Cultivation and Molecular Studies to Reveal the Microbial Communities of Groundwaters Discharge Located in Hungary

In the present study 12 water samples of five sampling sites (Tatabánya, Dandár, Szentendre, Szent Flórián and Ciprián groundwaters) known as nutrient-depleted aquatic environments were studied using amplicon sequencing (NGS) and cultivation techniques. Diversity indices and cell counts were determined to assess the species richness in relation to the cell counts within the samples, and the oligocarbophile growth capability of the isolated bacteria was tested in microtiter plates. Altogether, 55 bacterial phyla were identified from the samples by amplicon sequencing. The microbial communities of the different sampling times of the same sites did not differ significantly. Patescibacteria and Proteobacteria were present in all samples. Ciprián sample was dominated by Bacteroidetes, while in Dandár sample a high ratio of Chloroflexi was detected. Rokubacteria and WOR-1 dominated Szent Flórián sample and Tatabánya had a high number of Epsilonbacteraeota. Nine archaeal phyla were also detected
the samples were characterized by the presence of unclassified archaea and Nanoarchaeota, among them Woesearchaeia, as the most dominant. Crenarchaeota and Altiarchaeota were detected in high ratios in Dandár water samples. Among Thaumarchaeota the family Nitrosopumilaceae, and orders of Nitrosotaleales and Nitrososphaerales appeared in Szent Flórián and Tatabánya samples. Key organisms of the different biogeochemical cycles were discovered in these nutrient-depleted environments: methanogenic archaea, methanotrophic bacteria, ammonia oxidizer, nitrate reducers, diazotrophs, sulfate reducers, and sulfur oxidizer. Diversity indices and cell counts of the samples show negative correlation in case of bacteria and positive in case of archaea in Ciprián sample. The high diversity indices in Szentendre samples are connected to low cell counts, most probably due to the vulnerability of the groundwaters to the external environment factors which lead to the infiltration of soil microbes and contaminants to the water. The isolated bacteria were affiliated into four phyla, most of them belonging to Proteobacteria (59%) followed by Actinobacteria (21%), Firmicutes (17%) and Verrucomicrobia (1%). The members of the facultative chemolithotrophic genera of Sphingobium, Sphingomonas, Sphingopyxis were characterizing only Szentendre, Szent Flórián and Tatabánya samples. Only 10% of the isolated species showed an obligate oligocarbophile character. From the samples, a high number of novel bacterial taxa were cultivated. As a conclusion, our results confirmed the predominance of unclassified and unknown taxa in subsurface water, pointing to the importance and necessity of further studies to characterize these microbial populations.