Growth and Protein Profiles of the Three Psychrophilic Microbes
Figure 1 presents the growth profile of the three psychrophilic microbes, Sporosarcina psychrophile strain (S1), Sporosarcina globispora strain (S2), and Polaromonas hydrogenivorans strain (S3), selected for this study, and their released protein concentration in the extracellular growth fractions after a regular interval of 3 days. As witnessed in Fig. 1 (a) to (c), cultures S1 and S2 grew faster compared to S3 to enter their exponential growth phase from the lag phase by day 6. In the case of S3, this phase shift only occurred on the 16th day of incubation. On a comparative note, S1 growth based on OD 600nm readings peaked on the 15th day, continued to the stationary phase till the 18th day, and immediately moved to the slow death phase. In the case of S2, peak growth was witnessed on the 9th day, and the stationary phase continued till the 18th day. After that, S2 bacteria shifted to the death phase. Interestingly, S3, under the same condition, started to grow exponentially only on the 15th day. Based on the results described in the later section, the growth phase was decided to continue for 24 days. In this period, S3 continued its exponential growth and showed no stationary phase.
Simultaneously, at a similar interval of growth phase monitoring, the extracellular supernatant was collected and tested to determine any effect on the thermal changes in porous media. We hypothesized, expected, and tested here that the microbes, while being grown under the cold temperature of 4 0C, would release ice crystal arresting proteins. It is important to note that the initial protein concentrations reflected in Fig. 1, until 6th day, should be the background proteins in the media. As all 3 strains were in their lag phase till then, we do not expect them to produce or release any psychrophilic proteins. Therefore, any effect of the solution on ice crystallization is understandably not validated until the 6th day. During the experimentations, the maximum protein concentrations in the extracellular supernatant for strains S1 (5.3 mg/ml), S2 (4.35 mg/ml), and S3 (2.43 mg/ml) were measured on the 15th, 21st, and 24th days, respectively. Coincidentally, for all three strains, the peak points of the maximum proteins in their culture supernatant were reached while they were in their late log or early phase of stationary growth.
Thermal Properties-Based Results
For this part of the experimentation, 20 µL of the supernatants collected on days 6, 9, 12, 15, 18, 21, and 24 from actively growing cultures of S1, S2, and S3 psychrophiles, respectively, were mixed with 10 mg of soil grains and tested for thermal characteristics during freezing and thawing. Figure 2 presents these thermal results along with the images taken using the microscope during the phase change (Fig. 2a to 2e) of porous media (liquid-to-ice crystal and ice crystal-to-liquid).
During the freezing stage, at 0 0C temperature, the water (control), S1, S2, and S3 did not change their physical state (liquid to frozen), as seen in Fig. 2 (a). As the temperatures decrease below 0 0C, the ice formation (Fig. 2 (b)) initiated on the specimen surface (i.e., -4.08 0C for water-soil mixture), which will be referred to as ‘Ice Nucleation Initiation’. In Fig. 2 (c), these ice formations start to take stable forms with polygonal shapes at much lower negative temperatures (i.e., -4.59 0C for water-soil mixture). After this point, no new crystal formation was observed, and this signifies the end of the freezing stage referred to as ‘Ice Nucleation Completion’. In Fig. 2 (d), the frozen crystals started to melt (i.e., -1.69 0C for water-soil mixture), which is referred to as ‘Thawing Initiation’. At this negative but higher temperature than the freezing point temperature, the ice crystals spontaneously melt and move from place to place. This signifies the potential increment of Gibbs free energy to melt the ice crystals by increasing enthalpy and entropy. Finally, Fig. 2 (e) indicates the end of the thawing point (i.e., 0.03 0C for water-soil mixture) where no ice crystal is visible for any water or supernatants mixed soil. This will be called the ‘Thawing Completion’ step.
Figure 3 provides a detailed account of the temperatures attributable to the phase change steps pictured in Fig. 2 as a crucial means for further understanding the thermal characteristics properties of the extracellular microbial solutions used for arresting ice crystallization of porous media in soil. The ice nucleation initiation stage illustrated in Fig. 2 represents the start of ice formation. This initiation was recorded at -4.08 ˚C for control sample (no psychrophile, just soil-water), but for the soils mixed with supernatants (S1, S2, and S3), the ice nucleation initiation (Tini) transpired at a comparatively lower temperature. As shown in Fig. 3 (a), for psychrophile S1, Tini was recorded at -6.99 0C on day 6 sample, where the microbe was still in its lag phase of growth (Fig. 1). The lowest Tini for S1 treated samples was observed on day 15, at -7.29 0C, which was 3.15 0C lower than the control. The depression recorded for Tini between day 6 and 15 reads minimal, but still, it is significant to believe that the S1 is active at lower temperatures depicting anti-freeze activities. For thawing initiation (Tti), the control sample started to thaw at -1.69 0C; for samples S1-soil mixtures, ice thawing was recorded with Tti being in the range of -3.63 0C on day 12, and − 3.89 0C on day 15. To note, for microbial culture S1, day 15 also marks the time when a maximum of the protein concentrations was recorded in the extracellular solution of culture.
The phase change diagram for S2 samples mixed soil samples over time of culture growth is shown in Fig. 3 (b). For S2 samples, the lowest Tini and Tti were recorded for day 15, with values of -7.30 0C and − 3.69 0C, respectively. Likewise, for Sample S1, the closest initiation temperature for both steps were observed for day 6 samples, the period triggering S1’s move from its lag phase to the exponential growth phase. For S3 samples’ the phase change diagram over its growth phases is shown in Fig. 3 (c). The lowest Tini and Tti can be seen registered with day 21 and 9 with values of -7.75 0C and − 3.68 0C, respectively. All three S1-S3 cultures depicted analogous freezing and thawing steps initiation temperature, depicting antifreeze activities of psychrophilic microbes.
Freezing Point (Tf)
In the ice nucleation completion step, the freezing point temperatures were observed for water, S1, S2, and S3 supernatant mixed with soil in Fig. 3. Here, the control test conducted using 20 µL water mixed with 10 mg silt showed an average freezing temperature of -4.59 0C. On the other hand, the same amount of soil combined with 20 µL bacterial extracellular solutions displayed depressions in the freezing temperatures, as shown in Fig. 3, with the depression correlating directly with the increment in the protein concentrations for different test days. With sample S1, the most significant freezing point depression was recorded for day 15 samples, where a freezing point of -8.54 0C was observed with respective protein content in the culture solution measured at 5.30 mg/ml. Subsequently, the freezing point depressions started to decrease, coinciding with the decrement of protein concentrations in the culture solution. Like the S1 culture solution, the lowest freezing point of -8.50 0C was observed for S2, on day 15, with protein content at 2.83 mg/ml. For the S3 sample culture supernatants, the lowest freezing point of -8.85 0C was observed on day 21, with the measured protein concentration in the sample being 1.55 mg/ml. Amongst the three psychrophilic strains tested, overall, S3 seemed to comprehend better control over the freezing point depression compared to microbes S1 and S2.
Thawing Point (Tt)
The thawing completion temperatures in Fig. 3 represent a complete unfrozen state of the control and bio-treated (S1, S2, S3 with soils). The control sample consisting of frozen water-soil thawed at 0.03 0C. However, the bio-treated soils (S1, S2, S3 with soils) thawed at negative temperatures. The thawing temperature for the S1-soil was between the range of -0.85 to -1.07 0C on days 15 and 24 respectively. Like S1, the thawing temperature for S2 treated soils was between the range of -0.96 to -1.15 0C, where the lowest temperature was observed on day 9 while the highest was observed on day 6. For S3, the thawing temperature was between − 1.14 to -1.32 0C, where the lowest temperature was observed on day 9, and the highest was observed on days 18 and 21.
Freezing Point Depression Difference (ΔTf)
Figure 4 presents the difference in the freezing point depression (ΔTf) between soil mixed with water and proteins over different days of the growth period by using Eq. (1). For S1 and S2, the maximum freezing point depression difference was observed on Day 15 with ΔTf = 3.95 0C and ΔTf = 3.91 0C, equivalent to depression of 85.98% and 85.26%, respectively. The lowest depression difference observed for them is on Day 6 and Day 24, respectively, which is still ΔTf >3 0C. A significant difference in performance was marked for S3 with ΔTf = 4.26 0C on Day 21 with 92.81% depression. Even the lowest ΔTf = 3.57 0C was found on Day 6 when the growth of bacteria or concentration of ice crystal inhibiting protein was minimal. The results conclude that some extracellular products secreted by the microbes lowered the porous media's freezing point and showed significant performance.
Thermal Hysteresis (TH) Activity
The effect of three psychrophilic microbes’ (S1, S2, and S3) extracellular solution on enhancing the thermal properties of frost-susceptible soils can be described more accurately with Thermal hysteresis (TH) activity. The TH is the thermal gap between freezing and thawing points which were recorded and calculated using Eq. (2). Its dependability on the tested samples is shown in Fig. 5. As expected, the lowest 4.62 0C TH activity was observed for the water-soil mixture. Compared, all the extracellular protein-treated samples registered higher TH activity. The TH activity for S1 ranged from 7.04 to 7.47 0C for different growth periods, with the maximum observed on Day 15. This day of maximum TH recorded for the bacterial sample S1 was the due the highest protein content recorded in its extracellular solution, i.e., 15th day for the S1 culture growth at 4 0C (Fig. 1). For S2, the maximum TH activity of 7.35 0C was also observed on Day 15, and the lowest activity was 6.89 0C on Day 24. For culture S3, the maximum TH activity of 7.71 0C (also the highest activity recorded among S1, S2, and S3) was seen on Day 21, and the lowest activity was 6.98 0C on Day 6.
Delayed Freezing and Premature Thawing Analysis
In the freezing point results, we have explained how AFP molecules adhere to the crystal's surface and inhibit ice crystal development. Thus, the freezing point of porous media is lowered to a new freezing point by preventing the creation of ice crystals. This is also true for the lower ice nucleation initiation temperature. Here, we quantify this observation with ‘Delayed Freezing (TD),’ where the difference between the ice nucleation initiation (Tini) and ice nucleation completion (Tinc) have been calculated by using Eq. (3). From Fig. 6, where the vertical axis shows the delayed freezing, the observations for the maximum delay in the freezing temperature for sample S1 can again be seen to be varying and peaking in for the day 15th with a value of 1.25 0C when the microbial culture S1 had a maximum of the protein concentrations recorded in the extracellular solution of culture. Comparatively, the water soil control had a TD of only 0.51 0C, which is a much faster process for freezing to complete. For culture S2, the maximum TD temperature of 1.20 0C was found for Day 15, whereas the maximum TD temperature for culture S3 was 1.15 0C on Day 18. Notably, for each S1, S2, and S3-mixed soil, the lowest freezing point and delayed freezing value were observed on the same day, which coincides with the knowledge that the AFPs control ice crystal growth to a much lower freezing point.
Similar to the delayed freezing, for the manuscript’s reference, the difference between the thawing initiation and thawing completion has been termed ‘Premature Thawing (TP)’ and has been calculated using Eq. (4). The water-soil control recorded a thermal gap of 1.72 0C between its thawing initiation at -1.69 0C and thawing completion at 0.03 0C (Fig. 6). Conversely, the culture test samples have higher Premature Thawing. For culture S1, each sample from a different growth phase showed a TP of 2.75 0C or above. The premature thawing characteristic for S2 showed a TP of 2.43 0C or above; for S3, the TP was recorded at 2 0C or above.