Isolation of chemolithoautotrophic bacteria from the surface sediments of an alpine oligotrophic pond

Benthic organisms have been reported to actively support primary production in lentic ecosystems, and their contribution may overcome those of phytoplankton in shallow, oligotrophic water bodies. A pivotal role in this sense is played by phototrophic organisms such as micro and macroalgae, diatoms, dinoflagellates and cyanobacteria, although freshwater sediments can harbour also chemolithoautotrophic prokaryotic populations, potentially involved in C-fixation as well as in biogeochemical cycling of different nutrients. In the framework of a broader study on the prokaryotic diversity of an alpine oligotrophic pond (Col d’Olen Rock Glacier Pond, Valle d’Aosta, Italy), we set up a procedure for the enrichment and isolation of chemolithoautotrophic benthic bacteria, in order to gain new insights on this important but still poorly known functional guild. Surface sediment samples were aseptically collected from the bottom of the pond, and enrichment cultures were set up aerobically in two different mineral media, at 4 and 25°C. Cultures were kept in the dark to exclude the growth of phototrophic microorganisms, and were refreshed at intervals of approximately three weeks. The presence of microorganisms harbouring ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) encoding genes (cbbL ‘red-like’ and ‘green-like’, cbbM) was tested by PCR. Aliquots of the positive enrichment cultures were plated on different agar media (the original mineral medium, Thiomonas medium, full-strength or diluted R2A) and incubated at 4 and 25°C, in both aerobic end anaerobic conditions, in order to increase the possibilities to isolate the target strains. The pure cultures isolated from solid media were tested for the presence of RubisCO genes, and positive strains were identified by sequencing the entire 16S rRNA gene. Five enrichment cultures, all of them set up in the same medium, gave a positive signal for the presence of RubisCO genes. In the three of them grown at 4°C RubisCO form II was detected, while the two grown at 25°C were positive for form I ‘red-like’. Plate counts revealed microbial abundances in the range of 105-106 CFU ml-1 in both aerobic/anaerobic conditions, suggesting that an important proportion of the enriched microorganisms may be microaerophiles or facultative anaerobes. Unfortunately, no isolates harbouring cbbM genes were recovered in any of the tested conditions. However, a bacterial strain identified as Dyella ginsengisoli, recurrent on Thiomonas medium agar plates in both aerobic and anaerobic conditions, was found to be positive for the presence of cbbL genes form ‘red-like’. Considering that, to our best knowledge, no previous observations on the autotrophic behaviour of this species has been reported in literature, further investigations on its actual C-fixation activity may add valuable information for future studies on the ecology of freshwater benthic ecosystems and soils, where D. ginsengisoli was first isolated.