IV. RESULTS AND DISCUSSION
2.3.2. Culturability changes in stress conditions
In this regard, it should be noted that due to the low buffering capacity of the used medium, the effect of bubbling with N2:CO2 in the cultures in order to achieve the oxygen depletion was also accompanied by a pH drop from 7.2 to 5.8. After different times under stress incubation, the dynamics of each strain under different stresses was followed by quantification of cells numbers, quantification of FISH-‐hybridized cells, and changes in the culturability.
2.3.1. Cells abundances and FISH-‐detectable cells numbers under stress conditions
As it can be seen in Fig. 19, both strains responded similarly and all stresses promoted a continuous decrease in the total cell numbers observed by DAPI, in some cases to nearly the half of the initial value. Oxygen depletion and dilution promoted a similar effect on the decrease of cell counts, whereas during temperature stress, this effect was less pronounced and cells did not always show an abrupt drop. As example, during the first two hours of treatment, the most remarkable changes were on M31 cells, which showed a much higher sensitivity to both stresses than M8. This decrease in the number of cells respect to the control condition was about 20% (M8) and 40% (M31) for oxygen depletion, and 40% (M8) and 60% (M31) for osmotic stress. At this point, it is important to note that the changes observed under anoxia may not be only due to the oxygen depletion, but also to the decrease of the pH value from 7.2 to 5.8 since in previous experiments S. ruber showed a decrease in the growth yields when the pH was reduced to 6.0 (Antón et al., 2002). On the other hand, during the first two hours of temperature stress, M8 cells showed a lower sensitivity than M31 cells (about 10% versus 30%), a trend that remained during all tested stress conditions (Fig. 19).
Although the number of cells decreased in all sampled points and stress conditions studied, the fraction of FISH detectable cells did not decrease so strongly, and in the worst case, the detection rates dropped about 13% with respect to the control condition (Fig. 19).
2.3.2. Culturability changes in stress conditions
The reduction in cell abundances observed by DAPI were mirrored by the culturable numbers (CFU), the last showing more abrupt changes. The differences between total cell counts and CFU were already apparent after 2 h under every stress conditions with respect to the control, with a significant loss of culturability.
During this period, the culturability of M8 cells decreased in each stress condition. M31 seemed to be mostly influenced by the dilution stress, being less susceptible to the oxygen and temperature conditions during the same period of incubation (Fig. 19).
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Actually, this strain has been shown to outcompete M8 in high salt concentrations, but less fit in lower ionic strength (Peña et al., 2010), a fact that would be in accordance with its higher sensitivity to the dilution of the culture media.
Dilution and oxygen promoted an abrupt drop in the culturability in both strains, which reached the minimal values after 40 h, but they retained similar FISH detection rates, as occurred in the exponential–stationary phase transition. However, the temperature effect showed a different dynamics. Both strains strongly reduced their culturability, reaching the minimal values just after 16 h, but this culturability reduction was recovered when the incubation time was prolonged for 40 h under low temperature, at which point the initial rates were not only recovered, but also increased to 100% of the DAPI counts (Fig. 18). Besides, this phenomenon was observed even when the incubation at low temperature was prolonged up to 30 days (see below).
Figure 19: Culturability and cell counts of M8 and M31 strains under different stress conditions. All points correspond to the average of two independent measurements. In the graph it is represented the number of total cells determined by DAPI staining and the FISH hybridized cells (expressed as cells mL-‐1 with time), and the number of cells grown on agar plates expressed as CFU mL-‐1 with time.
2.3.4. Cells numbers, FISH counts, and culturability changes during prolonged temperature stress conditions Both strains responded similarly to the prolonged temperature stress, promoting a slight decrease of total cell numbers observed by DAPI which was not accompanied by a decrease in the fraction of FISH-‐detectable cells (Fig. 20).
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Contrary to previous observations, the prolonged incubation at 4ºC promoted an increase in the number of FISH hybridized cells respect to control condition (Fig. 20). On the other hand, the culturability under prolonged low temperature showed the same behavior observed at 40 h of stress, increasing the culturability to 100% (Fig. 20). The minor changes in the number of cells, accompanied with an increase in the ribosome content as well as the recovery of culturability under low temperature, supports the previous hypothesis that under low temperature incubation, S. ruber may generate a survival strategy to re-‐adapt to low temperatures probably by adopting a dormant state.
As mentioned before, cells could display increased stability after extended cold incubation (Weichart &
Kjelleberg, 1996), which could promote the change to cultivable phenotype as observed in Figs 19 and 20. A recovery of the culturable fraction is a phenomenon that has been observed in species such as Vibrio parahaemolyticus (Coutard et al. 2007) in which a proportion of viable but non-‐culturable cells (VBNC) subjected to low temperatures remained viable after a temperature upshift, suggesting the re-‐growth of these cells rather than resuscitation of all bacteria of the initial inoculum (Coutard et al. 2007). In addition, the use of exponential cultures to study the stress response could have effects in the culturability, since the growth phase prior to cold incubation has been identified as the major determinant for maintenance the culturability at low temperatures in species as Vibrio vulnificus (Oliver et al., 1991). Thus, S. ruber may generate a survival strategy to re-‐adapt to the new environmental conditions, which would promote a readjustment of their cellular machinery to shift the non-‐culturable state to a culturable state.
Figure 20: Culturability and cell counts of M8 and M31 strains under prolonged temperature decrease (30 days). All points correspond to the average of two independent measurements, and represent the number of total cells determined by DAPI staining and FISH (expressed as cells mL-‐1 vs time), and the number of cells grown on agar plates (expressed as CFU mL-‐1 vs time).
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2.4. Metabolome comparisons
As in the previous chapter, in order to study the changes during the different phases of growth and the response to adverse conditions, standard microbiological techniques were supplemented with a metabolomic study of chemical extracts of M8 and M31 strains by using high-‐field ICR-‐FT/MS. Different metabolomic comparisons were performed in order to characterize the metabolic response in both strains.
2.4.1. Common metabolome composition in both strains along the different phases of the growth curve