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.

Bacterial Diversity of a High-Altitude Permafrost Thaw Pond Located on Ojos del Salado (Dry Andes, Altiplano-Atacama Region).

Microbial ecology of permafrost, due to its ecological and astrobiological importance, has been in the focus of studies in past decades. Although permafrost is an ancient and stable environment, it is also subjected to current climate changes. Permafrost degradation often results in generation of thaw ponds, a phenomenon not only reported mainly from polar regions but also present in high-altitude permafrost environments. Our knowledge about microbial communities of thaw ponds in these unique, remote mountain habitats is sparse. This study presents the first culture collection and results of the next-generation DNA sequencing (NGS) analysis of bacterial communities inhabiting a high-altitude permafrost thaw pond. In February 2016, a permafrost thaw pond on the Ojos del Salado at 5900 m a.s.l. (meters above sea level) was sampled as part of the Hungarian Dry Andes Research Programme. A culture collection of 125 isolates was established, containing altogether 11 genera belonging to phyla Bacteroidetes, Actinobacteria, and Proteobacteria. Simplified bacterial communities with a high proportion of candidate and hitherto uncultured bacteria were revealed by Illumina MiSeq NGS. Water of the thaw pond was dominated by Bacteroidetes and Proteobacteria, while in the sediment of the lake and permafrost, members of Acidobacteria, Actinobacteria, Bacteroidetes, Patescibacteria, Proteobacteria, and Verrucomicrobia were abundant. This permafrost habitat can be interesting as a potential Mars analog.

Deinococcus fonticola sp. nov., isolated from a radioactive thermal spring in Hungary.

A Gram-stain-negative, aerobic, non-motile and coccus-shaped bacterium, designated strain FeSDHB5-19T, was isolated from a biofilm sample collected from a radioactive thermal spring (Budapest, Hungary), after exposure to 5 kGy gamma radiation. A polyphasic approach was used to study the taxonomic properties of strain FeSDHB5-19T, which had highest 16S rRNA gene sequence similarity to Deinococcus antarcticus G3-6-20T (96.5 %). The 16S rRNA gene sequence similarity to type strains of other Deinococcus species were 93.0 % or lower. The DNA G+C content of the draft genome sequence, consisting of 3.9 Mb, was 63.9 mol%. Strain FeSHDB5-19T was found to grow at temperatures of 10-32 °C (optimum, 28 °C) and pH 5-10 (pH 6.5-7.5) and tolerated up to 1.5 % NaCl (w/v) with optimum growth at 0-0.5 % NaCl. The predominant fatty acids (>10 %) were C16 : 0 and C16 : 1ω7c. The cell-wall peptidoglycan type was A3ß l-Orn-Gly1-2. The whole-cell sugars were glucose and low amounts of galactose. Strain FeSDHB5-19T possessed MK-8 as the predominant respiratory quinone, typical of the genus Deinococcus. The polar lipid profile contained unidentified phosphoglycolipids and unidentified glycolipids. The isolate was found to be highly resistant to gamma (D10<8 kGy) and UV (D10~800 J m-2) radiation. According to its genotypic, phenotypic and chemotaxonomic characteristics, strain FeSDHB5-19T represents a novel species in the genus Deinococcus, for which the name Deinococcusfonticola sp. nov. is proposed. The type strain is FeSDHB5-19T (=NCAIM B.02639T=DSM 106917T).

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.

Biofilm forming bacteria and archaea in thermal karst springs of Gellért Hill discharge area (Hungary).

The Buda Thermal Karst System (BTKS) is an extensive active hypogenic cave system located beneath the residential area of the Hungarian capital. At the river Danube, several thermal springs discharge forming spring caves. To reveal and compare the morphological structure and prokaryotic diversity of reddish-brown biofilms developed on the carbonate rock surfaces of the springs, scanning electron microscopy (SEM), and molecular cloning were applied. Microbial networks formed by filamentous bacteria and other cells with mineral crystals embedded in extracellular polymeric substances were observed in the SEM images. Biofilms were dominated by prokaryotes belonging to phyla Proteobacteria, Chloroflexi and Nitrospirae (Bacteria) and Thaumarchaeota (Archaea) but their abundance showed differences according to the type of the host rock, geographic distance, and different water exchange. In addition, representatives of phyla Acidobacteria, Actinobacteria, Caldithrix, Cyanobacteria, Firmicutes Gemmatimonadetes, and several candidate divisions of Bacteria as well as Crenarchaeota and Euryarchaeota were detected in sample-dependent higher abundance. The results indicate that thermophilic, anaerobic sulfur-, sulfate-, nitrate-, and iron(III)-reducing chemoorganotrophic as well as sulfur-, ammonia-, and nitrite-oxidizing chemolithotrophic prokaryotes can interact in the studied biofilms adapted to the unique and extreme circumstances (e.g., aphotic and nearly anoxic conditions, oligotrophy, and radionuclide accumulation) in the thermal karst springs.

Deinococcus budaensis sp. nov., a mesophilic species isolated from a biofilm sample of a hydrothermal spring cave.

Following the exposure of a biofilm sample from a hydrothermal spring cave (Gellért Hill, Budapest, Hungary) to gamma radiation, a strain designated FeSTC15-38T was isolated and studied by polyphasic taxonomic methods. The spherical-shaped cells stained Gram-negative, and were aerobic and non-motile. The pH range for growth was pH 6.0-9.0, with an optimum at pH 7.0. The temperature range for growth was 20-37 °C, with an optimum at 28 °C. Phylogenetic analysis based on the 16S rRNA gene sequence of the isolate indicated that the organism belongs to the genus Deinococcus. The highest sequence similarities appeared with Deinococcus hopiensis KR-140T (94.1 %), Deinococcus aquaticus PB314T (93.3 %) and Deinococcus aerophilus 5516T-11T (92.7 %). The DNA G+C content of the novel strain was 68.2 mol%. The predominant fatty acids (>10 %) were iso-C16 : 0 and C16 : 1ω7c, and the cell-wall peptidoglycan type was A3ß l-Orn-Gly2-3, corroborating the assignment of the strain to the genus Deinococcus. Strain FeSTC15-38T contained MK-8 as the major menaquinone and several unidentified phospholipids, glycolipids and phosphoglycolipids. Resistance to gamma radiation (D10) of strain FeSTC15-38T was <3.0 kGy. According to phenotypic and genotypic data, strain FeSTC15-38T represents a novel species for which the name Deinococcus budaensis sp. nov. is proposed. The type strain is FeSTC15-38T (=NCAIM B.02630T=DSM 101791T).

Diversity of extremophilic bacteria in the sediment of high-altitude lakes located in the mountain desert of Ojos del Salado volcano, Dry-Andes.

Ojos del Salado, the highest volcano on Earth is surrounded by a special mountain desert with extreme aridity, great daily temperature range, intense solar radiation, and permafrost from 5000 meters above sea level. Several saline lakes and permafrost derived high-altitude lakes can be found in this area, often surrounded by fumaroles and hot springs. The aim of this study was to gain information about the bacterial communities inhabiting the sediment of high-altitude lakes of the Ojos del Salado region located between 3770 and 6500 m. Altogether 11 sediment samples from 4 different altitudes were examined with 16S rRNA gene based denaturing gradient gel electrophoresis and clone libraries. Members of 17 phyla or candidate divisions were detected with the dominance of Proteobacteria, Acidobacteria, Actinobacteria and Bacteroidetes. The bacterial community composition was determined mainly by the altitude of the sampling sites; nevertheless, the extreme aridity and the active volcanism had a strong influence on it. Most of the sequences showed the highest relation to bacterial species or uncultured clones from similar extreme environments.

Thermophilic prokaryotic communities inhabiting the biofilm and well water of a thermal karst system located in Budapest (Hungary).

In this study, scanning electron microscopy (SEM) and 16S rRNA gene-based phylogenetic approach were applied to reveal the morphological structure and genetic diversity of thermophilic prokaryotic communities of a thermal karst well located in Budapest (Hungary). Bacterial and archaeal diversity of the well water (73.7 °C) and the biofilm developed on the inner surface of an outflow pipeline of the well were studied by molecular cloning method. According to the SEM images calcium carbonate minerals serve as a surface for colonization of bacterial aggregates. The vast majority of the bacterial and archaeal clones showed the highest sequence similarities to chemolithoautotrophic species. The bacterial clone libraries were dominated by sulfur oxidizer Thiobacillus (Betaproteobacteria) in the water and Sulfurihydrogenibium (Aquificae) in the biofilm. A relatively high proportion of molecular clones represented genera Thermus and Bellilinea in the biofilm library. The most abundant phylotypes both in water and biofilm archaeal clone libraries were closely related to thermophilic ammonia oxidizer Nitrosocaldus and Nitrososphaera but phylotypes belonging to methanogens were also detected. The results show that in addition to the bacterial sulfur and hydrogen oxidation, mainly archaeal ammonia oxidation may play a decisive role in the studied thermal karst system.

Diversity and morphological structure of bacterial communities inhabiting the Diana-Hygieia Thermal Spring (Budapest, Hungary).

The Buda Thermal Karst System is an active hypogenic karst area that offers possibility for the analysis of biogenic cave formation. The aim of the present study was to gain information about morphological structure and genetic diversity of bacterial communities inhabiting the Diana-Hygieia Thermal Spring (DHTS). Using scanning electron microscopy, metal accumulating and unusual reticulated filaments were detected in large numbers in the DHTS biofilm samples. The phyla Actinobacteria, Firmicutes and Proteobacteria were represented by both bacterial strains and molecular clones but phyla Acidobacteria, Chlorobi, Chlorofexi, Gemmatimonadetes, Nitrospirae and Thermotogae only by molecular clones which showed the highest similarity to uncultured clone sequences originating from different environmental sources. The biofilm bacterial community proved to be somewhat more diverse than that of the water sample and the distribution of the dominant bacterial clones was different between biofilm and water samples. The majority of biofilm clones was affiliated with Deltaproteobacteria and Nitrospirae while the largest group of water clones was related to Betaproteobacteria. Considering the metabolic properties of known species related to the strains and molecular clones from DHTS, it can be assumed that these bacterial communities may participate in the local sulphur and iron cycles, and contribute to biogenic cave formation.