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

Abstract

"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 werestudied using amplicon sequencing (NGS) and cultivation techniques. Diversity indices and cellcounts were determined to assess the species richness in relation to the cell counts within thesamples, and the oligocarbophile growth capability of the isolated bacteria was tested in microtiterplates. Altogether, 55 bacterial phyla were identified from the samples by amplicon sequencing. Themicrobial communities of the different sampling times of the same sites did not differ significantly.PatescibacteriaandProteobacteriawere present in all samples. Ciprián sample was dominated byBacteroidetes, while in Dandár sample a high ratio ofChloroflexiwas detected.Rokubacteriaand WOR-1dominated Szent Flórián sample and Tatabánya had a high number ofEpsilonbacteraeota. Ninearchaeal phyla were also detected"," the samples were characterized by the presence of unclassifiedarchaea andNanoarchaeota, among themWoesearchaeia,as the most dominant.CrenarchaeotaandAltiarchaeotawere detected in high ratios in Dandár water samples. AmongThaumarchaeotathe familyNitrosopumilaceae, and orders ofNitrosotalealesandNitrososphaeralesappeared in Szent Flórián andTatabánya samples. Key organisms of the different biogeochemical cycles were discovered in thesenutrient-depleted environments: methanogenic archaea, methanotrophic bacteria, ammonia oxidizer,nitrate reducers, diazotrophs, sulfate reducers, and sulfur oxidizer. Diversity indices and cell countsof the samples show negative correlation in case of bacteria and positive in case of archaea in Cipriánsample. The high diversity indices in Szentendre samples are connected to low cell counts, mostprobably due to the vulnerability of the groundwaters to the external environment factors which leadto the infiltration of soil microbes and contaminants to the water. The isolated bacteria were affiliatedinto four phyla, most of them belonging toProteobacteria(59%) followed byActinobacteria(21%),Firmicutes(17%) andVerrucomicrobia(1%). The members of the facultative chemolithotrophic generaofSphingobium,Sphingomonas,Sphingopyxiswere characterizing only Szentendre, Szent Flórián andTatabánya 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 resultsconfirmed the predominance of unclassified and unknown taxa in subsurface water, pointing to theimportance and necessity of further studies to characterize these microbial populations."