INFLUENCE OF PH AMMONIA ACCUMULATION AND TOXICITY IN HALOPHILIC, METHYLOTROPHIC METHANOGENS

We studied the effects of pH and ammonia concentration on the growth o f three methanogens. These three halophilic, methylotrophic methanogen s, Methanolobus bombayensis, Methanolobus taylorii, and Mathanohalophi lus zhilinaeae, grew at environmental pH ranges that overlapped with e ach other and spanned the pH range from 7.0 to 9.5. During growth they had reversed membrane pH gradients (Delta pH) at all pH values tested . The Delta pH was in the range of -0.4 to -0.9 pH units, with the cyt osol being more acidic than the environmental pH. Methanohalophilus zh ilinaeae had the most negative Delta pH (-0.9 pH units). These negativ e pH gradients resulted in the accumulation of ammonium (NH4+), and wh en grown at the highest external ammonia concentrations that allowed g ood growth, cells had cytosolic NH4+ concentrations as high as 180 mM. The high concentrations of cytosolic NH4+ were accompanied by greater Delta pH and lower concentrations of the major cytosolic cation K+ (c ompared with cells grown in medium with only 5 mM ammonia). Methanolob us bombayensis and Methanolobus taylorii were more sensitive to total external ammonia at higher external pH values, but the inhibitory conc entration of un-ionized ammonia that resulted in a 50% reduction of th e growth rate was about 2 to 5 mM, regardless of the pH. This is consi stent with growth inhibition by ammonia in other bacteria. However, Me thanohalophilus zhilinaeae was more resistant to un-ionized ammonia th an any other known organism, It had a 50% inhibitory concentration for un-ionized ammonia of 13 mM at pH 8.5 and 45 mM at pH 9.5. We examine d the effects of pH on three ammonia-assimilating activities (glutamin e synthetase, glutamate dehydrogenase, and alanine dehydrogenase) in c ell lysates and found that the pH ranges were consistent with the obse rved ranges of intracellular pH.