New cultural approaches for microaerophilic hyperthermophiles.

This article reports on a new culture system designed for studying the effects of nutritional factors on the growth of hyperthermophilic and chemolithotrophic microorganisms. The system comprises 5-l stainless steel jars, an automatic gas dispenser, propylene microplates, and a robotic platform. The culture system was validated using Aquifex aeolicus, a hyperthermophilic, chemolithotrophic, and microaerophilic bacterium, which requires hydrogen, oxygen, CO₂, and minerals for growth. We demonstrated that the cell densities measured on 147 cultures of A. aeolicus microplated in jar at 80°C under partial pressures (in kPa) of water vapor (47), H₂ (117.7), O₂ (28.1), CO₂ (31.4), and N₂ (3.9), followed a normal distribution, with a mean of 0.72 and a standard deviation of 0.04 (variation coefficient: 5.7%). In addition, cross-comparison of the growth kinetics of A. aeolicus in serum bottles and in a jar system highlighted similar kinetics patterns (both mean growth rates were 0.18 and 0.17 h-1, respectively), whereas the maximum cell densities reached were slightly lower in jar than in bottle (0.73 vs. 0.88 OD units, respectively). Furthermore, these results showed that, contrary to bottles, the total pressure of gas in jars remained constant throughout the biotic experiments, even with seven microplates completely filled with grown cultures. In addition, this system has been validated also for hyperthermophilic strictly anaerobes such as Thermotoga maritima or aerobes such as Sulfolobus solfataricus. This new culture system offers an interesting alternative for cultivating hyperthermophiles, using gas as substrate under constant pressure, thus making it possible to miniaturize experiments and study a large number of nutritional factors in one experimental run.