Seasonal dynamics of dissolved organic matter and microbial activity in the coastal North Sea

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1 The following supplement accompanies the article Seasonal dynamics of dissolved organic matter and microbial activity in the coastal North Sea Eva Sintes 1, 2, *, Karen Stoderegger 1, Veronica Parada 1, Gerhard J. Herndl 1, 2 1 Department of Biological Oceanography, Royal Netherlands Institute for Sea Research, PO Box 59, 1790 AB, Den Burg, The Netherlands 2 Department of Marine Biology, Faculty Center of Ecology, University of Vienna, Althanstr. 14, 1090 Vienna, Austria * eva.sintes@univie.ac.at Aquatic Microbial Ecology 60:85 95 (2010) Supplement 1 EXTENDED MATERIALS AND METHODS Detailed information on materials and methods is provided here for the parameters shown as complimentary data, such as inorganic nutrient concentrations and phytoplanktonic primary production. Additionally, supplementary information on the recovery efficiency of dissolved proteins is provided as background to the data on protein concentrations reported in the main body of the paper. where PP d is the daily primary production, PP inc is the primary production measured during the incubation, I PARinc is the measured photosynthetically available radiation (PAR) intensity during the incubation and I 0PARdaily is the accumulated PAR over the day. The value of the attenuation coefficient K PAR (m 1 ) was empirically derived from the Secchi depth (d Secchi ; m) according to Tillmann et al. (2000) in the German Wadden Sea: K PAR = (1.242) / d Secchi Inorganic nutrients The concentrations of dissolved inorganic nutrients (NH 4 +, NO 3, NO 2, PO 4 3 ) were determined on 0.2 µm filtered (Acrodisc, Gelman Science) samples in a TRAACS 800 autoanalyzer system. NH 4 + was detected with the indo-phenolblue method (ph 10.5) at 630 nm (Helder & De Vries 1979). NO 2 was detected after diazotation with sulfanilamide and N-(1-naphthyl)-ethylene diammonium-dichloride as the reddish-purple dye complex at 540 nm. NO 3 was reduced in a copper cadmium coil to nitrite and then measured as nitrite. PO 4 3 was determined via the molybdenum blue complex at 880 nm. Phytoplankton and primary production Depth-integrated primary production was calculated following (Philippart et al. 2007). Briefly, assuming that measured production is linearly related to in situ light intensity, a fixed factor of 7% for daylight reflection at the water surface (Højerslev 1978), and that the production-light curve is roughly linear over the range of relevant light intensities in the water column, as well as over the range from 0 to light intensity during incubation, then it can be deduced that: PP d = PP inc I PARinc 1 K PAR 1 I 0PARdaily 0.93 (S1) Dissolved protein recovery The percent recovery was not measured, but was probably <100%. Powell & Timperman (2005) measured a recovery of 55 to 60% using a surface blocking agent (SBA) and protein concentrations above 2 µg l 1. We can assume that our measurements resulted in a recovery efficiency at least as high as that of Powell & Timperman (2005) because, although we did not use SBA, we pre-conditioned the system by allowing 100 to 500 ml pre-filtered sample to run through and discarding the permeate and retentate prior to processing the actual sample, thus minimizing losses due to adsorption (Buesseler et al. 1996). Additionally, we used filters with smaller molecular weight cut-off (MWCO) than those used by Powell & Timperman (2005), which should minimize losses of smaller proteins through the ultrafiltration membrane. Also, our active membrane area of both the pre-filter and the ultrafiltration membrane (200 cm 2 each) was much smaller than that of Powell & Timperman (2005), minimizing losses of proteins by adsorption onto the ultrafiltration membranes. LITERATURE CITED Buesseler KO, Bauer JE, Chen RF, Eglinton TI, and others (1996) An intercomparison of cross-flow filtration techniques used for sampling marine colloids: overview and organic carbon results. Mar Chem 55:1 31

2 2 Supplement 1 (continued) Helder W, De Vries RTP (1979) Automatic phenol-hypochlorite method for the determination of ammonia in sea and brackish waters. Neth J Sea Res 13: Højerslev NK (1978) Daylight measurements appropriate for photosynthetic studies in natural seawaters. J Cons Int Explor Mer 38: Philippart CJM, Beukema JJ, Cadée GC, Dekker Rand others (2007) Impacts of nutrient reduction on coastal communities. Ecosystems 10: Powell MJ, Timperman AT (2005) Quantitative analysis of protein recovery from dilute, large volume samples by tangential flow ultrafiltration. J Membrane Sci 252: Tillmann U, Hesse KJ, Colijn F (2000) Planktonic primary production in the German Wadden Sea. J Plankton Res 22: Fig. S1. Respiration measured in the large (LF) and small free-living (SF) bacteria over the seasonal cycle

3 Supplement 1 (continued) 3 Fig. S2. (A) Annual dynamics of total flagellate and virus-like particle (VLP) abundance, total and free-living bacterial abundance also plotted as reference. (B) Dynamics in viral production and total (free-living and attached) bacterial carbon production Fig. S3. Seasonal dynamics of water temperature (Temp), salinity (Sal) and wind speed (Wind) at the NIOZ jetty at the western entrance of the North Sea into the Dutch Wadden Sea

4 4 Supplement 1 (continued) Table S1. Organic nutrient concentration over the annual cycle in coastal North Sea waters: DOC = dissolved organic carbon; DON = dissolved organic nitrogen; DOP = dissolved organic phosphorus; contribution of DOP and DON to total dissolved phosphorus (TDP) and nitrogen (TDN), respectively, and total nitrogen to total phosphorus (TDN:TDP) ratios are indicated; Prot = dissolved protein concentration; % prot-n to DON = contribution of protein-n to DON assuming 16% N-content; % prot-c to DOC = contribution of protein-c to DOC assuming 50% C-content; BD = below detection limit Date DOC DON DOP DOC: DON: DOC: %DOP %DON TDN: Prot % prot-n % prot-c µm µm µm DON DOP DOP of TDP of TDN TDP µg l 1 to DON to DOC 18-Dec BD Jan BD Jan Jan Jan Feb Feb Feb Feb Mar Mar Mar Mar Mar Mar Mar Apr Apr Apr Apr Apr Apr Apr May May May May Jun Jun Jun Jun Jul Jul Aug Aug Aug Aug Sep Sep Sep Sep Sep Oct Oct Oct Nov Nov Nov Nov Dec Dec

5 Supplement 1 (continued) 5 Table S2. Bacterial growth efficiency (BGE), cell-specific bacterial heterotrophic production (BHP, fmol C cell 1 d 1 ) and cell-specific oxygen consumption (fmol O 2 cell 1 d 1 ) for the different size-fractions of the bacterial community over the annual cycle: total, attached, total free-living (tfl, <3 µm), large free-living (LF, µm) and small free-living (SF, <0.8 µm) Date BGE Cell-specific BHP Cell-specific respiration tfl LF SF Total Attached tfl LF SF tfl LF SF 18-Dec Jan Jan Jan Feb Feb Feb Feb Mar Mar Mar Mar Mar Mar Apr Apr Apr Apr Apr Apr Apr May May May May Jun Jun Jun Jun Jun Jul Aug Aug Aug Aug Sep Sep Sep Sep Sep Oct Oct Oct Nov Nov Nov Nov Dec Dec

6 6 Supplement 1 (continued) 3 Table S3. Pearson regression coefficient (r) between nutrients (Si: silicate, PO 4 : phosphate, + NH4 : ammonium, NO2 : nitrite, NO3 : nitrate, IN: inorganic nitrogen, DOC: dissolved organic carbon, DON: dissolved organic nitrogen, DOP: dissolved organic phosphorus, TDP: total dissolved phosphorus, TDN: total dissolved nitrogen, Prots: proteins) and salinity (Sal) and phytoplankton parameters (Chl a: chlorophyll a, Phaeo: total Phaeocystis ml 1, Diat: diatoms ml 1, Algae: total phytoplankton cells ml 1, PPP: particulate phytoplankton production mmol C m 2 d 1, PER: phytoplanktonic extracellular release (mmol C m 2 d 1 ). Winter: 18 December to 4 March, Spring: 10 March to 16 June, Summer: 23 June to 22 September, Fall: 29 September to 12 December. Significant relationships (p < 0.05) are marked in bold Winter Spring Summer Fall Sal Chl a Phaeo Diat Algae PPP PER Sal Chl a Phaeo Diat Algae PPP PER Sal Chl a Phaeo Diat Algae PPP PER Sal Chl a Phaeo Diat Algae PPP PER µm Si nd nd 3 µm PO nd nd + µm NH nd nd µm NO nd nd µm NO nd nd µm IN nd nd µm DOC nd nd µm DON nd nd µm DOP nd nd µm TP nd nd µm TN nd nd Prots µg l nd nd Table S4. Pearson correlation coefficient between different bacterial parameters and environmental and other biological parameters over the annual cycle. BA: bacterial abundance, R: bacterial respiration, BHP: bacterial heterotrophic production, BGE: bacterial growth efficiency, BCD: bacterial carbon demand, GR: bacterial growth rate, Chl a: chlorophyll a, PPP: particulate phytoplankton production, PER: photosynthetic extracellular release, %PER: % of photosynthetic extracellular release from PPP, FA: flagellate abundance, VA: viral abundance, VP: viral production. SF: small free-living (<0.8 µm), LF: large free-living (0.8 3 µm), tfl: total free-living bacteria (<3 µm), Attached: attached bacteria (>3 µm), Total: total bacteria (unfiltered). Significant correlations (p < 0.05) are indicated in bold BA (cells ml 1 ) R (µmol O 2 l 1 d 1 ) BHP (µmol C l 1 d 1 ) BGE (%) BCD (µmol GR (d 1 ) C l 1 d 1 ) SF LF tfl Attached Total SF LF tfl SF LF tfl Attached Total SF LF tfl SF tfl SF LF tfl Attached Total Temperature (ºC) DOC µm DON µm DOP µm Protein (µg l 1 ) DOC:DON DON:DOP DOC:DOP Chla (mg m 3 ) Algae (cells ml 1 ) Phaeocystis (cells ml 1 ) Diatoms (cells ml 1 ) Other Phyto (cells ml 1 ) PPP (mg C m 2 d 1 ) PER (mg C m 2 d 1 ) % PER FA total (cells ml 1 ) FA (<3 µm) (cells ml 1 ) VA (VLP ml 1 ) VP (VLP ml 1 d 1 )