Decreased microgel equilibrium size (black circles) and bound Ca²⁺ (blue triangles) with concomitant increase in hydrophobicity (red squares).

<p>The non-linear rate of declining microgel size with increased temperature indicates potential cooperativity; around 32˚C all three parameters experienced the most pronounced associative effect—a major drop in microgel size and bound Ca²⁺, with a concomitant rise in hydrophobicity.</p

DOM assembly monitored with temperature and pH reveals that, as either pH decreases or temperature increases, microgel equilibrium size and assembly rates decrease at a non-linear rate.

<p>a. DOM assembly at three temperatures—22˚C (black circles), 30˚C (blue triangles), 32˚C (red squares)—over time at three pH units. Each data point represents (mean ± SD) of six measurements made in each of six replicate samples. b. DOM assembly at three pHs—8.0 (black circles), 7.7 (blue triangles), 7.5 (red squares)—over time at three constant temperature incubations. Microgels assembled in identical pH conditions showed equilibrium size reduction and decelerated non-linear assembly rates when exposed to increased temperature. Each data point represents the mean (+/− SD) of six measurements made in each of six replicate samples. Shaded windows represent an average microgel equilibrium size range (4–6 μm) at 22˚C and pH 8.</p

Microgel assembly / dispersion are temperature dependent.

<p>1-a. Microgel assembly rate and equilibrium size decreases with increased temperature. Samples were incubated at 22˚C (black circles), 32˚C (blue triangles) and 35˚C (red squares) for 24 hours, then stored in the dark at 22˚C for the remainder of the experiment. Assembly was measured using dynamic laser scattering at 22˚C. Each data point represents (mean ± SD) of six measurements made in each of six replicate samples. Data highlight that short-term temperature exposure above 35˚C confers significant DOM assembly loss with no obvious recovery. 1-b. Microgel dispersion depends on temperature variation. Self-assembled microgels (size ~ 6 μm) were incubated at various temperatures (from 22˚C to 40 ˚C) for 24 hours. The equilibrium microgel sizes were monitored with dynamic laser scattering spectroscopy. Each data point represents six replicate samples. Non-linear temperature responses of microgels were observed—particularly for microgels incubated at temperatures above 32˚C, which showed a marked size decrease.</p

EPS Assembly analysis.

<p>EPS Assembly analysis.</p

Chemical analysis of EPS.

<p>*Zhange et al., (2008) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021865#pone.0021865-Zhang1" target="_blank">[40]</a>.</p

Assembly kinetics of EPS monitored with DLS.

<p>(A) Assembly kinetics of EPS of <i>Amphora sp.</i> (B) Assembly kinetics of EPS of <i>Ankistrodesmus angustus</i> (C) Assembly kinetics of EPS of <i>Phaeodactylum tricornutum</i> EPS assembly in Ca²⁺-free ASW (black) was monitored to investigate assembly kinetics with decreased divalent ion availability. Different concentrations of ENs (polystyrene nanoparticles): 0 (red), 10 (green) and 100 ppb (blue), were added to investigate the effect of ENs on EPS microgel formation.</p

Fluorescence images of EPS and ENs-induced EPS microgels.

<p>Nile Red was used to determine the microgel morphology. Green fluorescent signals indicated the fluorescent ENs. From the overlay images, results showed that the ENs incorporated within EPS matrixes. Scale bar is 10 µm.</p

ESEM images of EPS microgel.

<p>(A) <i>Amphora sp.</i> (Scale Bar = 4 µm) (B) <i>Ankistrodesmus angustus</i> (Scale Bar = 5 µm) (C) <i>Phaeodactylum tricornutum</i> (Scale Bar = 5 µm).</p