Identification of physiological and environmental factors that limit efficient growth of hyperthermophiles is important for practical application of these organisms to the production of useful enzymes or metabolites. During fed-batch cultivation of Sulfolobus solfataricus in medium containing L-glutamate, we observed formation of L-pyroglutamic acid (PGA). PGA formed spontaneously from L-glutamate under culture conditions (78°C and pH 3.0), and the PGA formation rate was much higher at an acidic or alkaline pH than at neutral pH. It was also found that PGA is a potent inhibitor of S. solfataricus growth. The cell growth rate was reduced by one-half by the presence of 5.1 mM PGA, and no growth was observed in the presence of 15.5 mM PGA. On the other hand, the inhibitory effect of PGA on cell growth was alleviated by addition of L-glutamate or L-aspartate to the medium. PGA was also produced from the L-glutamate in yeast extract; the PGA content increased to 8.5% (wt/wt) after 80 h of incubation of a yeast extract solution at 78°C and pH 3.0. In medium supplemented with yeast extract, cell growth was optimal in the presence of 3.0 g of yeast extract per liter, and higher yeast extract concentrations resulted in reduced cell yields. The extents of cell growth inhibition at yeast extract concentrations above the optimal concentration were correlated with the PGA concentration in the culture broth. Although other structural analogues of L-glutamate, such as L-methionine sulfoxide, glutaric acid, succinic acid, and L-glutamic acid ␥-methyl ester, also inhibited the growth of S. solfataricus, the greatest cell growth inhibition was observed with PGA. We also observed that unlike other glutamate analogues, N-acetyl-L-glutamate enhanced the growth of S. solfataricus. This compound was stable under cell culture conditions, and replacement of L-glutamate with N-acetyl-L-glutamate in the medium resulted in increased cell density.Recently, hyperthermophiles have attracted the attention of many workers in the biotechnology research community because of the potential industrial applications of these organisms and because they may provide a better understanding of how cell components are stabilized when they are heated (1,13,18,28,29). Despite the biotechnological potential of hyperthermophiles, thus far the uses of these organisms have been limited because of the low cell yields when they are cultivated, which are attributed mainly to a lack of knowledge concerning the physiological characteristics and high-temperature cultivation techniques (5, 17). Therefore, identification of physiological and environmental factors that limit efficient growth of hyperthermophiles and development of strategies to obtain high cell densities under high-temperature conditions are particularly important for increasing the biomass yields of these microorganisms in environments different from their natural habitats.Sulfolobus solfataricus P2 is a hyperthermophilic archaeon which normally grows at 75 to 85°C and pH 2.0 to 4.0 (4, 7, 32). Since phy...

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