In the present study the microbial communites at a shallow submarine hydrothermal vent in the Aegean Sea near the island of Milos (Greece) were investigated. In particular, the relation between the composition and the structure of the microbial communities and the changing environmental conditions along a transect from the center of the vent to the surrounding area was studied. Synecological studies were carried out by 16S rRNA based techniques in parallel to autecological studies with bacteria isolated from the habitat.

1. A trend from a diverse range of bacterial populations which were present in approximately equal abundance in the transition zone from the strongly hydrothermally-influenced sediments to normal sedimentary conditions, to a community dominated by few populations close to the center of the vent was revealed. Temperature was found to be an important parameter in determining this trend. This supported the hypothesis that extreme environmental conditions will lead to a community structure with few dominating populations.

2. In total 91 unique bacterial populations could be detected. The sequencing of excised and reamplified bands from denaturing gradient gel electrophoresis gels revealed the presence of a wide spectrum of bacterial lineages. However, only a low percentage of the sequences was closely related to the sequences of cultivated bacteria. Because of the input of allochtonous organic matter the microbial community seemed to be net hetero-trophic. However, autotrophic populations were also detected and they contributed to the new production of organic matter.

3. The concentration of rRNA in general decreased sharply with sediment depth and the concentration was higher in regions closer to the vent center. This indicates that the microbial activity was stimulated by the hydrothermalism. Bacteria made up at least 78% (average 95%) of the prokaryotic rRNA. Along the steepest temperature gradient the proportion of archaeal rRNA was increasing. Nevertheless, even in the hottest sediment layer where a quantification was possible (in situ temperature 82 °C) archaeal rRNA made up only 11.9% of the prokaryotic rRNA. This suggests that Archaea were generally a minor component of the microbial community at this vent site

4. Viable cell counts of specific physiological groups of bacteria (autotrophic sulfur oxidizers, sulfate reducers, and iron reducers), which were determined by using the most-probable number (MPN) technique, constituted at most about 4 % of the total cell counts as determined by the acridine-orange-direct-count (AODC) method. Both counting procedures revealed highest cell numbers in a transition zone from the strongly hydrothermally-influenced sediments to normal sedimentary conditions.

5. Like at deep-sea hydrothermal vents, members of the sulfur-oxidizing genus Thiomicrospira constituted an important component of the microbial communities. One type (Milos-T2) that was isolated from high dilutions was shown to be dominant. This also demonstrated the rare occurence of a congruence between results of molecular based analysis and cultivation based analysis.

6. The diversity among the sulfur-oxidizing bacterial community was high. New species that belong to the genera Thiomicrospira and Halothiobacillus, both of which have been described at other vent sites before, could be isolated. In addition, five sulfur-oxidizing bacteria that form new lineages among the g-subclass of the Proteobacteria and are most closely related to symbionts, could be isolated. Some of these are able to oxidize reduced sulfur compounds anaerobically using nitrate as electron acceptor. Important parameters that appear to govern the distribution of the sulfur-oxidizing bacteria are the in situ temperature, the substrate concentration, i.e. hydrogen sulfide, and the availability of oxygen.

7. A thermophilic sulfate-reducing bacterium could be isolated (strain MT-96), which represents a new species of the genus Desulfacinum. In contrast to the most closely related thermophilic species Desulfacinum infernum and Thermodesulforhabdus norvegicus, which were isolated from oil field waters, the completely oxidizing strain MT-96 was isolated from a geothermally influenced habitat containing no oil related substrates.

8. Experiments to investigate the fractionation of the stable sulfur isotopes by strain MT-96 were carried out. The hydrogen sulfide produced in the experiments was enriched in 32S by approximately 19‰ compared to sulfate, which indicates that stable isotope discrimination by this thermophile is within the range found previously for mesophilic sulfate-reducing bacteria and only slightly higher than observed for the thermophilic Gram-positive Desulfotomaculum nigrificans. Field data on the sulfur isotope partitioning between sulfate and reduced sulfur species at hydrothermal vents from Milos are close to the results observed in the experiments of the present study.