Common Presence of Phototrophic Gemmatimonadota in Temperate Freshwater Lakes

Members of the bacterial phylum Gemmatimonadota are ubiquitous in most natural environments and represent one of the top 10 most abundant bacterial phyla in soil. Sequences affiliated with Gemmatimonadota were also reported from diverse aquatic habitats; however, it remains unknown whether they are native organisms or represent bacteria passively transported from sediment or soil. To address this question, we analyzed metagenomes constructed from five freshwater lakes in central Europe. Based on the 16S rRNA gene frequency, Gemmatimonadota represented from 0.02 to 0.6% of all bacteria in the epilimnion and between 0.1 and 1% in the hypolimnion. These proportions were independently confirmed using catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). Some cells in the epilimnion were attached to diatoms (Fragilaria sp.) or cyanobacteria (Microcystis sp.), which suggests a close association with phytoplankton. In addition, we reconstructed 45 metagenome-assembled genomes (MAGs) related to Gemmatimonadota. They represent several novel lineages, which persist in the studied lakes during the seasons. Three lineages contained photosynthesis gene clusters. One of these lineages was related to Gemmatimonas phototrophica and represented the majority of Gemmatimonadota retrieved from the lakes’ epilimnion. The other two lineages came from hypolimnion and probably represented novel photoheterotrophic genera. None of these phototrophic MAGs contained genes for carbon fixation. Since most of the identified MAGs were present during the whole year and cells associated with phytoplankton were observed, we conclude that they represent truly limnic Gemmatimonadota distinct from the previously described species isolated from soils or sediments

A freshwater radiation of diplonemids

Diplonemids are considered marine protists and have been reported among the most abundant and diverse eukaryotes in the world oceans. Recently we detected the presence of freshwater diplonemids in Lake Biwa, Japan. However, their distribution and abundances in freshwater ecosystems remain unknown. We assessed abundance and diversity of diplonemids from several geographically distant deep freshwater lakes of the world by amplicon-sequencing, shotgun metagenomics and CARD-FISH. We found diplonemids in all the studied lakes, albeit with low abundances and diversity. We assembled long 18S rRNA sequences from freshwater diplonemids and showed that they form a new lineage distinct from the diverse marine clades. Freshwater diplonemids are a sister-group to marine isolates from coastal and bay areas, suggesting a recent habitat transition from marine to freshwater habitats. Images of CARD-FISH targeted freshwater diplonemids suggest they feed on bacteria. Our analyses of 18S rRNA sequences retrieved from single cell genomes of marine diplonemids shows they encode multiple rRNA copies that may be very divergent from each other, suggesting that marine diplonemid abundance and diversity both have been overestimated. These results have wider implications on assessing eukaryotic abundances in natural habitats by using amplicon-sequencing alone

Biodegradation of microcystins during gravity-driven membrane (GDM) ultrafiltration

Gravity-driven membrane (GDM) ultrafiltration systems require little maintenance: they operate without electricity at ultra-low pressure in dead-end mode and without control of the biofilm formation. These systems are already in use for water purification in some regions of the world where adequate treatment and distribution of drinking water is not readily available. However, many water bodies worldwide exhibit harmful blooms of cyanobacteria that severely lower the water quality due to the production of toxic microcystins (MCs). We studied the performance of a GDM system during an artificial Microcystis aeruginosa bloom in lake water and its simulated collapse (i.e., the massive release of microcystins) over a period of 21 days. Presence of live or destroyed cyanobacterial cells in the feed water decreased the permeate flux in the Microcystis treatments considerably. At the same time, the microbial biofilms on the filter membranes could successfully reduce the amount of microcystins in the filtrate below the critical threshold concentration of 1 µg L(-1) MC for human consumption in three out of four replicates after 15 days. We found pronounced differences in the composition of bacterial communities of the biofilms on the filter membranes. Bacterial genera that could be related to microcystin degradation substantially enriched in the biofilms amended with microcystin-containing cyanobacteria. In addition to bacteria previously characterized as microcystin degraders, members of other bacterial clades potentially involved in MC degradation could be identified

High-resolution metagenomic reconstruction of the freshwater spring bloom

Background The phytoplankton spring bloom in freshwater habitats is a complex, recurring, and dynamic ecological spectacle that unfolds at multiple biological scales. Although enormous taxonomic shifts in microbial assemblages during and after the bloom have been reported, genomic information on the microbial community of the spring bloom remains scarce. Results We performed a high-resolution spatio-temporal sampling of the spring bloom in a freshwater reservoir and describe a multitude of previously unknown taxa using metagenome-assembled genomes of eukaryotes, prokaryotes, and viruses in combination with a broad array of methodologies. The recovered genomes reveal multiple distributional dynamics for several bacterial groups with progressively increasing stratification. Analyses of abundances of metagenome-assembled genomes in concert with CARD-FISH revealed remarkably similar in situ doubling time estimates for dominant genome-streamlined microbial lineages. Discordance between quantitations of cryptophytes arising from sequence data and microscopic identification suggested the presence of hidden, yet extremely abundant aplastidic cryptophytes that were confirmed by CARD-FISH analyses. Aplastidic cryptophytes are prevalent throughout the water column but have never been considered in prior models of plankton dynamics. We also recovered the first metagenomic-assembled genomes of freshwater protists (a diatom and a haptophyte) along with thousands of giant viral genomic contigs, some of which appeared similar to viruses infecting haptophytes but owing to lack of known representatives, most remained without any indication of their hosts. The contrasting distribution of giant viruses that are present in the entire water column to that of parasitic perkinsids residing largely in deeper waters allows us to propose giant viruses as the biological agents of top-down control and bloom collapse, likely in combination with bottom-up factors like a nutrient limitation. Conclusion We reconstructed thousands of genomes of microbes and viruses from a freshwater spring bloom and show that such large-scale genome recovery allows tracking of planktonic succession in great detail. However, integration of metagenomic information with other methodologies (e.g., microscopy, CARD-FISH) remains critical to reveal diverse phenomena (e.g., distributional patterns, in situ doubling times) and novel participants (e.g., aplastidic cryptophytes) and to further refine existing ecological models (e.g., factors affecting bloom collapse). This work provides a genomic foundation for future approaches towards a fine-scale characterization of the organisms in relation to the rapidly changing environment during the course of the freshwater spring bloom

Different components of interconnected karst environment select for highly distinct microbial communities

Karst environments, formed by dissolution of rocks, present a unique opportunity to study groundwater and surface water as a single dynamic system. We aimed to explore the hydrological connections and identify shared microbiome between different components of this complex landscape (terrestrial, surface-subsurface freshwater networks and two different lake strata) on example of karst landscape at north shore of Lake Thun (CH).52 samples from soil and 87 from the freshwater network were collected during low hydrological conditions. Additionally, 43 samples from 13 different lakes across Europe were included in the analysis. A 16S rRNA gene amplicon analysis revealed highly homogenous lake communities dominated by typical lake microbes represented by members of Alphaproteobacteria, Gammaproteobacteria and Actinobacteria. On the contrary, communities in soils and surface and subsurface water were observed to be highly heterogenous within the karst landscapes. Soil samples built two robust groups that corresponded to alkaline and acidic pH values associated with two types of bedrocks. Alkaline soils had higher abundance of Gammaproteobacteria and Alphaproteobacteria, while acidic soils were dominated by Acidobacteria. Alkaline cluster was better connected with aquatic environments and shared genotypes from the families Nitrosomonadaceae and Nitrospiraceae with cave sediments and lake hypolimnia, that implied ammonia and nitrite oxidation as important chemotrophic processes in light depleted environments. Rhodoferax, Limnohabitans and Sediminibacterium were shared within subsurface network and lakes. Single Rhodoferax genotype was detected in all aquatic environments. Additionally, we observed that lineages from the families Pirellulaceae and Gemmataceae (phylum Planctomycetota) were alternatively distributed in both soil clusters and lakes. A similar pattern was observed in subsurface aquatic components and deeper lake strata within family Methylophilaceae, where Methylotenera was extensively replaced by Methylopumilus. This indicates that despite high hydrological connectedness between different karst landscape components, deterministic selective factors seem to prevail in the community assemblies

Groundwater ecosystems in changing times

Waterbodies worldwide undergo changes and this influences the ecosystems with the resident communities. Groundwater is no exception. However, few studies have so far focused on how the combined effects from catchment management and a changing climate impact the communities living in the groundwater. In 2019-2021 in southwestern Czech Republic, we sampled fauna and microorganisms in 37 wells that had been monitored by CHMI for up to 40 years and that varied in the trends in chemical and physical properties. The wells tapped the shallow quaternary and deeper aquifers of seven major hydrogeological zones. As expected, more quaternary than deeper wells showed - stronger - trends in physical and chemical properties of the groundwater. The chemical property changing significantly in the highest proportion of wells, i.e. 100% of the wells sampled for fauna, was silica. The trend of silica was increasing in all cases. Faunal numbers were lowest in the wells with the highest silica values. This may be an indirect effect of the interaction between changing agricultural practices and climate change, and may have implications for ecosystem functions, such as the use of groundwater for drinking water production

Bacterial and eukaryotic small-subunit amplicon data do not provide a quantitative picture of microbial communities, but they are reliable in the context of ecological interpretations

High-throughput sequencing (HTS) of gene amplicons is a preferred method of assessing microbial community composition, because it rapidly provides information from a large number of samples at high taxonomic resolution and low costs. However, mock community studies show that HTS data poorly reflect the actual relative abundances of individual phylotypes, casting doubt on the reliability of subsequent statistical analysis and data interpretation. We investigated how accurately HTS data reflect the variability of bacterial and eukaryotic community composition and their relationship with environmental factors in natural samples. For this, we compared results of HTS from three independent aquatic time series (n = 883) with those from an established, quantitative microscopic method (catalyzed reporter deposition-fluorescence in situ hybridization [CARD-FISH]). Relative abundances obtained by CARD-FISH and HTS disagreed for most bacterial and eukaryotic phylotypes. Nevertheless, the two methods identified the same environmental drivers to shape bacterial and eukaryotic communities. Our results show that amplicon data do provide reliable information for their ecological interpretations. Yet, when studying specific phylogenetic groups, it is advisable to combine HTS with quantification using microscopy and/or the addition of internal standards

Ecological Traits of the Algae-Bearing Tetrahymena utriculariae (Ciliophora) from Traps of the Aquatic Carnivorous Plant Utricularia reflexa

Trap fluid of aquatic carnivorous plants of the genus Utricularia hosts specific microbiomes consisting of commensal pro- and eukaryotes of largely unknown ecology. We examined the characteristics and dynamics of bacteria and the three dominant eukaryotes, i.e. the algae-bearing ciliate Tetrahymena utriculariae (Ciliophora), a green flagellate Euglena agilis (Euglenophyta), and the alga Scenedesmus alternans (Chlorophyta), associated with the traps of Utricularia reflexa. Our study focused on ecological traits and life strategies of the highly abundant ciliate whose biomass by far exceeds that of other eukaryotes and bacteria independent of the trap age. The ciliate was the only bacterivore in the traps, driving rapid turnover of bacterial standing stock. However, given the large size of the ciliate and the cell-specific uptake rates of bacteria we estimated that bacterivory alone would likely be insufficient to support its apparent rapid growth in traps. We suggest that mixotrophy based on algal symbionts contributes significantly to the diet and survival strategy of the ciliate in the extreme (anaerobic, low pH) trap-fluid environment. We propose a revised concept of major microbial interactions in the trap fluid where ciliate bacterivory plays a central role in regeneration of nutrients bound in rapidly growing bacterial biomass

Distribution and ecological preferences of the freshwater lineage LimA (genus Limnohabitans) revealed by a new double hybridization approach

The ecological relevance and factors shaping dynamics of Limnohabitans sp. have been largely studied by fluorescence in situ hybridization with a 16S rRNA probe targeting the R-BT group (lineages LimBCDE), but not lineage LimA. Consequently, ecology and distribution of LimA remained unknown. We developed a double hybridization strategy using a novel 23S rRNA probe specifically targeting LimA and LimE that in combination with the existing R-BT probe can discriminate LimA populations. This technique was applied for more than 1000 samples from 46 freshwater sites including long-term data sets from oligo-mesotrophic Lake Zurich, CH and meso-eutrophic Římov reservoir, CZ. LimA was ubiquitously distributed and highly abundant. Observed ecological preferences of LimA in Lake Zurich were in general similar to already reported for Limnohabitans with highest numbers in surface waters during growing seasons. Three times higher densities of LimA were detected in Římov reservoir, where they were significantly more abundant at the riverine zone especially after flood events that introduced fresh terrestrial DOM (dissolved organic matter). Moreover, statistical analyses of biological and physicochemical parameters obtained from small dynamic water bodies confirmed a correspondence between LimA and allochthonous DOM, in opposite to R-BT that was more related to algal primary production