Sampling and cultivation

Samples of possible HT containing hot waters, soils, rock and sediments may serve as primary material to set-up enrichment cultures in the laboratory. Special care has to be taken to avoid contamination of the sample by oxygen. In the presence of high temperatures of growth, it is toxic to anaerobic HT. In contrast, at low temperatures (for example: 4 °C) in the presence of oxygen, anaerobic HT may survive for years. Transportation and shipping can be performed at ambient temperature. In the lab, anaerobic samples (in tightly stoppered 100 ml storage bottles and stored at 4 °C) can be used for successful enrichment cultures at least for 10 years.

Enrichment cultures can be obtained by simulating the varying geochemical and geophysical composition of the environments. Various plausible electron donors and acceptors may be used under anaerobic, microaerophilic, or aerobic culture conditions. Depending on the (unknown) initial cell concentration and the doubling time of the organism, positive enrichment cultures of HT can be identified by microscopy within 1-7 days. For a deeper understanding of the organisms, the study of pure cultures is required. Due to the high incubation temperatures, the traditional manner of cloning by plating does not perform well in HT (even by using heat stable polymers like Gelrite). Therefore, we developed a new procedure for cloning single cells under the laser microscope, by employing optical tweezers (Ashkin and Dziedzic, 1987; Huber et al., 1995). Large cell masses are required for biochemical and biophysical investigations. For mass culturing of HT, a new type of high temperature fermentor was invented in collaboration with an engineering

Fig. 7.3. Fermentation plant, University of Regensburg. Partial view, showing two 300 l fermenters and one 130 l fermenter for cultivation of HT.

company (Figure 7.3). Its steel casing is enamel-protected in order to resist the highly corrosive culture conditions. Sharp-edged parts like stirrers, gasing and sampling pipes and condensers are made of titanium. The cell yield of a 300 l fermentation may vary from approximately 3 g to 2 kg (wet weight), depending on the HT isolate.

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