilization in the glacial Antarctic: Evidence
from a new method for diatom-bound N isotopic analysis, Paleoceanography, 19(3), PA3001,
doi:3010.1029/2003PA000996.
Robinson, R. S., and D. M. Sigman (2008), Nitrogen isotopic evidence for a poleward decrease
in surface nitrate within the ice age Antarctic, Quaternary Science Reviews, 27(9-10), 10761090.
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