TY - JOUR
AU - Ziervogel,, Kai
AB - Abstract Small-scale turbulence in the surface ocean is ubiquitous, influencing phytoplankton dynamics with consequences for energy flow. The underlying mechanisms that drive changes in phytoplankton dynamics under turbulence are not well constrained. We investigated growth of four phytoplankton species at different turbulence levels in oscillating grid tanks. We also measured transparent exopolymer particles (TEP) from phytoplankton exudates, which play a major role in biogeochemical fluxes in the ocean. Turbulence levels in the tanks reflected in situ conditions in surface waters from the open ocean to higher turbulent environments such as estuaries. Growth rates were unaffected by turbulence while TEP concentrations as xanthan gum (XG) equivalents normalized to algal cells showed generally higher levels in the high turbulence compared to the low turbulence treatments particularly during initial algal growth. Results from a mixing experiment without algal cells and XG also revealed enhanced formation of TEP-like particles under high mixing conditions, indicating that TEP formation in the phytoplankton turbulence treatments was mainly driven by physical processes, such as enhanced encounter rates of TEP-precursors under high mixing. Our results underline the importance of small-scale turbulence on TEP formation with possible consequences for particle aggregation and vertical carbon fluxes in the ocean. INTRODUCTION Phytoplankton form the base of the marine food web and play an integral role in the sequestration of atmospheric carbon to the deep ocean through a mechanism known as the biological pump (DeVries et al. 2012). The suite of biogeochemical processes that determine phytoplankton dynamics are in part affected by physical properties of the surface ocean (Kiørboe, 1993). For instance, wind-driven water mixing and turbulence, meaning secondary motion caused by moving fluids, can at times enhance nutrient supply to the upper ocean (Fan et al., 2013; Hemer et al., 2013a, 2013b). Larger scale water mixing also affects turbulence on the scale of individual planktonic cells. As demonstrated by numerical models, the region of lower nutrient concentration around planktonic cells can be perturbed by the laminar shear derived from small-scale turbulence and positively impact nutrient assimilation into the cell (Karp-Boss et al., 1996; Wolf-Gladrow and Riebesell, 1997; Barton et al., 2014). Fluid dynamic theory predicts a size threshold (>60 μm) for open ocean planktonic cells above which organisms approach the length of the smallest turbulent eddies, called Kolmogorov length scales (Kolmogorov, 1962), or are impacted by the shear derived from these eddies, and thus ‘feel’ the effects of small-scale turbulence (Karp-Boss et al., 1996). However, experimental studies on the effects of small-scale turbulence on phytoplankton dynamics are often inconsistent with fluid dynamic theory. While some studies show a positive correlation between turbulence and nutrient uptake and growth of planktonic cells below the Kolmogorov length scale (Arin et al., 2002; Hondzo and Wüest, 2009; Iversen et al., 2010), others demonstrate growth inhibition of small planktonic cells under turbulence (Thomas and Gibson, 1990; Hondzo and Lyn, 1999; Peters et al., 2006). Moreover, some mesocosm experiments with natural planktonic assemblages show that turbulence enhances phytoplankton growth and increases exudation of organic matter, resulting in the formation of transparent exopolymer particles (TEP, Beauvais et al., 2006; Iversen et al., 2010). TEP are surface-active (sticky) particles, accelerating the formation of sinking organic matter aggregates, also known as marine snow (e.g. Alldredge and Silver, 1988). TEP form abiotically from organic precursors (i.e. organic macromolecules forming microgels) that are excreted by many marine organisms (Decho, 1990; Decho and Gutierrez, 2017 and references therein). The main source of TEP precursors in the surface ocean are phytoplankton metabolites that are often excreted in copious amounts in response to environmental stress such as nutrient limitation, increased temperature and, in some species, high pCO2 (Mari et al., 2017 and references therein). Table I Experimental conditions and results of biogeochemical parameters Experiment. Turbulence levels, Hz Algal growth rate, d−1 Photosynthetic efficiency, Fv/Fm Bacterial biomass production, μg C L−1 Algal growth phase TEP-C, μg C L−1 Thalassiosira sp. 2.0 (HT) 0.38 0.6 66 EE 201 ± 14 ME 566 ± 40 S 512 ± 6 1.5 (MT) 0.41 0.6 300 EE 170 ± 72 ME 542 ± 52 S 504 ± 23 1.0 (LT) 0.42 0.6 318 EE 216 ± 47 ME 523 ± 74 S 503 ± 17 Chaetoceros sp. 2.0 (HT) 0.97 n.a. 351 EE 397 ± 38 ME 549 ± 58 LE 856 ± 52 1.0 (LT) 0.94 n.a. 528 EE 371 ± 19 ME 510 ± 116 LE 859 ± 97 T. pseudonana 2.0 (HT) 1.02 0.6 228 EE 83 ± 25 ME 85 ± 14 LE 331 ± 83 S 485 ± 124 1.0 (MT) 1.02 0.6 267 EE 105 ± 35 ME 110 ± 15 LE 285 ± 52 S 653 ± 107 0.5 (LT) 0.99 0.6 213 EE 104 ± 36 ME 130 ± 16 LE 212 ± 36 S 504 ± 67 P. globosa 2.0 (HT) 0.79 0.5 162 EE 237 ± 31 ME 231 ± 7 S 389 ± 23 1.0 (MT) 0.80 0.5 9 EE 206 ± 17 ME 202 ± 19 S 156 ± 8 0.5 (LT) 0.75 0.5 3 EE 193 ± 9 ME 250 ± 26 S 392 ± 19 Experiment. Turbulence levels, Hz Algal growth rate, d−1 Photosynthetic efficiency, Fv/Fm Bacterial biomass production, μg C L−1 Algal growth phase TEP-C, μg C L−1 Thalassiosira sp. 2.0 (HT) 0.38 0.6 66 EE 201 ± 14 ME 566 ± 40 S 512 ± 6 1.5 (MT) 0.41 0.6 300 EE 170 ± 72 ME 542 ± 52 S 504 ± 23 1.0 (LT) 0.42 0.6 318 EE 216 ± 47 ME 523 ± 74 S 503 ± 17 Chaetoceros sp. 2.0 (HT) 0.97 n.a. 351 EE 397 ± 38 ME 549 ± 58 LE 856 ± 52 1.0 (LT) 0.94 n.a. 528 EE 371 ± 19 ME 510 ± 116 LE 859 ± 97 T. pseudonana 2.0 (HT) 1.02 0.6 228 EE 83 ± 25 ME 85 ± 14 LE 331 ± 83 S 485 ± 124 1.0 (MT) 1.02 0.6 267 EE 105 ± 35 ME 110 ± 15 LE 285 ± 52 S 653 ± 107 0.5 (LT) 0.99 0.6 213 EE 104 ± 36 ME 130 ± 16 LE 212 ± 36 S 504 ± 67 P. globosa 2.0 (HT) 0.79 0.5 162 EE 237 ± 31 ME 231 ± 7 S 389 ± 23 1.0 (MT) 0.80 0.5 9 EE 206 ± 17 ME 202 ± 19 S 156 ± 8 0.5 (LT) 0.75 0.5 3 EE 193 ± 9 ME 250 ± 26 S 392 ± 19 Bacterial biomass production is estimated from changes in cell concentration over time. EE: early exponential, ME: mid exponential, LE: late exponential, S: stationary; n.a.—not available; n.d.—not detectable. View Large Table I Experimental conditions and results of biogeochemical parameters Experiment. Turbulence levels, Hz Algal growth rate, d−1 Photosynthetic efficiency, Fv/Fm Bacterial biomass production, μg C L−1 Algal growth phase TEP-C, μg C L−1 Thalassiosira sp. 2.0 (HT) 0.38 0.6 66 EE 201 ± 14 ME 566 ± 40 S 512 ± 6 1.5 (MT) 0.41 0.6 300 EE 170 ± 72 ME 542 ± 52 S 504 ± 23 1.0 (LT) 0.42 0.6 318 EE 216 ± 47 ME 523 ± 74 S 503 ± 17 Chaetoceros sp. 2.0 (HT) 0.97 n.a. 351 EE 397 ± 38 ME 549 ± 58 LE 856 ± 52 1.0 (LT) 0.94 n.a. 528 EE 371 ± 19 ME 510 ± 116 LE 859 ± 97 T. pseudonana 2.0 (HT) 1.02 0.6 228 EE 83 ± 25 ME 85 ± 14 LE 331 ± 83 S 485 ± 124 1.0 (MT) 1.02 0.6 267 EE 105 ± 35 ME 110 ± 15 LE 285 ± 52 S 653 ± 107 0.5 (LT) 0.99 0.6 213 EE 104 ± 36 ME 130 ± 16 LE 212 ± 36 S 504 ± 67 P. globosa 2.0 (HT) 0.79 0.5 162 EE 237 ± 31 ME 231 ± 7 S 389 ± 23 1.0 (MT) 0.80 0.5 9 EE 206 ± 17 ME 202 ± 19 S 156 ± 8 0.5 (LT) 0.75 0.5 3 EE 193 ± 9 ME 250 ± 26 S 392 ± 19 Experiment. Turbulence levels, Hz Algal growth rate, d−1 Photosynthetic efficiency, Fv/Fm Bacterial biomass production, μg C L−1 Algal growth phase TEP-C, μg C L−1 Thalassiosira sp. 2.0 (HT) 0.38 0.6 66 EE 201 ± 14 ME 566 ± 40 S 512 ± 6 1.5 (MT) 0.41 0.6 300 EE 170 ± 72 ME 542 ± 52 S 504 ± 23 1.0 (LT) 0.42 0.6 318 EE 216 ± 47 ME 523 ± 74 S 503 ± 17 Chaetoceros sp. 2.0 (HT) 0.97 n.a. 351 EE 397 ± 38 ME 549 ± 58 LE 856 ± 52 1.0 (LT) 0.94 n.a. 528 EE 371 ± 19 ME 510 ± 116 LE 859 ± 97 T. pseudonana 2.0 (HT) 1.02 0.6 228 EE 83 ± 25 ME 85 ± 14 LE 331 ± 83 S 485 ± 124 1.0 (MT) 1.02 0.6 267 EE 105 ± 35 ME 110 ± 15 LE 285 ± 52 S 653 ± 107 0.5 (LT) 0.99 0.6 213 EE 104 ± 36 ME 130 ± 16 LE 212 ± 36 S 504 ± 67 P. globosa 2.0 (HT) 0.79 0.5 162 EE 237 ± 31 ME 231 ± 7 S 389 ± 23 1.0 (MT) 0.80 0.5 9 EE 206 ± 17 ME 202 ± 19 S 156 ± 8 0.5 (LT) 0.75 0.5 3 EE 193 ± 9 ME 250 ± 26 S 392 ± 19 Bacterial biomass production is estimated from changes in cell concentration over time. EE: early exponential, ME: mid exponential, LE: late exponential, S: stationary; n.a.—not available; n.d.—not detectable. View Large Fig. 1 View largeDownload slide Timecourses of relative fluorescence units (RFU) during (A) Thalassiosira sp., (B) Chaetoceros sp., (C) T. pseudonana and (D) Phaeocystis globose experiments. HT—high turbulence (black diamonds), MT—medium turbulence (open rectangles) and LT—low turbulence (open diamonds). Vertical bars indicate sampling at early exponential (EE), ME, late exponential (LE), stationary (S) growth phase. Note that in the Phaeocystis MT experiment, EE and ME sampling was conducted at 7 (*) and 9 (**) days, respectively. Also note the different scales of the y-axis. Fig. 1 View largeDownload slide Timecourses of relative fluorescence units (RFU) during (A) Thalassiosira sp., (B) Chaetoceros sp., (C) T. pseudonana and (D) Phaeocystis globose experiments. HT—high turbulence (black diamonds), MT—medium turbulence (open rectangles) and LT—low turbulence (open diamonds). Vertical bars indicate sampling at early exponential (EE), ME, late exponential (LE), stationary (S) growth phase. Note that in the Phaeocystis MT experiment, EE and ME sampling was conducted at 7 (*) and 9 (**) days, respectively. Also note the different scales of the y-axis. Fig. 2 View largeDownload slide TEP and algal cells for (A) Thalassiosira sp., (B) Chaetoceros sp., (C) T. pseudonana and (D) P. globosa. Error bars are standard deviations of analytical replicates. Note the different scales of the secondary y-axis. Fig. 2 View largeDownload slide TEP and algal cells for (A) Thalassiosira sp., (B) Chaetoceros sp., (C) T. pseudonana and (D) P. globosa. Error bars are standard deviations of analytical replicates. Note the different scales of the secondary y-axis. Fig. 3 View largeDownload slide TEP normalized to algal cells for (A) Thalassiosira sp., (B) Chaetoceros sp., (C) T. pseudonana and (D) P. globosa. Error bars are standard deviations of analytical replicates. Note the different scales of the y-axis. Fig. 3 View largeDownload slide TEP normalized to algal cells for (A) Thalassiosira sp., (B) Chaetoceros sp., (C) T. pseudonana and (D) P. globosa. Error bars are standard deviations of analytical replicates. Note the different scales of the y-axis. Turbulence may alter formation of TEP through (i) modified nutrient fluxes and phytoplankton growth, and (ii) physical processes affecting encounter rates of TEP precursors (Beauvais et al., 2006; Pedrotti et al., 2009; Iversen et al., 2010). However, the results from the few previous studies (mesocosms with natural assemblages) are somewhat inconclusive since most of the biogeochemical changes during phytoplankton growth were observed between treatments of high and no turbulence (still water control), which is a rather untypical state of the surface ocean. We conducted laboratory experiments with phytoplankton cultures of varying cell sizes to determine the effects of small-scale turbulence (deliberately avoiding non-turbulent treatments) as a potential stressor on cell growth and TEP formation. Our major objective was to investigate if cell growth and TEP formation changes as a function of small-scale turbulence. Phytoplankton-turbulence experiments were conducted under nutrient-replete conditions in tanks equipped with oscillating grids, i.e. one of the most commonly used apparatuses to study the effects of small-scale turbulence on planktonic organisms (Alldredge et al., 1990; Hondzo and Lyn, 1999; Peters et al., 2006; Guadayol et al., 2009; Hondzo and Wüest, 2009). The four types of phytoplankton (Thalassiosira sp.; Chaetoceros sp.; Thalassiosira pseudonana; Phaeocystis globosa) were chosen because they are ubiquitous in the oceans and incorporate a range of cell size and morphometry, which makes them suitable for turbulence studies. Members of the genera Thalassiosira, Chaetoceros and Phaeocystis are known to produce and exude copious amounts of biomacromolecules that form TEP (Riebesell et al., 1995; Corzo et al., 2000; Passow, 2002a, 2002b; Mari et al., 2005). Moreover, T. pseudonana is a well-studied diatom and is used as a model species in biogeochemical studies (Armbrust et al., 2004). We used non-axenic phytoplankton cultures (i.e. in the presence of phytoplankton-associated bacteria) as previous studies demonstrated the importance of phytoplankton-bacterial interactions in TEP dynamics in the ocean (Passow, 2002a; Grossart et al., 2006; Gärdes et al., 2011). Heterotrophic bacteria contribute to the pool of TEP precursors and TEP in the ocean (Sugimoto et al., 2007) through either dissolved organic matter (DOM) excretion (Decho, 1990), transformation of phytoplankton-derived macromolecules which then form TEP abiotically (Smith et al., 1995), or colonization and degradation of TEP (Passow and Alldredge, 1994; Mari and Kiørboe, 1996; Pedrotti et al., 2009; Bar-Zeev and Rahav, 2015; Taylor and Cunliffe, 2017). To evaluate the role of bacteria in the formation of TEP, we followed bacterial growth and activities during the turbulence incubations. Rates of bacterial activities were measured by means of two hydrolytic enzymes, i.e. the main tools of heterotrophic bacteria to access and degrade organic macromolecules (Arnosti, 2011). We monitored activities of ß-glucosidases (BGases) and leucine-aminopeptidases (LAPase) that hydrolyze carbohydrates and peptides, respectively, which are abundant components of phytoplankton exudates and TEP (Mari et al., 2017, and references therein). Hydrolytic enzyme activities were used as an indicator for microbial degradation of organic matter including TEP compounds during the turbulence incubations. METHOD Experimental set-up Growth experiments with phytoplankton cultures were conducted in cylindrical acrylic tanks (height: 195 mm; diameter: 140 mm). The tanks were equipped with circular stainless steel grids (diameter: 125 mm) that oscillate at specific frequencies in the lower part of the tank (between 20 and 70 mm above the bottom), generating small-scale turbulence in the water (Guadayol et al., 2009). Oscillating frequencies ranged between 0.5 Hz and 2 Hz (Table I), corresponding to a range of turbulent energy dissipation rates (ɛ) of 0.02 cm2 s−3 to 1.2 cm2 s−3 (Guadayol et al., 2009). These values are at the higher end of naturally occurring ɛ (10−6 cm2 s−3—102 cm2 s−3), corresponding to wind velocities between 12 m s−1 and 47 m s−1 (Table III). Separate oscillating grid experiments were conducted with three diatoms (Thalassiosira sp.; Chaetoceros sp.; T. pseudonana) and one haptophyte (P. globosa) (Table I). Thalassiosira sp. [UNC 1203; cell length: 18–20 μm; biovolume: 3054–4 189 μm3 assuming sphere-shaped cells (Hillebrand et al., 1999)] and Chaetoceros sp. (UNC 1201; 14–16 μm; 1 440–2 145 μm3) were isolated in 2012 from Station P8 along the Line P transect in the Northeast Pacific Ocean (Ellis et al., 2017). T. pseudonana (CCMP 1335; 6–8 μm; 113–268 μm3) and P. globosa (CCMP 2754; 4–7 μm; 34–180 μm3) were obtained from the National Center for Marine Algae and Microbiota at the Bigelow Laboratory for Ocean Sciences. Stock cultures were maintained in sterile Aquil medium (Price et al., 1989) under non-axenic conditions. The growth medium was amended with 50 μΜ of nitrate (NO3−), 200 μΜ of silicic acid (Si(OH)4 and 10 μΜ of phosphate (PO43−) (all final concentrations). The experimental set-up consisted of two (in the case of Chaetoceros sp.) or three oscillating grid tanks (in the case of Thalassiosira sp., T. pseudonana, P. globosa) set to different oscillating frequencies (Table I). Prior to the turbulence experiments, we determined the lowest mixing frequency for each of the monocultures at which most of the cells stayed in suspension throughout the entire growth period (data not shown). To initiate growth in the experimental tanks, 8 mL of non-axenic maintenance culture in mid-exponential (ME) growth was added to 2.3 L of microwave sterilized Aquil medium. Oscillating grids were then turned on and the tanks were incubated under constant light (120–140 μmol photons m−2 s−1) and temperature (12°C for Thalassiosira sp., T. pseudonana, Chaetoceros sp. and 19°C for P. globosa according to the temperature of the environment that the cells were isolated from; unchanged from stock culture) until the cells reached S growth (14 days for Thalassiosira sp., 10 days for T. pseudonana and up to 12 days for P. globosa; note that Chaetoceros sp. did not reach S growth at the end of the incubation after 10 days; Fig. 1). The experimental design used in this study enabled us to attribute changes in cell growth and metabolism over time to the effects of turbulence since other environmental factors (light, temperature) were kept constant, and initial biochemical conditions (nutrient levels, physiological state of the inoculum) were very similar among the different turbulence treatments. We also conducted an abiotic turbulence experiment with a xanthan gum (XG) solution in artificial seawater (Instant Ocean) incubated under high and low turbulence (LT) conditions (2 and 1 Hz, respectively) over a time course of 6 hours (XG final conc. in the tanks: 3 g L−1) to evaluate physical effects on particle formation. XG was chosen as a model substrate as it forms gel-like particles in solution like TEP (Passow and Alldredge, 1995). The XG solution was prepared as described in Passow and Alldredge (1995) using a tissue grinder to break apart visible particles prior to the oscillating grid experiment. Abiotic formation of TEP was determined as described below. Analytical methods Phytoplankton cell growth in the turbulence tanks was monitored daily by means of in vivo chlorophyll a fluorescence expressed as RFUs (Fig. 1) using a Turner design 10-AU fluorometer (in vivo chlorophyll optical kit). Intrinsic growth rates were obtained by plotting the natural log (ln) of daily RFU during EE to ME growth phase against time (Monod, 1949; Brand et al., 1981). For Thalassiosira sp., growth rates were calculated from Day 4 to Day 9; growth rates for the Chaetoceros sp. and P. globosa incubations were calculated from Day 5 to Day 8 and Day 3 to Day 5, respectively. For P. globosa, RFU values from Day 5 to Day 8 were used to calculate growth rates in the medium turbulence (MT) tank, and from Day 6 to Day 9 for the LT and high turbulence (HT) tanks. To investigate the effects of photosynthetic efficiency, active chlorophyll fluorescence techniques were used in the Thalassiosira sp., T. pseudonana, and P. globosa experiments to measure the ratio of variable to maximum photochemical yield of photosystem II (Fv/Fm) (Geider et al., 1993a, 1993b), following the procedure described in Cohen et al. (2017) using a Satlantic FIRe system. Fv/Fm measurements were taken on dark-adapted cells during ME growth. Thalassiosira sp., Chaetoceros sp. and P. globosa cells were counted using a 1-mL Sedgewick-Rafter chamber on a light microscope (Accu-scope; at least 300 cells or 30 fields of view for each sample). Cells were fixed with 5% Lugol’s solution and stored in the dark at room temperature prior to microscopic analysis. Note that cell counts for P. globosa were only obtained for the first and last sampling time point in the MT tank and for the first and second sampling time point in the LT tank. A linear regression between RFU and existing cell concentration was applied to obtain cell numbers for the other sampling time points (y = 5 507x—1947, R2 = 0.98; y = cell concentration, x = RFU). A flow cytometer (Becton Dickinson FACSCalibur) was used for T. pseudonana cell counts (fluorescence peak: 670 nm). Table II Results from two-way ANOVAs and t-tests comparing cell-specific TEP and volumetric TEP values One-way ANOVA Cell-specific TEP, XG eq. cell−1 Tukey HSD Volumetric TEP, XG eq. L−1 Tukey HSD Thalassiosira sp. EE 0.07 - 0.55 - ME 0.01 HT = MT > LT 0.66 - S 2E-04 HT > MT = LT 0.76 - T. pseudonana EE 0.008 MT ≥ HT ≥ LT 0.65 - ME LE 0.002 0.07 LT > MT = HT - 0.03 0.82 LT ≥ MT ≥ HT - S 0.25 - 0.17 - Phaeocystis globosa EE ME S 3E-04 2E-04 2E-05 HT > LT = MT HT > MT > LT HT = LT > MT 0.09 0.05 5E-06 -—LT = MT > HT Student’s t-test Cell-specific TEP, XG eq. cell−1 volumetric TEP, XG eq. L−1 Chaetoceros sp. EE 0.004; HT > LT 0.36 ME 0.25 0.63 LE 0.05 0.96 XG 2 hrs n/a 0.07 3 hrs n/a 0.08 6 hrs n/a 0.04; HT > LT One-way ANOVA Cell-specific TEP, XG eq. cell−1 Tukey HSD Volumetric TEP, XG eq. L−1 Tukey HSD Thalassiosira sp. EE 0.07 - 0.55 - ME 0.01 HT = MT > LT 0.66 - S 2E-04 HT > MT = LT 0.76 - T. pseudonana EE 0.008 MT ≥ HT ≥ LT 0.65 - ME LE 0.002 0.07 LT > MT = HT - 0.03 0.82 LT ≥ MT ≥ HT - S 0.25 - 0.17 - Phaeocystis globosa EE ME S 3E-04 2E-04 2E-05 HT > LT = MT HT > MT > LT HT = LT > MT 0.09 0.05 5E-06 -—LT = MT > HT Student’s t-test Cell-specific TEP, XG eq. cell−1 volumetric TEP, XG eq. L−1 Chaetoceros sp. EE 0.004; HT > LT 0.36 ME 0.25 0.63 LE 0.05 0.96 XG 2 hrs n/a 0.07 3 hrs n/a 0.08 6 hrs n/a 0.04; HT > LT Italicized P-values indicate a significant difference among analytical replicates (P < 0.05). Growth phases are EE—early exponential, ME—mid exponential, LE—late exponential and S—stationary. View Large Table II Results from two-way ANOVAs and t-tests comparing cell-specific TEP and volumetric TEP values One-way ANOVA Cell-specific TEP, XG eq. cell−1 Tukey HSD Volumetric TEP, XG eq. L−1 Tukey HSD Thalassiosira sp. EE 0.07 - 0.55 - ME 0.01 HT = MT > LT 0.66 - S 2E-04 HT > MT = LT 0.76 - T. pseudonana EE 0.008 MT ≥ HT ≥ LT 0.65 - ME LE 0.002 0.07 LT > MT = HT - 0.03 0.82 LT ≥ MT ≥ HT - S 0.25 - 0.17 - Phaeocystis globosa EE ME S 3E-04 2E-04 2E-05 HT > LT = MT HT > MT > LT HT = LT > MT 0.09 0.05 5E-06 -—LT = MT > HT Student’s t-test Cell-specific TEP, XG eq. cell−1 volumetric TEP, XG eq. L−1 Chaetoceros sp. EE 0.004; HT > LT 0.36 ME 0.25 0.63 LE 0.05 0.96 XG 2 hrs n/a 0.07 3 hrs n/a 0.08 6 hrs n/a 0.04; HT > LT One-way ANOVA Cell-specific TEP, XG eq. cell−1 Tukey HSD Volumetric TEP, XG eq. L−1 Tukey HSD Thalassiosira sp. EE 0.07 - 0.55 - ME 0.01 HT = MT > LT 0.66 - S 2E-04 HT > MT = LT 0.76 - T. pseudonana EE 0.008 MT ≥ HT ≥ LT 0.65 - ME LE 0.002 0.07 LT > MT = HT - 0.03 0.82 LT ≥ MT ≥ HT - S 0.25 - 0.17 - Phaeocystis globosa EE ME S 3E-04 2E-04 2E-05 HT > LT = MT HT > MT > LT HT = LT > MT 0.09 0.05 5E-06 -—LT = MT > HT Student’s t-test Cell-specific TEP, XG eq. cell−1 volumetric TEP, XG eq. L−1 Chaetoceros sp. EE 0.004; HT > LT 0.36 ME 0.25 0.63 LE 0.05 0.96 XG 2 hrs n/a 0.07 3 hrs n/a 0.08 6 hrs n/a 0.04; HT > LT Italicized P-values indicate a significant difference among analytical replicates (P < 0.05). Growth phases are EE—early exponential, ME—mid exponential, LE—late exponential and S—stationary. View Large Fig. 4 View largeDownload slide Abiotic TEP formation in a XG–ASW solution. Bars indicate differences in absorbance readings at 787 nm relative to initial readings before the start of the turbulence incubation. HT—high turbulence (2 Hz); LT—low turbulence (1 Hz). Error bars are standard deviations of analytical replicates. Fig. 4 View largeDownload slide Abiotic TEP formation in a XG–ASW solution. Bars indicate differences in absorbance readings at 787 nm relative to initial readings before the start of the turbulence incubation. HT—high turbulence (2 Hz); LT—low turbulence (1 Hz). Error bars are standard deviations of analytical replicates. Fig. 5 View largeDownload slide Enzyme activities and bacterial cells during (A) Thalassiosira sp., (B) Chaetoceros sp., (C) T. pseudonana and (D) P. globosa experiments. LAPase—leucine aminopeptidase, BGase—b-glucosidase; note that BGase was not measured during (B). Error bars are standard deviations of analytical replicates. n.a. means not available, n.d. means not detectable; note the different scales of the secondary y-axis. Fig. 5 View largeDownload slide Enzyme activities and bacterial cells during (A) Thalassiosira sp., (B) Chaetoceros sp., (C) T. pseudonana and (D) P. globosa experiments. LAPase—leucine aminopeptidase, BGase—b-glucosidase; note that BGase was not measured during (B). Error bars are standard deviations of analytical replicates. n.a. means not available, n.d. means not detectable; note the different scales of the secondary y-axis. Table III Calculated physical conditions in the oscillating grid tanks Turbulence levels/. Oscillating frequency, Hz Dissipation rate, cm2 s−3 Wind speed, m s−1 Kolmogorov length scale, μm 12 ° C, 35 ppt 19 °C, 35 ppt HT/2 1.2 47 370 320 MT/1–1.5 0.16–0.52 24–36 450–610 400–540 LT/0.5 0.02 12 1 020 890 Turbulence levels/. Oscillating frequency, Hz Dissipation rate, cm2 s−3 Wind speed, m s−1 Kolmogorov length scale, μm 12 ° C, 35 ppt 19 °C, 35 ppt HT/2 1.2 47 370 320 MT/1–1.5 0.16–0.52 24–36 450–610 400–540 LT/0.5 0.02 12 1 020 890 Dissipation rates (ɛ) were calculated according to eq. 11 in Guadayol et al. (2009). Wind speed that would result in (ɛ) at 5 m water depth from the surface, according to eq. 1 in MacKenzie and Leggett (1993). Kolmogorov length scales were estimated as (υ3 ɛ−1)0.25, where υ is the fluid viscosity (0.01 at 35 ppt); Kolmogorov length scales were calculated for 19°C (P. globosa) and 12°C (Thalassiosira sp., Chaetoceros sp., T. pseudonana). View Large Table III Calculated physical conditions in the oscillating grid tanks Turbulence levels/. Oscillating frequency, Hz Dissipation rate, cm2 s−3 Wind speed, m s−1 Kolmogorov length scale, μm 12 ° C, 35 ppt 19 °C, 35 ppt HT/2 1.2 47 370 320 MT/1–1.5 0.16–0.52 24–36 450–610 400–540 LT/0.5 0.02 12 1 020 890 Turbulence levels/. Oscillating frequency, Hz Dissipation rate, cm2 s−3 Wind speed, m s−1 Kolmogorov length scale, μm 12 ° C, 35 ppt 19 °C, 35 ppt HT/2 1.2 47 370 320 MT/1–1.5 0.16–0.52 24–36 450–610 400–540 LT/0.5 0.02 12 1 020 890 Dissipation rates (ɛ) were calculated according to eq. 11 in Guadayol et al. (2009). Wind speed that would result in (ɛ) at 5 m water depth from the surface, according to eq. 1 in MacKenzie and Leggett (1993). Kolmogorov length scales were estimated as (υ3 ɛ−1)0.25, where υ is the fluid viscosity (0.01 at 35 ppt); Kolmogorov length scales were calculated for 19°C (P. globosa) and 12°C (Thalassiosira sp., Chaetoceros sp., T. pseudonana). View Large TEP was measured colorimetrically in triplicate samples by filtering 5 mL tank water onto 0.4 μm polycarbonate filters that were subsequently stained with Alcian blue (Passow and Alldredge, 1995). TEP absorbance was measured at 787 nm using a Spectronic 601 spectrophotometer (Milton Roy). XG was used as a standard solution and TEP concentrations are reported as XG equivalents per unit water volume (XG eq. L−1) and normalized to algal cell counts (XG eq. cell−1); the latter makes a comparison between the turbulence treatments more accurate. TEP carbon content (TEP-C, μg C L−1) was calculated using the concentration-dependent relationship proposed by Engel and Passow (2001), where TEP (XGeq L−1) is multiplied by 0.75. TEP and TEP-C are also expressed as concentration normalized to algal cells, enabling a direct comparison of the different turbulence treatments. Dissolved inorganic nitrate was analyzed in glass microfiber filter (GF/F) filtered tank water on a Smartchem Chemistry Analyzer using standard colorimetric methods (EPA, 2013) (detection limit: 0.3 μmol DIN L−1). The filtrate was stored at −20°C until analysis. Bacterial cell counts were obtained with a flow cytometer (Becton Dickinson FACSCalibur) on 1.5 mL of tank water fixed with 0.1% glutaraldehyde (final concentration). Samples were stored frozen at −80°C until analysis. Thawed samples were stained with SYBR Green I (Molecular Probes) for 15 min prior to injection. Cells were enumerated according to their right-angle scatter and green fluorescence (fluorescence peak: 575 nm) using the FloJo 7.6.1 software. Bacterial carbon production was estimated from changes in bacterial cell numbers between the initial and the final sampling time multiplied by a bacterial cell carbon content of 30 fg (Fukuda et al., 1998). We used the average carbon content for coastal bacterial communities assuming similar physiological conditions in the bloom-like situations that were simulated in our experiments. Bacterial enzyme activities were measured using 4-Methylumbelliferyl (MUF)-β-D-glucopyranoside and L-leucine-methylcoumarin (MCA) hydrochloride as fluorogenic substrate proxies for BGase and LAPase, respectively (Hoppe, 1983). This approach measures activities of exo-acting enzymes cleaving terminal units from polymers. The substrate proxies were used at the enzyme-saturating level (400 μM final concentration). Triplicates live and autoclaved (killed control) tank water was added to 96-well plates to which peptide and glucose substrates were added (final volume: 200 μL). Fluorescence was measured using a plate reader (Tecan Spectrafluor Plus) at specific time points throughout the plate incubations (3–6 hours). Fluorescence changes were calibrated using standard solutions of the respective fluorophores (MUF and MCA) in tank water and used to calculate enzymatic activities. Minor changes in fluorescence over time were detected in killed controls and used to correct enzymatic hydrolysis rates in live tanks for abiotic substrate hydrolysis. Statistical analysis TEP concentrations and enzyme activities are given as their statistical mean (n ≥ 3) ± standard deviation. Differences between average TEP values of three groups per turbulence treatment were assessed using an analysis of variance (one-way ANOVA) with Tukey honestly significant difference (HSD) post hoc pairwise comparisons of means at the 5% significant level (P = 0.05). Differences between average TEP values of two groups per turbulence treatment were assessed using Student’s t-test (P = 0.05). All statistical analysis was performed in Excel® using the data analysis tool pack (open source add-in). RESULTS Phytoplankton growth Thalassiosira sp. growth rates were very similar among the turbulence treatments at 0.38 day−1 (HT), 0.41 day−1 (MT) and 0.42 day−1 (LT) (Table I). Fv/Fm values in the three Thalassiosira sp. treatments were 0.6. Changes in Thalassiosira sp. cell numbers showed comparable patterns among the three turbulence treatments, increasing by one order of magnitude between the first sampling time (EE growth) and ME growth (Fig. 2A). Cell numbers during S growth were 2.8 × 107 cells L−1 (HT), 3.3 × 107 cells L−1 (MT) and 3.5 × 107 cells L−1 (LT), and thus up to three times higher compared to ME growth.. For Chaetoceros sp., growth rates ranged between 0.97 day−1 (HT tank) and 0.94 day1 (LT tank). Cell concentrations in the two turbulence tanks gradually increased by one order of magnitude throughout the time course of the incubation (Fig. 2B). T. pseudonana growth rates ranged between 1.02 day−1 (HT, MT) and 0.99 day−1 (LT) with Fv/Fm values of 0.6 for all three tanks. Cell concentrations gradually increased by up to two orders of magnitude between EE and S growth in the three tanks (Fig. 2C). P. globosa growth rates reached 0.75 day−1 in the LT, 0.80 day−1 in the MT, and 0.75 day−1 in the HT tank with Fv/Fm values at 0.5 for all three tanks. Initial cell numbers of P. globosa gradually increased two orders of magnitude until the end of the incubation (Fig. 2D). In all four cultures, DIN levels were below detection limit at the final sampling time. Transparent exopolymer particles Volumetric TEP during the Thalassiosira sp. incubations increased by a factor of 3 (HT, MT) and 2.4 (LT) between EE and ME growth (Fig. 2A). TEP remained somewhat constant in the three turbulence treatments until the end of the incubation when cells were in S growth. Average TEP normalized to algal cells during EE growth was about 1.5 times higher in the HT treatment compared to lower turbulence tanks (Fig. 3A). Subsequently, cell-specific TEP decreased in all treatments by one order of magnitude, remaining significantly higher in the HT compared to the LT tank until the end of the incubation (ME: F2,6 = 10.48, P = 0.01; S: F2,6 = 46.15, P < 0.01; Table II). During growth of Chaetoceros sp., initial volumetric TEP in the two turbulence treatments were almost double those of the Thalassiosira sp. experiment at the first sampling point (Fig. 2B). Volumetric TEP values in both treatments more than doubled until LE growth (Fig. 2B). Cell-specific TEP during Chaetoceros sp. growth was significantly higher in the HT compared to the LT treatment during EE growth (Fig. 3B; Table II). In both turbulence tanks, cell-specific TEP decreased by factors of 4 (HT) and 2.5 (LT) until the end of the incubations. For T. pseudonana, volumetric TEP remained relatively constant between early and ME growth in all three tanks (Fig. 2C) at levels that were in the same range than the initial levels of the Thalassiosira sp. experiment. Subsequently, TEP increased by a factor of about 4 (HT), 3 (MT) and 2 (LT) until the end of the incubation (S growth). Cell-specific TEP during EE growth of T. pseudonana was lowest in the LT tank (F2,6 = 11.92, P < 0.01; Table II). In contrast to the other three cultures, volumetric TEP in the P. globosa treatments showed only a minor increase between exponential and S growth (Fig. 2D). Cell-specific TEP showed similar patterns compared to the other three cultures with highest values in the HT treatment during EE (F2,6 = 44.15, P < 0.01) and ME growth (F2,6 = 52.40, P < 0.01; Table II). In all three treatments, cell-specific TEP decreased by one order of magnitude between early and ME growth. Results from the abiotic turbulence experiment revealed an increase in XG particles in the HT relative to the LT tank throughout the 6-hour incubation (Fig. 4; Table II). Bacterial cell abundance and enzymatic activities Bacterial cell numbers in the MT and LT tanks of the Thalassiosira sp. experiment gradually increased throughout the incubations, reaching up to one order of magnitude higher concentrations at the final sampling time compared to the initial time point (Fig. 5A). LAPase activities increased by one order of magnitude between the first and second sampling time and remained constant until the end of the incubations (Fig. 5A). BGase activity was more variable and lower compared to LAPase in all the turbulence treatments. For the Chaetoceros sp., bacterial cell concentrations in the HT and LT treatments were low during the first and second sampling and increased more than one order of magnitude until the end of the incubations (Fig. 5B). LAPase activities followed a different trend as activities in both treatments more than doubled between the first and second time point, remaining at similar levels until the end of the incubation. Bacterial cell numbers in the tanks with T. pseudonana gradually increased throughout the time course of the three turbulence treatments, reaching about 10 times higher concentrations at the last compared to the first sampling time (Fig. 5C). LAPase activities gradually increased in the three treatments, reaching up to two times higher activities at the end of the incubation compared to initial levels. BGase activities were highest in the HT treatments throughout the incubation; activities in each of the three treatments remained somewhat constant between the sampling times. In the treatments with P. globose, bacterial cell concentrations increased threefold in the HT tank, while cell numbers in the MT and LT treatments showed only minor variations with time (Fig. 5D). LAPase activities increased in all three treatments throughout the incubation. BGase activities were either not detectable or at the detection limit. DISCUSSION This study subjected cultured phytoplankton to varying levels of small-scale turbulence to study cell growth and the formation of TEP under controlled laboratory conditions. The gradient of turbulent levels (dissipation rates) ranged over two orders of magnitude (Table III), reflecting in-situ conditions in surface waters from the open ocean (LT tanks) to higher turbulent environments such as estuaries (MT tanks; Petersen et al., 1998; Kahl et al., 2008). Storm events with wind speeds up to 20 m s−1 to 40 m s−1 may trigger turbulent mixing in open ocean environments similar to those in the MT and HT tanks (Alldredge et al., 1990; MacKenzie and Leggett, 1993; Kahl et al., 2008; Guadayol et al., 2009). The wide range of turbulence tested here did not affect phytoplankton photosynthetic efficiency and growth as reflected by constant Fv/Fm ratios and minor variations in growth rates at the different turbulence levels (Thalassiosira sp.: 10%; Chaetoceros sp.: 3%; T. pseudonana: 3%; P. globosa: 5%; Table I). Dell’Aquila et al. (2017) also reported similar growth rates of Chaetoceros decipiens in a turbulent and still treatment. Other studies found stimulating effects of turbulence on phytoplankton cell growth, arguing that chain formation and aggregation of cells could alter cell shape and nutrient uptake, and thus phytoplankton growth in a turbulent environment (Arin et al., 2002; Iversen et al., 2010). Cell aggregation is facilitated by TEP (Passow, 2002b), which also formed during our turbulence treatments. Peak values of TEP (per unit volume) were in the same range as previously reported TEP concentrations from diatom batch cultures incubated under no turbulence. For instance, literature values of TEP for Thalassiosira weissflogii batch cultures range between 500 μg XG eq. L−1 (Gärdes et al., 2011) and 3 000 μg XG eq. L−1 (Passow, 2002a). Passow (2002a) found a similar range of TEP for two Chaetoceros sp. whereas Corzo et al. (2000) reported one order of magnitude higher TEP levels in a Chaetoceros sp. batch culture grown under different levels of dissolved nitrogen. Volumetric TEP concentrations in our P. globosa experiment were an order of magnitude lower than those reported for a colony-forming P. globosa culture (Mari et al., 2005) and for Phaeocystis antarctica (Hong et al., 1997). Peak levels of TEP in the field are often found at the decline of a phytoplankton bloom when cells become nutrient limited (Passow and Alldredge, 1994; Engel, 2000). However, the release of TEP precursors by phytoplankton is not limited to their S growth phase as actively growing cells release copious amounts of organic matter (Penna et al., 1999) that can form TEP during exponential cell growth (Corzo et al., 2000; Passow, 2002a). We found similar patterns as levels of TEP, both volumetric and normalized to algal concentration, were substantial during EE growth in all four cultures. A major goal of this study was to determine possible effects of turbulence on the formation of TEP from phytoplankton DOM produced at different growth phases. Levels of TEP normalized to algal cells in the LT treatments were generally lower compared with the HT treatments (Table II). Given that turbulence was the only variable that differed among the treatments, we assume that enhanced TEP formation in the higher turbulence treatments was driven by physical rather than metabolic processes. Turbulent shear flow has been demonstrated to affect dynamics of polymers (LeDuc et al., 1999) with possible consequences for the formation of TEP precursors in the ocean. Moreover, small-scale turbulence increases encounter rates of suspended particles in a wide range of sizes (Jackson, 1990; Hill et al., 1992; Brunk et al., 1998; Kiørboe, 1998), including TEP precursors that range in size from colloidal to several micrometers (Verdugo et al., 2004). Thus, higher collision rates of sticky organic exudates possibly resulted in enhanced formation of TEP particles in the HT treatments. This assumption is supported by the results from our abiotic experiment in which enhanced coagulation due to mixing was the main driver for particle formation (Fig. 4), underscoring the importance of physical processes in the formation of TEP. Similar conclusions were drawn by Beauvais et al. (2006) who found a positive correlation between TEP sizes and turbulence in a mesocosm experiment with natural planktonic assemblages. The overall decrease of cell-specific TEP with time indicates that the positive effects of turbulence with respect to TEP formation were diminished in the later stages of the incubations. Several reasons could have caused the observed decrease in cell-specific TEP during phytoplankton growth, including metabolic responses of phytoplankton causing reduced production of TEP precursors during the later growth phases (e.g. Passow, 2002a) and/or variations in the pool of inorganic compounds such as sodium and calcium ions that play an important role in the formation of TEP precursors (Meng and Liu, 2016). Additionally, bacterial degradation of organic compounds within the TEP matrix could have influenced the fate of TEP in our turbulence treatments. TEP-C was likely a major source for bacterial growth during our turbulence experiments as estimated levels of bacterial carbon production were well within the levels of the organic carbon in the TEP pool (Table I). Moreover, substantial LAPase activities during the latter part of the incubations may indicate enzymatic degradation of the TEP matrix as previous studies demonstrated that TEP can scavenge organic substances such as amino acids and peptides (Schuster et al., 1998; Passow, 2002a; Zhang et al., 2008). The fact that activities of BGase, which mainly hydrolyzes neutral sugars (Christian and Karl, 1995), were orders of magnitude lower than LAPase underlines the notion that neutral polysaccharides are less abundant within the TEP pool (Mopper et al., 1995). CONCLUSION The effects of turbulence on phytoplankton growth and TEP formation are complex and not well understood because results from laboratory studies are often inconsistent. Our culture experiments indicate no measurable effects of turbulence on cell growth under replete nutrient conditions, thus supporting the theoretical assumption that turbulence does not affect nutrient uptake and cell growth of planktonic cells that are smaller in size than the smallest turbulent eddy. Turbulence may, however, enhance TEP formation as mixing stimulates coagulation of phytoplankton exudates. Our results further indicate that microbial degradation of phytoplankton organic matter affected the fate of TEP in our turbulence tanks. Enzymatic accessibility of TEP is unknown and may be restricted due to the cross linking and entanglement between polymers in a TEP matrix. However, our results suggest that activities of exo-enzymes, such as LAPase, make TEP polymers more susceptible to physical fragmentation in a more turbulent environment. 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TI - Enhanced formation of transparent exopolymer particles (TEP) under turbulence during phytoplankton growth
JF - Journal of Plankton Research
DO - 10.1093/plankt/fbz018
DA - 2019-05-31
UR - https://www.deepdyve.com/lp/oxford-university-press/enhanced-formation-of-transparent-exopolymer-particles-tep-under-1OmmcZ7B1Q
SP - 349
VL - 41
IS - 3
DP - DeepDyve
ER -