<rahmann et al. 
nitrogen (PN), filters were exposed to fuming hydrochloric acid 
for 12 h to remove carbonate and subsequently dried (60°C, 
12h). Analyses were carried out with a Euro EA elemental 
analyzer calibrated with an acetanilide standard. Particulate 
organic phosphorus (POP) collected on GF/F filters was 
determined colorimetrically as ortho-phosphate after potassium 
peroxydisulfate digestion following the method of Hansen and 
<oroleff (1999). Biogenic silica (BSi) was determined from 
material filtered onto cellulose acetate filters (0.8 wm), dissolved 
with 25 ml NaOH (0.1 M) at 85°C for 2 h 15 min in a 
shaking water bath and analyzed after cooling as Si(OH)4 
according to the method by Hansen and Koroleff (1999). 
Biogenic opal was calculated assuming a water content of —10% 
(Mortlock and Fröhlich, 1989). 
Samples for phytoplankton pigment concentrations were 
collected by filtration of seawater from the CTD/rosette through 
GF/F filters, and stored at —80°C immediately after filtration. 
Pigments were extracted by adding 2 ml acetone (90%) to each 
filter sample followed by homogenization in a cell mill (Edmund 
3uehler GmbH). Each sample was then centrifuged and the 
supernatant was filtered through a 0.2 Wm Teflon filter and 
analyzed by High-Performance Liquid Chromatography (HPLC) 
after Franz et al. (2012b). Seawater samples (4 ml) were collected 
for analyses of the phytoplankton community composition by 
flow cytometry to complement phytoplankton pigment data, 
fixed with hexamine/formalin solution and stored at —80°C. 
Transparent exopolymer particles (TEP) and Coomassie 
stainable particles were filtered under low pressure (<150 mbar) 
onto 25 mm Nucleopore polycarbonate membrane filters (0.4 um 
pore size, Whatman Ltd.) and stained with Alcian Blue and 
Coomassie Brilliant Blue, respectively. Each filter was placed on 
the white side of a semi-transparent glass slide (Cytoclear®©) 
and stored frozen at —20°C until analysis. TEP and CSP 
were determined by microscopy and subsequent image analysis 
(Engel, 2009). 
Export flux of particles was characterized using surface- 
tethered sediment traps (Engel et al., 2017), with Particle 
interceptor-Traps (PIT) following Knauer et al. (1979). Each PIT 
had an inside diameter of 7 cm, an outside diameter of 7.6 cm 
and a height of 53 cm, leading to an aspect ratio of 7.5. PITs were 
covered with a baffle system consisting of smaller acrylic tubes 
attached to the top end and filled with a 0.2 wm filtered brine 
solution containing 50 g 17! sodium chloride to reduce drag 
induced movement within the trap. For preservation, formalin 
(2% final concentration) was added to the brine solution. 
Recovered samples were, at first, passed through a 500 wm nylon 
mesh. Swimmers were removed from the mesh using forceps 
under a binocular microscope. The remaining particles, which 
stuck to the mesh, were transferred back to the sample. 
Dissolved Organic Matter, Cell Abundance, 
Extracellular Enzyme Rates, and Bacterial Production 
For dissolved organic carbon (DOC) and total dissolved nitrogen 
(TDN), samples (20 ml) were collected in duplicate on a number 
of cruises (Engel et al., 2021; see Table 2 and Supplementary 
Table 16), filtered through combusted (8 h, 500°C) GF/F filters 
or through syringe filters (0.45 wm glass microfiber GD/X 
membrane, WhatmanTM) that were rinsed with 50 ml sample 
"rontiers in Marine Science | www.frontiersin.ore 
SFB754 Data Legacy 
and filled into combusted (8 h, 500°C) glass ampoules. Samples 
were acidified with 80 ul of 85% phosphoric acid or 20 ul of 30% 
ultrapure hydrochloric acid, heat-sealed immediately and stored 
at 4°C in the dark until analysis. DOC samples were analyzed by 
high-temperature catalytic oxidation (TOC-VCSH, Shimadzu), 
as described in more detail in Engel and Galgani (2016). 
Samples for the analysis of dissolved amino acids, which 
are a combination of free and hydrolysable amino acids (DAA, 
4 ml), and dissolved combined carbohydrates (DCHO, —16 ml) 
were filtered through rinsed Acrodisc® 0.45 Wm GHP membrane 
{Pall) in combusted vials (8 h, 500°C) and stored at —20°C, 
tespectively. Prior to analysis DAA were hydrolyzed using 
6 N HCI at 100°C for 20 h. Determination of DAA was 
carried out on a 1260 HPLC system (Agilent), following the 
methods described by Lindroth and Mopper (1979) and Dittmar 
et al. (2009), with modifications as described in Engel and 
Galgani (2016). For the analysis of free amino acids (FAA) the 
same samples were processed without hydrolyzation. DCHO 
samples were desalted by membrane dialysis (1 kDa, Spectra 
Por) and hydrolyzed using 1 M HCI for 20 h at 100°C prior 
to analyses. Samples were analyzed after Engel and Händel 
2011) with a high-performance anion exchange chromatography 
(HPAEC) (DIONEX ICS3000DC). More detail on molecular 
DOM composition may be found in Loginova et al. (2019), 
Maßmig et al. (2020), and Maßmig and Engel (2021). 
Bacterial abundance was determined by flow cytometry on a 
FACS Calibur (Becton Dickinson) after Gasol and del Giorgio 
(2000) from 1.6 ml sample, fixed with 0.75 u1 25% glutaraldehyde 
on board and stored at —80°C until analyses. To 400 ul sample 
10 ul Flouresbrite® fluorescent beads (Polyscience, Inc.) and 
10 w1 Sybr Green (Invitrogen) were added. 
For the extracellular enzymes leucine aminopeptidase and 
B-glucosidase, potential hydrolytic rates were determined 
after Hoppe (1983). L-leucine-7-amido-4-methylcoumarin 
(Sigma Aldrich) and 4-methylumbelliferyl-B-D-glucopyranoside 
(Acros Organics) were used as fluorescent substrate analogs 
and added in final concentrations of 1, 5, 10, 20, 50, 
80, 100, and 200 uwmol 17! in 69 well plates (Costar). 
Afterward, 200 ul sample were added and fluorescence 
was measured with a plate reader fluorometer (FLUOstar 
Optima, BMG Ilabtech) (excitation: 355 nm; emission: 
160 nm) after 0 and 12 h of incubation. For details about 
incubation conditions and subsequent calculations see 
Vaßmig et al. (2020). 
Bacterial production was determined by measuring the 
'ncorporation of labeled leucine (31H) that was added at a 
saturating final concentration of 20 nmol (specific activity 100 
Ci mmol-!, Biotrend) in 1.5 ml of sample (Kirchman et al., 
1985; Smith and Azam, 1992). After 3 h of incubation, samples 
were extracted via the centrifugation method according to Smith 
and Azam (1992) and measured with a liquid scintillation 
counter (Hidex 300 SL, Triathaler!M, FCI). For the estimation 
of incorporated carbon, a conversion factor of 1.5 kg C mol! 
leucine was used (Simon and Azam, 1989). For further details 
about incubation conditions, sample treatment, and subsequent 
calculations see Maßmig et al. (2020). 
FDOM samples were filtered through 0.2 m polyethersulfone 
syringe filters (CHROMAPHIL® Xtra PES—45/25) and stored 
3eptember 2021 | Volume 8 | Article 72330«&