Improved Methods for Cultivation of the Extremely Thermophilic Bacterium Thermotoga neapolitana

Abstract

Growth medium components and cultivation conditions for the extremely thermophilic bacterium Thermotoga neapolitana were optimized. A defined marine salts medium was formulated. Trace amounts of iron stimulated growth of T. neapolitana , while zinc inhibited growth at concentrations exceeding 11.1 μM. Other trace metals had no effect on its growth. Of the vitamins tested, only biotin was required for optimal growth. A defined mineral medium containing 5 g of carbohydrates per liter as the carbon source and 0.5 g of cysteine per liter as the sulfur source and reductant supported growth. Growth was stimulated by inclusion of vitamin-free Casamino Acids. Elemental sulfur, cystine, and dimethyl disulfide in the growth medium enhanced growth. Elemental sulfur and cystine relieved growth inhibition by hydrogen. T. neapolitana formed colonies in 2 days on plates of complex medium solidified with gellan gum and in 4 days on defined medium. The efficiency of plating was determined when growing cultures were sampled both aerobically and anaerobically and plated under aerobic and anaerobic conditions. Mean plating efficiencies were improved by sampling the growing cultures under strictly anaerobic conditions. Little or no improvement was obtained by inoculating plates inside an anaerobic chamber. Plating efficiencies of approximately 80% were obtained. Polycarbonate jars with aluminum lids withstood repeated incubation at 77°C without significant deterioration of the anaerobic seal and provided the most consistent results.