Uncoupling by Acetic Acid Limits Growth of and Acetogenesis by Clostridium thermoaceticum

Abstract

When cells of the anaerobic thermophile Clostridium thermoaceticum grow in batch culture and homoferment glucose to acetic acid, the pH of the medium decreases until growth and then acid production cease, at about pH 5. We postulated that the end product of fermentation limits growth by acting as an uncoupling agent. Thus, when the pH of the medium is low, the cytoplasm of the cells becomes acidified below a tolerable pH. We have therefore measured the internal pH of growing cells and compared these values with those of nongrowing cells incubated in the absence of acetic acid. Growing cells maintained an interior about 0.6 pH units more alkaline than the exterior throughout most of batch growth (i.e., ApH = 0.6). We also measured the transmembrane electrical potential (A*,), which decreased from 140 mV at pH 7 at the beginning of growth to 80 mV when the medium had reached pH 5. The proton motive force, therefore, was 155 mV at pH 7, decreasing to 120 mV at pH 5. When further fermentation acidified the medium below pH 5, both the ApH and the A* collapsed, indicating that these cells require an internal pH of at least 5.5 to 5.7. Cells harvested from stationary phase and suspended in citrate-phosphate buffer maintained a ApH of 1.5 at external pH 5.0. This ApH was dissipated by acetic acid (at the concentrations found in the growth medium) and other weak organic acids, as well as by ionophores and inhibitors of glycolysis and of the H+-ATPase. Nongrowing cells had a A*

Metabolism and energy generation in homoacetogenic clostridia

“…The magnitude of the A p was independent of the temperature as measured at 25 and 50°C. These observations axe in accordance with Ap measurements in whole cells of C. thermoaceticum [36]. With H 2 as electron donor, low but significant Ap values up to 50 mV were found at pH > 8.0 when benzyi viologen was used as electron acceptor [35].…”

Bioenergetics of lactic acid bacteria: cytoplasmic pH and osmotolerance

“…Cessation of growth and metabolism occur at a time when the cytoplasmic pH (pHi) reaches a given value, which is characteristic of the organism. Thus, in Clostridium thermoaceticum, a homoacetogenic anaerobe, the pH i did not decrease below pH 5.6 during growth [58]. In oral streptococci, the pH i at the end of batch culture was approximately 5.7 [55].…”

“…However, many bacteria do not maintain a constant pH i. In clostridia [46,58,61,62], many streptococci [55,35,41,38], and lactobacilli (see above), as the pH o decreases because of the accumulation of acidic fermentation end-products, the pH i also decreases, although it remains more alkahne than the pHo; i.e., the cells maintain a A pH, alkahne inside. Thus, unlike many aerobes [30,34], the anaerobic fermenters tolerate fairly wide variations in internal proton concentration.…”

“…The importance of the H +-ATPase in pH control is also shown by the isolation of mutants which grow well at pH 7.5, but cannot grow at pH 6. The mutants apparently are defective in the pH-regulatory system, as they cannot maintain a normal ApH [44,58]. The mutant cell membranes are normally proton-im-permeable, but show a deficient ATPase activity.…”

The fermentation pathways of Escherichia coli

“…The build up of acidic products, particularly acetic acid, eventually inhibits further growth of anaerobic cultures, probably by acting as a weak uncoupler [92]. At pH 4.5 the level of acetate inside the cell is several times its external concentration, at least in Clostridia [93].…”