Effect of salinity and pH on V.harveyi adaptation.
Supervisor: Vladimir Kaberdin (UPV/EHU)
Global warming and ongoing climate change caused by human activities dramatically affect the global characteristics of ocean by increasing the sea surface temperature, decreasing pH and changing the gradients of salinity. There has been limited information available on the combined impact of salinity and pH on free-living marine microorganisms in the context of climate change. Vibrio species have widely been used to assess the physiological, phenotypical and gene expression responses of marine microorganisms to changing climate.  To characterize the combined impact of salinity and pH on Vibrio adaptation and fitness, we analyzed the pH-dependent adaptation of Vibrio harveyi at different salinities(5‰, 15‰, 25‰ or 35‰) that resemble those occurring in natural ecosystems. Experiments were carried out at pH 7 and 8that mimic the pH of the world ocean in the present time and near future, respectively. Comparison of V. harveyi growth at 25⁰C demonstrates that, compared to neutral pH (i.e., pH 7),alkaline pH8 promotes the logarithmic growth of V. harveyi in nutrient-rich environments. Moreover, a decrease in salinity negatively impacts cells growth at both pH 7 and pH8.In addition, survival tests carried out in artificial seawater for 21 days under limitation of nutrients revealed that the culturability of V. harveyi declined gradually at both pHs and a decrease in salinity leads to a more profound loss of culturability reaching the minimal values at 5‰ salinity. Moreover, there was a more significant loss in culturability at lower pH (i.e.,pH 7)than at pH 8. Further analysis of stressed cell morphology and size distribution using epifluorescent microscopy reveals that adaptation of V. harveyi to starvation is associated with a continuous reduction of cell size to acquire coccoid-like morphology. Furthermore, this morphological change occurs faster at pH 8 over the range of salinities and is generally dependent on the particular salinity-pH combination. In addition, we found that V. harvey starvation in oligohaline environment (5‰) and neutral pH led to a more rapid and irreversible loss of V. harveyi culturability and total number of cells at pH 7 compared to pH 8, thereby indicating that pH 7creates the more hostile environment for V. harveyi. In other words, the hypotonic (5‰) condition has a strong negativeimpact on V. Harveyi adaptation at both pHs, especially at more acidic pH.

Keywords: climate change; ocean acidification; osmotic stress; V. harveyi; bacterial growth; microbial adaptation and survival; phenotypical change