Two of the most common stresses in natural environments, as well as in bioreactors, that are of importance in biofuel or bioterrorism contexts are pH and temperature stresses. Superoxide generation has been suspected as a possible reason for the effects of the above stresses, and superoxide is suspected to play a role in cell signaling. But, no information is available on a direct relationship between environmental stress and intracellular superoxide levels. We hypothesize that the exposure of cells to pH stress induces superoxide radicals, which leads to oxidative cellular damage, and thus it affects cells in culture. We tested the hypothesis on a model organism, Bacillus subtilis, which is well studied, and has relevance in biofuel and bioterrorism contexts. Bacillus subtilis cells were exposed to different pH-stress conditions (pH 5, 6, 8, or 9) and temperature-stress conditions (42, 45, 48, and 55 degrees C). Specific superoxide levels at pH 8 and 9 were 22% and 160%, respectively, higher than that of normal growth pH (pH 7), which was 6.48 +/- 0.37 mmol (10(12) live cells)(-1) Superoxide generation due to pH stress has not been reported thus far in the literature. Although detection of superoxide induced by temperature stress has been reported, it has not been quantified. When Bacillus subtilis cells were exposed to 42, 45, 48, and 55 degrees C for 1 h, the specific intracellular superoxide levels were 52%, 150%, 220%, and 314%, respectively, higher than the value at normal growth temperature, 37 degrees C. The extent of intracellular lipid peroxidation was higher under the stressed conditions. The mechanism for alkaline pH-stress-induced superoxide generation seems to be the continuation of metabolic activities even though the growth is arrested. The mode of temperature-stress-mediated cell death is necrosis, and the extent of cell death is correlated with the specific intracellular superoxide level.