Alkaline phosphatase (AP) activity was measured using the fluorogenic substrate 4-methylumbelliferyl phosphate (MUF-P) (Sigma-Aldrich, St. Louis, MO) and its reference standard, methylumbelliferone (MUF). Fluorescence was measured using black, flat bottom, 96-well microplates in a Spark 10M Multimode Microplate Reader (Tecan, M\u00e4nnedorf, Switzerland). Fluorescence of MUF is greatest at pH 10, therefore 25 \u03bcL of 0.4 M NaOH was added to the wells (final concentration 40 mM) to be read. 25 \u03bcL of 1M EDTA was added as well (100 mM final concentration) to prevent precipitation of c 439 arbonate from sampled veins.
\nFluorescence was measured with an excitation wavelength of 380 nm and emission of 454 nm for all substrates and standards. One cm3 powdered rock was mixed with 5 cm3 of sterileartificial seawater (ASW) in a 8 mL serum vial with 90:5:5 N2:CO2:H2 headspace. 700 \u03bcL of each slurry was withdrawn with a sterile syringe to a 1.5 mL Eppendorf tube after setup but before sealing the vial; this sample served as T0, with triplicate 200 \u03bcL technical replicates. These 700 \u03bcL samples were briefly centrifuged (60 sec. at 2500 rpm) and the supernatant used for the T0 analyses.
\nTwo additional samples were taken using the same methods as for T0 after at least 2 weeks and then again after 4-6 weeks to generate a slope of activity. Incubations were kept at 10\u02daC, the inferred in situ temperature, for the duration of each assay. Autoclaved, powderized rock from each of the samples was tested to determine the amount of fluorophore adsorbance to rock powder.
\nAdsorbance was found to behave in a systematic manner, resulting in a straight line when comparing fluorescence standards in artificial seawater (ASW) alone with fluorescence standards plus rock powder in ASW, although this relationship was found to be different when measured at 4 hours versus days later. Therefore, a correction factor for adsorbance was applied to the enzyme data for the initial measurement (t0, y=1.90x-676), taken <2 hours after experiment initiation, versus the second and third measurements (t1 and t2, y = 4.64x - 303), taken days to weeks later. Negative controls consisting of the same ASW used for the sample incubations plus substrate, but no sample, were consistently below detection. The limit of quantification for the AP assay, defined as 3X the standard deviation of the blank, was 0.0242 pmol cm-3 rock hour-1 based on analysis of eight blanks.
Supplementary Table 3B: Overview of archaeal and bacterial lipid biomarkers and cell counts. Replicate cell counts for the 11 samples and alkaline phosphatase activity measurements available for any of the 11 samples. Samples were taken on board of the JOIDES Resolution\u00a0 between November 30, 2015 and January 30, 2016 in the SW Indian Ridge.
BCO-DMO processing notes:
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