Group member: Dr. Harald Huber, Prof. Dr. Michael Thomm


Isolation, cultivation, and characterization of novel hyperthermophilic Archaea from high temperature ecosystems


Hyperthermophilic members of the Archaea (growing optimally at temperatures above 80 °C) have been isolated from numerous continental high temperature ecosystems, like Iceland, Italy, or the Yellowstone National Park or from submarine hydrothermal systems at the Mid Atlantic Ridge or the South Pacific Ridge. They represent deep branching lineages in the universal tree of life and are therefore highly relevant for the evolution of life. The main topics of our research activities are the enrichment of such microorganisms, the development of new cultivation techniques, the physiological, biochemical, and molecular characterization of the isolates, and the determination of their position in the universal phylogenetic tree of life. These organisms are also cultivated in large scale (up to 300 l) in our fermentation plant, producing cell masses for molecular investigations. Furthermore, optimization of the culture conditions and the culture media (e.g. by the use of ICP-analyses) are carried out.

 

Biotops:

Lesbos

Prof. Thomm

 

Lesbos

Fig. 1: Sampling at the hot springs of Polichnitos, Lesbos Island, Greece (Temperatures up to 88 °C)

 

Cultivation of extremely anaerobic microorganisms:

For the cultivation of extremely anaerobic microorganisms anoxic culture media are necessary. Such media are produced aerobically. Then at a gas station the major part of the dissolved oxygen is removed by stripping the medium with molecular nitrogen (N2). By the addition of Na2S the remaining oxygen is chemically removed. In an anaerobic chamber (Fig. 2) – the oxygen concentration within the chamber is kept at approx. 5 ppm – the culture media are filled into serum bottles which are subsequently sealed with rubber stoppers. Outside the anaerobic chamber the serum bottles are gassed three times with the desired gas mixture (e.g. H2/CO2 in a proportion of 80:20 for methane-producing Archaea). Finally the culture media can be autoclaved. If necessary, the media are supplemented with organics (e.g. anaerobic yeast extract).

 

Anaerobic chamber Fig. 2: Anaerobic chamber

 

Isolation of hyperthermophilic and extremely anaerobic microorganisms:

To obtain pure cultures a novel isolation technique was developed which avoids platting on solid surfaces, since most hyperthermophiles cannot be grown on plates. This technique is based on the use of a laser microscope and the “optical tweezers”.
This micromanipulation, named either “optical tweezers“, “laser tweezers“ or “laser trap“ consists of a modified computer-controlled inverse microscope equipped with a strongly focused infrared laser (Fig. 3). The laser beam creates a so called “light pressure” which allows the fixation and separation of a single cell under permanent optical control. In the laser focus the cell can be manipulated in all three dimensions. For this “trapping“ inside the laser beam (diameter around 1 µm) a phase contrast objective with high numerical aperture is necessary.

Schematic compisition of the "laser-microscope" Fig. 3: Schematic composition of the “laser-microscope“

For the isolation of a single cell by the “optical tweezers” a special separation unit was developed. It consists of a rectangular glass capillary (dimensions of the inner side: 0.1 x 1 mm; length 10 cm), which is connected with a tube to a needle of a 1 ml plastic syringe (Fig. 4a; 1-4). A cutting line separates the glass capillary (microslide) into two equal compartments (Abb. 4a, 5).

The capillary is usually filled inside an anaerobic chamber. First, about 90 % of the volume of the capillary is filled with sterile medium from the open end. Then about 1 µl of the culture medium (mixed culture) is soaked into the microslide. Now the separation unit is fixed on the microscope stage (Fig. 3). Under optical control a single cell from the mixed culture can be selected and fixed by activation of the laser beam in the laser focus over the objective (Fig. 4a). By horizontal movement of the microscope stage this fixed cell is now separated from the mixed culture. After the sterile compartment beyond the cutting line is reached (separation length about 6 – 7 cm; Fig. 4b; duration 3 to ten minutes)) the capillary is broken and the single cell is transferred into a sterile serum bottle with the corresponding culture medium .

Scheme of the single cell isolation
Fig. 4: Scheme of the single cell isolation.
a: Isolation unit filled with medium and culture. One single cell is fixed with the laser beam over the objective. b: At the end of the separation procedure the single cell is in the sterile culture medium of the capillary. 1: glass capillary; 2: tube; 3: needle; 4: syringe; 5: cutting line. 6: mixed culture; 7: objective .