Phototrophic consortia are known since the beginning of the 20th century (55) and were discovered in the chemoclines of freshwater lakes worldwide (78). Although mass accumulations of phototrophic consortia have been reported (32), aspects concerning the ecological significance, the physiology and the selective advantage of phototrophic consortia in the natural environment remained obscure.
In consequence, the first aim of the present study was to gain a deeper understanding of the physicochemical and biological factors providing a selective advantage of "P. roseum" under natural conditions. This approach was based on the characterization of "P. roseum" population in the chemocline of Lake Dagow (78). The rather small, mesophilic Lake Dagow is located approximately 100 km in the north of Berlin (Germany) and develop a stratified water column each summer. Subsequently a large population of "P. roseum" occurs in the anoxic hypolimnion. Depending on year and seasonal development the consortia are found between a depths of 6.6 to 7.4 m (78, Chapter 3). From microscopic studies of chemocline samples it became clear that a uniform community of phototrophic bacteria, mainly consisting of "P. roseum", dominated the chemocline of Lake Dagow (78). Although the ecological niche of "P. roseum" was inferred from a two year study of the chemocline and literature data (78), the vertical distribution, relative abundance and diversity of epibiotic green sulfur bacteria in Lake Dagow remained obscure. Our approach involved the analysis of the numerical significance and phylogenetic diversity of epibiotic and free-living green sulfur bacteria in the chemocline of Lake Dagow by applying specific fluorescent oligonucleotide probes (99) and 16S rRNA gene specific primers for green sulfur bacteria (77).
The dominance of "P. roseum" over other phototrophic bacteria made the chemocline of Lake Dagow an ideal model system to study "P. roseum" under natural conditions. Therefore, the second aim focused on the in situ physiology of "P. roseum" epibionts with respect for their carbon substrates. Although green sulfur bacteria are obligate photoautotrophs, organic substrates may play an important role for the growth of green sulfur bacteria under conditions when electron donors become limiting (9). Clearly, sulfide concentrations in the chemocline and the flux of sulfide from the hypolimnion into the chemocline were not sufficient to explain the large biomass accumulation of "P. roseum" in Lake Dagow (78). Because a syntrophic interaction based on a closed sulfur cycle became unlikely due to the phylogenetic position of the central bacterium (28) electron donors are presumably a limiting factor in the chemocline and organic substrates may be important for the growth of the epibionts of "P. roseum" in situ. Biomarkers from green sulfur bacteria differ significantly in the
composition of stable carbon isotopes (91, 100) and can therefore be used to determine the in situ carbon substrate. For this investigation, the community of green sulfur bacteria had to consist of almost only epibionts of "P. roseum". The obstacle of this study was the absence of the typical biomarker for brown colored Chlorobiaceae, isorenieratene. Therefore, a search for new biomarkers was necessary to be able to investigate the in situ physiology of "P. roseum" epibionts. Because of their high abundance in green sulfur bacteria, derivatives of bacteriochlorophylls have been used as biomarkers previously (39, 43). The esterifying alcohols of bacteriochlorophyll e provide useful biomarkers for stable carbon isotope analysis and the investigation of the in situ physiology of brown-colored Chlorobiaceae, but have not been investigated to a sufficient extent before. The detailed investigation of biomarkers derived from photosynthetic pigment of green sulfur bacteria in general and "P. roseum" epibionts in particular was followed by the in situ study of stable carbon isotope fractionation in the esterifying alcohols of bacteriochlorophylls and fractions of particulate and dissolved organic and dissolved inorganic carbon. 14C-labled bicarbonate was incubated with
chemocline samples to assess the extent of photosynthetic activity in the bacterial community of the chemocline and, hence, the photosynthetic activity of the epibiotic green sulfur bacteria in Lake Dagow.
Enrichments of "C. aggregatum" could only be established in the presence of 2-oxoglutarate (27). Therefore, it remained an open question whether 2-oxoglutarate is important for other consortia, as "P. roseum" and if organic substrates are important for the growth of phototrophic consortia and especially for the central bacterium in nature. Consequently, the third aim of this study was to investigate the effect of environmental stimuli like organic substrates or electron donors on the chemotaxis of "P. roseum". Because the attraction of "C. aggregatum" by sulfide and 2-oxoglutarte was known from studies with enrichment cultures it was important to test whether substrates like 2-oxoglutate function as attractant of "P. roseum" and substrate of the central bacterium under in situ conditions. Initial experiments with enrichments of "C. aggregatum" suggested a strong physiological interaction and a rapid signal transfer between epibionts and central bacterium, which presumably act as a selective factor under natural conditions (27). The chemotaxis towards sulfide and 2-oxoglutarate and the scotophobic response as observed in "C. aggregatum" (27) might represent a reaction to stimuli encountered in nature that may help to gain and maintain the optimum position in the chemocline. In situ experiments would provide valuable information on the importance and nature of interaction for the search of substrates under natural conditions. Therefore, chemotaxis assays were performed in situ and the uptake of
14C-labled 2-oxoglutarate by "P. roseum" was analyzed by microautoradiography in
chemocline samples.
The phylogenetic position of the epibionts from a limited number of phototrophic consortia was analyzed previously and determined as unique phylotypes within the radiation of green sulfur bacteria (28, 99). Because morphologically identical phototrophic consortia from different lakes represented unique phylotypes a non-ubiquitous distribution of phototrophic consortia was suggested (28). The major focus of the fourth aim of this study was the detailed analysis of the phylogenetic position of the epibiotic green sulfur bacteria from most of the morphologically described phototrophic consortia. The obtained data would also provide the basis to elucidate the global diversity among these morphotypes. Therefore, chemocline samples containing phototrophic consortia from 14 Lakes of 6 geographical regions were studied. The assessment of the global diversity of epibiotic green sulfur bacteria will furthermore provide (i) answers to the question if this type of symbiosis has developed only once in the evolution of green sulfur bacteria or arose convergently among various phylogenetic lines of green sulfur bacteria; (ii) data to investigate if competitive exclusion exists between different morphological types of phototrophic consortia and (iii) insight into the question if endemic populations exists among epibiont phylotypes. Again, a culture independent approach was chosen to determine the diversity of epibionts from chemocline samples. Phototrophic consortia were separated by micromanipulation and subsequently 16S rRNA gene fragments were amplified with oligonucleotide primers specific for green sulfur bacteria (77). The amplified 16S rRNA gene fragments were analyzed by denaturing gradient gel electrophoresis and subsequently sequenced. The phylogenetic analysis of the partial 16S rRNA gene sequences provided the information to answer the questions asked previously.