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 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.

Archaea and their interactions with bacteria in a karst ecosystem

“…Nitrosopumilaceae were reported to live in the active and fossil parts of the Dupnisa Cave System with the relative abundance >5% ( Doğruöz-Güngör, 2020 ). Nitrosopumilaceae preferred aquatic niches (DW) in the Heshang Cave, which might process ammonia-oxidation in water samples ( Toumi et al, 2021 ). Aenigmarchaeales, found in ice-capped lake and wheat-maize rotation soils, has never been reported in karst ecosystem ( Wan et al, 2021 ; Vigneron et al, 2022 ).…”

Antiquaquibacter oligotrophicus gen. nov., sp. nov., a novel oligotrophic bacterium from groundwater

“…pH was adjusted to pH 7.0–7.2 and tap water was added to the medium until the final volume of 1 l was reached, and then finally autoclaved at 121 °C for 20 min. The media were solidified with either agar or gellan gum, respectively [6]. Isolation happened randomly after enrichment of 50 ml water sample in 250 ml of M5 media for 2 weeks using polyurethane foam-based traps [7].…”

“…Possesses the following properties in addition to those given for the genus: grows well on R2A agar and M5 media [6]. Colonies on R2A agar are small, circular and light yellow in colour.…”

Microbial Community Associated with Groundwaters Discharge in Transylvania (Romania) and Balaton Highland (Hungary)

“…Has the capacity to thrive in harsh environments (Verma et al, 2020), commonly isolated also from freshwater and marine habitats and many are facultatively chemolithotrophs, often producing H 2 during their metabolism (Toumi et al, 2021).…”

“…Known taxa a liations from OTU taxon classi cation were condensed to display the percentage of that taxon in the dataset.Woesarchaeota, presented in a relatively large proportion of this microbial dataset (10 %), especially in Szent Jakab and Kossuth Lajos water samples with a percentage of 16.92 % and 17.55 %, respectively. They are part of DPANN superphylum, previous studies based on genome analyses and metabolic reconstructions propose that they are anaerobic heterotrophic organisms with a potential of symbiotic relationships especially with methanogens(Toumi et al, 2021) (Liu et al, 2018). This feature can remove the thermodynamic bottlenecks and enables several metabolic reactions under nutrient-depleted conditions(Lau et al, 2016).…”