Fig 1.
Temperature profile set points of the cooling plate for ice recrystallization inhibition (IRI) activity assessment.
Images are taken at time points 154 and 354 s, highlighted in green and red accordingly. The microscope images shown are for the 35% sucrose solution at −6.8 °C.
Fig 2.
Graphical assessment of ice recrystallization inhibition (IRI) activity.
Data represents mean values with standard deviation error bars (n = 3). The IRI activity is expressed as Dk50%, calculated from the quadratic polynomial trend line (blue dotted line). The green marker at 32 × dilution indicates a plateau in ice crystal growth rates across the dilution series.
Fig 3.
Comparison of two experiments assessing the growth and ice recrystallization inhibition (IRI) activity of Pseudomonas fluorescens AQP671 cultivated with different nitrogen sources following a temperature shift-down to 5 °C.
In Experiment 1, ammonium chloride (NH₄Cl) was used as the nitrogen source throughout cultivation (biomass (X): yellow line; IRI activity: blue line). In Experiment 2, yeast extract was added as the nitrogen source after the temperature shift-down (biomass (X): black line; IRI activity: green line). The bioreactor temperature set point is shown by the orange line.
Fig 4.
Change in the optical density during the growth of P. fluorescens AQP671 in the base medium containing a variety of amino acids or yeast extract as a nitrogen source.
L-asparagine (green) is highlighted as the nitrogen source giving the highest IRI activity by the end of the experiment. Data represent mean values with standard deviation error bars (n = 3).
Fig 5.
The effect of nitrogen source (L-asparagine (N), yeast extract (YE), L-proline (P), L-valine (V), L-isoleucine (I), L-arginine (R), L-glutamine (Q), L-serine (S), L-alanine (A), L-threonine (T), L-methionine (M) on P. fluorescens AQP671 IRI activity (blue bars) after 48 h incubation at 5 °C.
Orange bars – the biomass concentration (OD 600 nm) at 48 h, grey bars – total protein concentration in the culture media at 168 h of incubation, yellow boxes - pH of the culture media after 168 h of incubation. Data represent mean values with standard deviation error bars (n = 3). The full data for each time point can be found in the supporting materials, S1 Table. * As a comparison, NH4Cl results correspond to the results from the experiment in the bioreactor at 71 h, shown in Fig 3, ∅ – no activity detected.
Fig 6.
The change in ice recrystallization inhibition activity in Pseudomonas fluorescens AQP671 culture supplemented with different nitrogen sources during incubation at 5 °C on shaker, 180 rpm.
a – high activity group: L-proline (P), L-valine (V), L-asparagine (N) and yeast extract (YE), b – low activity group: L-isoleucine (I), L-alanine (A), L-arginine (R) and L-methionine (M), L-threonine (T), L-glutamine (Q), L-serine (S). Data represent mean values with standard deviation error bars (n = 3).
Fig 7.
Ice recrystallization inhibition activity in P. fluorescens AQP671 culture supernatant.
Data represents mean values with standard deviation error bars (n = 3). Lowercase letters indicate significant differences between samples according to the t-test values (p < 0.05) (left). Microscopic images of the ice crystals in the culture supernatant (2x diluted) at different pH values. The scale vars are 50 μm (right).
Fig 8.
Total protein concentration normalized to cell density (gray), biomass concentration at 24 h (blue), IRI activity normalized to biomass concentration (orange) and IRI activity normalized to total protein concentration in the culture media of P. fluorescens AQP671 after incubation for 24 h at 5, 10, 15 and 20 °C.
Data represent mean values with standard deviation error bars (n = 3). N – not detected.