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Freshwater network ecosystems consist of interconnected lotic and lentic habitats within the same catchment area. The hydrologic and biogeochemical changes that occur during runoff in lotic systems significantly affect the succession of microbial communities in these systems [1]. Similarly, surface runoff from lotic systems has a significant impact on the environmental conditions and diversity of microbial communities in lentic systems [1]. Therefore, hydrology, system location, and local environmental conditions, along with community assembly processes [2], are the most important factors affecting microbial community formation in an interconnected freshwater network ecosystem. Using Plitvice Lakes as an example, we studied changes in environmental conditions and microbial communities (bacteria and fungi) that occur with runoff. Water samples from tributaries, interlake streams, and the Korana River were collected in three different seasons (spring, summer and autumn). Physio-chemical parameters were measured in situ (temperature and dissolved oxygen concentration) and subsequently from filtered water samples (DOC, Ca2+, NO3 - and Clconcentrations). The microbial communities of the water samples were characterized by amplicon sequencing of bacterial 16S rRNA and fungal ITS2 genes. All statistical analysis was performed using R software (R Core Team, 2021). The obtained results showed differences among three stream types and three different seasons, both in environmental conditions and diversity of microbial communities. They also showed seasonal differences in the bacterial community assembly processes, while fungal community assembly was not dominated by any single process. Despite the microbial diversity among different stream types and seasons, we proved the stability of the Plitvice lakes by determining the core microbiome, which accounted for the majority of the microbial communities in all stream types and seasons.

Background: Microbial communities in natural geothermal springs represent an excellent example of microorganisms inhabiting extreme environments. Geothermal ecosystems present poorly investigated habitats in Croatia. The biodiversity in such environments depends on temperature and pH, but also different nutrient concentrations. Nitrogen and sulfur, in particular, are often crucial factors in determining microbial community structure. Objectives: The primary objective was to analyze the diversity of thermotolerant and thermophilic microorganisms in the geothermal springs of Daruvarske Toplice through different seasons, and to identify abiotic factors that shape the microbial community composition. Methods: Two hot springs (Antunovo vrelo and Ivanovo vrelo) and one well (Marijino vrelo) located in Daruvarske Toplice were selected for sample collection during three seasons. Physical parameters were measured in situ, as well as H2S concentrations. Other parameters were analyzed in the laboratory immediately after field sampling. After water filtering, DNA extraction, and PCR amplification, we analyzed the 16S rRNA gene diversity obtained using high throughput sequencing techniques. All statistical analyses were performed in the R environment. Results: Antunovo vrelo had the most stable community structure during all three sampling seasons and was dominated by microorganisms affiliated with the phyla Crenarchaeota, Proteobacteria, and Nitrospirota. In contrast, most bacterial ASVs in Ivanovo vrelo were affiliated to Bateroidota and Proteobacteria, while the bacterial community at Marijino vrelo was most diverse, with sequences of the phylum Proteobacteria dominating during all three seasons. On the genus level, the most abundant were ammonia-oxidizing archaea of the genera Candidatus Nitrosotenuis and Candidatus Nitrososphaera, as well as nitrite-oxidizing Nitrospira and sulfur-oxidizing Sulfuricurvum. Our results indicated that inorganic nitrogen and sulfur compound concentrations, along with correlating temperature and pH were the main drivers for microbial community composition.

Defined clines in anchialine ecosystems act as selection barriers that influence species distributions. Along with light resources and nutrient limitations, cline areas are recognized promoters of microbial evolution in these complex habitats. To reveal microbial diversity and its potential ecological role in biogeochemical cycles along a salinity gradient, we sampled different water masses and analyzed microbial diversity using 16S rRNA amplicon sequencing along with physicochemical parameters during two seasons (spring and summer). Our results showed that a well-defined halocline maintained the stratification of the water column in both seasons, with a slight shift in the position of the halocline. The stabile halocline promoted the accumulation of the sulfide in anoxic (euxinic) water layers situated between the oxic surface layer (characterized by lower salinity and impacted by precipitation) and the seawater layer (characterized by oxygen saturated seawater of high salinity entering the cave). The oxic and anoxic layers exhibited notable differences in microbial community composition at all depths. Interestingly, the oxygenic and brackish depths were predominantly inhabited by bacterial lineages, while the saline and anoxic depths were dominated by archeal lineages. In terms of average relative abundance, Nanoarchaeota were the most abundant archaeal lineage, accounting for more than 40% of ASVs in the anoxic depths and exhibiting high diversity in 16S rRNA gene sequences. During summer, bacterial lineages of Campylobacterota co-occurred with Nanoarchaeota, with the highest abundance of ~50% in the photic surface layer.

Microbial communities in geothermal waters are composed of various, phylogenetically diverse microorganisms adapted to life under extreme conditions such as temperature and pH. Many previous studies have established that the abundance and diversity of microorganisms are usually reduced when compared to other aquatic habitats [1]. The primary objective was to identify, enrich and characterize nitrifying microorganisms (ammonia and nitrite oxidizing, and comammox microorganisms) from Croatian geothermal springs and wells. We used amoA gene quantitative PCR to quantify ammonia oxidizer communities in geothermal samples, and thereafter select localities for nitrifier enrichment experiments. One spring (Antunovo vrelo) and two wells (TS Stubicke and Krecaves) have been selected for sample collection, and enrichment experiments. AOA of the genera Candidatus Nitrosotenuis and Candidatus Nitrososphaera as well as comammox Nitrospira were detected to occur at high abundance in the selected samples, based on 16S rRNA gene amplicon sequencing and amoA gene qPCR data. However, enrichment procedures showed only limited success in respect to AOA enrichment, while comammox Nitrospira did seem to prevail and grow in a fraction of enrichment cultures. Interestingly, ammonia oxidation also occurred in enrichments in which amoA gene qPCR consistently yielded negative results, indicating a enrichment of novel, hitherto unknown ammonia oxidizers. Further exploration of the enrichment cultures by metagenome sequencing is underway.