Nonfermentative Thermoalkaliphilic Growth Is Restricted to Alkaline Environments

Caldalkalibacillus thermarum strain TA2.A1 grew in pH-controlled batch culture containing a fermentable growth substrate (i.e., sucrose) from pH 7.5 to 10.0 with no significant change in the specific growth rate, suggesting that this bacterium was a facultative alkaliphile. However, when strain TA2.A1 was grown on a nonfermentable carbon source, such as succinate or malate, no growth was observed until the external pH was >9.0, suggesting that this bacterium was an obligate alkaliphile. Succinate transport and sucrose transport by strain TA2.A1 showed pH profiles similar to that of growth on these carbon sources, and the molar growth yield on sucrose was higher at pH 9.5 than at pH 7.5, despite the increased energy demands on the cell for intracellular pH regulation. Succinate transport, succinate-dependent oxygen consumption, and succinate dehydrogenase and F₁F_o-ATPase specific activities were all significantly lower in cultures of strain TA2.A1 grown at pH 7.5 than in those cultured at pH 9.5. No significant ATP synthesis via the F₁F_o-ATP synthase was detected until the external pH was >8.5. On the basis of these results, we propose that nonfermentative thermoalkaliphilic growth is specialized to function at high pH values, but not at pH values near neutral pH.Alkaliphilic microorganisms have been isolated from a diverse range of environments and have traditionally been classified into two distinct groups based on their pH profile for growth (8). Bacteria that grow across a broad pH range from 7.0 to 11.0 have been classified as facultative alkaliphiles (e.g., Bacillus pseudofirmus OF4) (28), and those that are able to grow only above pH 9.0 have been classified as obligate alkaliphiles (e.g., Bacillus alcalophilus) (4). The reasons why obligate alkaliphiles fail to grow below pH 9.0 remain speculative.While the classification of alkaliphilic bacteria based on pH profiles for growth has gained universal acceptance, it does not consider the nature of the carbon source that is used to grow the cells, and for aerobic alkaliphiles, this may have important consequences. For example, growth on succinate in aerobic bacteria is strictly coupled to oxidative phosphorylation and ATP is produced in the cell via the membrane-bound F₁F_o-ATP synthase. Growth on fermentable carbon sources, such as glucose, allows the cells to bypass this machinery, as ATP can be produced via substrate-level phosphorylation and incomplete oxidation of glucose to acetate can occur.A thermoalkaliphilic bacterium, Bacillus sp. strain TA2.A1, capable of optimal aerobic growth at a temperature of 65°C at pH 9.5 was isolated from an alkaline thermal bore at Mt. Te Aroha, New Zealand (19). The 16S rRNA gene sequence of strain TA2.A1, compared with those available in the EMBL database, shows 99.5% similarity to Caldalkalibacillus thermarum strain HA6^T, an aerobic, heterotrophic, thermophilic bacterium isolated from an alkaline hot spring in China (30). On the basis of the similarity of its phenotypic and genotypic characteristics to those of strain HA6^T, we assign strain TA2.A1 to the genus and species Caldalkalibacillus thermarum. C. thermarum strain TA2.A1 grows on sucrose, common C₄-dicarboxylates, glutamate, pyruvate, and trehalose; however, glucose and fructose fail to support growth (19). We originally described strain TA2.A1 as a facultative alkaliphile based on its pH profile for growth on glutamate or sucrose (18-20); however, both are substrates whose metabolism is not strictly coupled to oxidative phosphorylation.In this communication, we determine the pH profile for growth of C. thermarum strain TA2.A1 on nonfermentable (i.e., succinate and malate) and fermentable carbon sources (i.e., sucrose) using pH-controlled batch culture and demonstrate that strain TA2.A1 was unable to grow below pH 9.0 in pH-controlled batch culture on succinate but grew from pH 7.5 to 10 on sucrose. The physiological and biochemical bases for this phenomenon were investigated.