Discovery of two novel highly tolerant NaHCO3 Trebouxiophytes: Identification and characterization of microalgae from extreme saline-alkali soil

Through identifying and characterizing microalgae from extreme saline-alkaline soils (pH > 10, Songnen Plain, China), which are rich in carbonate (NaHCO3 and Na2CO3), we aimed to explore the microalgae carbonate stress mechanism and acquire possible extreme saline-alkali microalgae as genetic resources for crop improvement and cultivation. We identified the microalgae based on optical microscopy (OM), scanning electron microscopy (SEM), and 18S rRNA sequence analysis. We determined strains' salt-tolerance abilities with a range of NaCl and NaHCO3 concentrations. The intracellular ultrastructure was observed with transmission electron microscopy (TEM) after NaCl and NaHCO3 treatments. We observed strain growth response by detecting dry weight and chlorophyll-a (chl-a) content at different NaHCO3 concentrations. We identified 20 novel strains of microalgae as 13 genera based on morphological and sequence analyses, and classified them into six groups by class level, including Chlorophyceae, Trebouxiophyceae, Ulvophyceae, Bacillariophyceae, Xanthophyceae, and Bangiophyceae; Chlorophyta occupied the most dominant algal groups. Most isolates possessed saline tolerance, especially two strains in the class Trebouxiophyceae, which could grow on solid media with high NaHCO3 concentrations. The TEM results showed cell walls of the two Trebouxiophyte strains (JB6 and 17) with more integrity than reference Chlorella, and a larger number of starch grains were accumulated under 300 mM NaCl and NaHCO3. Further analysis showed that the dry weights and chl-a contents of two Trebouxiophyte strains were much better than the reference Chlorella in liquid media with different concentrations of NaHCO3. The two Trebouxiophyte strains had optimal growth at 100 and 400 mM NaHCO3 concentrations, and both survived at 1000 mM NaHCO3. The results revealed that the two Trebouxiophyte strains showed extreme tolerance to high concentrations of NaHCO3.