In situ modelling of biofilm formation in a hydrothermal spring cave.

Attachment of microorganisms to natural or artificial surfaces and the development of biofilms are complex processes which can be influenced by several factors. Nevertheless, our knowledge on biofilm formation in karstic environment is quite incomplete. The present study aimed to examine biofilm development for a year under controlled conditions in quasi-stagnant water of a hydrothermal spring cave located in the Buda Thermal Karst System (Hungary). Using a model system, we investigated how the structure of the biofilm is formed from the water and also how the growth rate of biofilm development takes place in this environment. Besides scanning electron microscopy, next-generation DNA sequencing was used to reveal the characteristic taxa and major shifts in the composition of the bacterial communities. Dynamic temporal changes were observed in the structure of bacterial communities. Bacterial richness and diversity increased during the biofilm formation, and 9-12 weeks were needed for the maturation. Increasing EPS production was also observed from the 9-12 weeks. The biofilm was different from the water that filled the cave pool, in terms of the taxonomic composition and metabolic potential of microorganisms. In these karstic environments, the formation of mature biofilm appears to take place relatively quickly, in a few months.

Radioactive environment adapted bacterial communities constituting the biofilms of hydrothermal spring caves (Budapest, Hungary).

The thermal waters of Gellért Hill discharge area of the Buda Thermal Karst System (Hungary) are characterized by high (up to 1000 Bq/L) 222Rn-activity due to the radium-accumulating biogeochemical layers. Samples were taken from these ferruginous and calcareous layers developed on spring cave walls and water surface. Accumulation of potentially toxic metals (e.g. As, Hg, Pb, Sn, Sr, Zn) in the dense extracellular polymeric substance containing bacterial cells and remains was detected by inductively coupled plasma mass spectrometry. The comparison of bacterial phylogenetic diversity of the biofilm samples was performed by high throughput next generation sequencing (NGS). The analysis showed similar sets of mainly unidentified taxa of phyla Chloroflexi, Nitrospirae, Proteobacteria, Planctomycetes; however, large differences were found in their abundance. Cultivation-based method complemented with irradiation assay was performed using 5, 10 and 15 kGy doses of gamma-rays from a 60Co-source to reveal the extreme radiation-resistant bacteria. The phyla Actinobacteria, Firmicutes, Proteobacteria (classes Alpha- Beta- and Gammaproteobacteria), Bacteriodetes and Deinococcus-Thermus were represented among the 452 bacterial strains. The applied irradiation treatments promoted the isolation of 100 different species, involving candidate novel species, as well. The vast majority of the isolates belonged to bacterial taxa previously unknown as radiation-resistant microorganisms. Members of the genera Paracoccus, Marmoricola, Dermacoccus and Kytococcus were identified from the 15 kGy dose irradiated samples. The close relatives of several known radiation-tolerant bacteria were also detected from the biofilm samples, alongside with bacteria capable of detoxification by metal accumulation, adsorption and precipitation in the form of calcium-carbonate which possibly maintain the viability of the habitat. The results suggest the establishment of a unique, extremophilic microbiota in the studied hydrothermal spring caves.

Natural radioactivity of thermal springs and related precipitates in Gellért Hill area, Buda Thermal Karst, Hungary.

The elevated radioactivity of the thermal waters of Buda Thermal Karst (BTK), Hungary is known and studied since the beginning of the 20th century. In the recent studies, the anomalous 222Rn/226Ra ratios have drawn the attention to the existence of local 222Rn source. Biogeochemical precipitates (i.e. biofilms) in spring caves were found to have high adsorption capacity, accumulating e.g. 226Ra. Biogeochemical precipitates are ubiquitous in the thermal springs of BTK, occurring in different amount and colours (dark grey, brown, red, white), and have different microbial communities and elemental composition. The detailed investigation of the radioactivity of spring waters highlighted the different 226Ra and 222Rn activity concentrations. The present study aimed to survey the radioactivity of the thermal springs of Gellért Hill area, together with the biogeochemical precipitates and air above the water level, and to assess the evolution of the radioactivity of known-aged precipitates, formed during in situ experiments. We found that the basic physicochemical parameters of the spring waters (field parameters, major ions) do not affect the adsorption capacity of biogeochemical precipitates. It was revealed by the conducted in situ experiments, that the flow conditions influence the evolution rate of precipitates, so their adsorption capacity. The 222Rn activity concentrations of spring waters are dependent on the area of the water surface, volume of air space above the water level, ventilation of the caves/channels and presence of calcite layer on the water surface. The latter has a blocking effect on degassing.