Aquatic Thermal Reservoirs of Microbial Life in a Remote and Extreme High Andean Hydrothermal System.

Hydrothermal systems are ideal to understand how microbial communities cope with challenging conditions. Lirima, our study site, is a polyextreme, high-altitude, hydrothermal ecosystem located in the Chilean Andean highlands. Herein, we analyze the benthic communities of three nearby springs in a gradient of temperature (42-72 °C represented by stations P42, P53, and P72) and pH, and we characterize their microbial diversity by using bacteria 16S rRNA (V4) gene metabarcoding and 16S rRNA gene clone libraries (bacteria and archaea). Bacterial clone libraries of P42 and P53 springs showed that the community composition was mainly represented by phototrophic bacteria (Chlorobia, 3%, Cyanobacteria 3%, at P42; Chlorobia 5%, and Chloroflexi 5% at P53), Firmicutes (32% at P42 and 43% at P53) and Gammaproteobacteria (13% at P42 and 29% at P53). Furthermore, bacterial communities that were analyzed by 16S rRNA gene metabarcoding were characterized by an overall predominance of Chloroflexi in springs with lower temperatures (33% at P42), followed by Firmicutes in hotter springs (50% at P72). The archaeal diversity of P42 and P53 were represented by taxa belonging to Crenarchaeota, Diapherotrites, Nanoarchaeota, Hadesarchaeota, Thaumarchaeota, and Euryarchaeota. The microbial diversity of the Lirima hydrothermal system is represented by groups from deep branches of the tree of life, suggesting this ecosystem as a reservoir of primitive life and a key system to study the processes that shaped the evolution of the biosphere.

Polyphenols obtained from Didymosphenia geminata (Lyngbye) Schmith altered the viability and proliferation of salmonids cells lines SHK-1 and CHSE-214.

Didymosphenia geminata (Lyngbye) Schmidt, also referred to as Didymo, is an invasive diatom that forms nuisance mats. Since it was first reported in our country in approximately 2010, Didymo has expanded and colonized different rivers in the Zona Austral region of Chile. Its biology and effects on ecosystems are still being studied because Didymo is an invasive algal mat that forms in a range of systems from oligotrophic austral rivers to more subtropical systems. We aimed to evaluate the viability of two salmonid cell lines, CHSE-214 and SHK-1 (somatic and embryonic cell lines, respectively), in dilutions of river water alone and in river water contaminated with Didymo or polyphenols extracted from Didymo under controlled conditions. We developed an artificial river system (2 aquariums/replicate) from five different rivers from the central area (Bio-Bio) and Patagonia area (Futaleufú) of Chile to maintain Didymo in the benthic phase. The Didymo populations were maintained for six months in the water from the rivers, after which samples were obtained. Following the extraction of polyphenols from the Didymo samples maintained in the artificial rivers, toxicity assays (10 assays) were performed to determine cell viability. Our results indicated that the CHSE-214 cells were highly sensitive to increasing concentrations of Didymo extracts. We observed a 50% reduction in cell viability after 24 h of exposure to a 0.01 V/V dilution, and this treatment further reduced the proliferative capacity by 70% after 120 h. The SHK-1 cells were less responsive, showing only a 20% decrease in viability at 24 h and a lower cell proliferation rate (45%) after 120 h, which remained higher than that of the CHSE-214 cells. We conclude that certain cell types are sensitive to Didymo in rivers, suggesting that there are chronic effects on several aquatic species following exposure to these diatom substances. These effects should be further studied using this laboratory model to understand the full impact of Didymo on river ecosystems.

Characterization and Salt Response in Recurrent Halotolerant Exiguobacterium sp. SH31 Isolated From Sediments of Salar de Huasco, Chilean Altiplano.

Poly-extremophiles microorganisms have the capacity to inhabit hostile environments and can survive several adverse conditions that include as variations in temperature, pH, and salinity, high levels UV light and atmospheric pressure, and even the presence of toxic compounds and the formation of reactive oxygen species (ROS). A halotolerant Exiguobacterium strain was isolated from Salar de Huasco (Chilean Altiplano), a well-known shallow lake area with variable salinity levels, little human intervention, and extreme environmental conditions, which makes it ideal for the study of resistant mechanisms and the evolution of adaptations. This bacterial genus has not been extensively studied, although its cosmopolitan location indicates that it has high levels of plasticity and adaptive capacity. However, to date, there are no studies regarding the tolerance and resistance to salinity and osmotic pressure. We set out to characterize the Exiguobacterium sp. SH31 strain and describe its phenotypical and genotypical response to osmotic stress. In this context, as a first step to characterize the response to the SH31 strain to salinity and to establish the bases for a molecular study, we proposed to compare its response under three salt conditions (0, 25, and 50 g/l NaCl). Using different physiology, genomic, and transcriptomic approaches, we determined that the bacterium is able to grow properly in a NaCl concentration of up to 50 g/l; however, the best growth rate was observed at 25 g/l. Although the presence of flagella is not affected by salinity, motility was diminished at 25 g/l NaCl and abolished at 50 g/l. Biofilm formation was induced proportionally with increases in salinity, which was expected. These phenotypic results correlated with the expression of related genes: fliG and fliS Motility); opuBA and putP (transport); glnA, proC, gltA, and gbsA (compatible solutes); ywqC, bdlA, luxS y pgaC (biofilm and stress response); and therefore, we conclude that this strain effectively modifies gene expression and physiology in a differential manner when faced with different concentrations of NaCl and these modifications aid survival.

The activity of nitrifying microorganisms in a high-altitude Andean wetland.

High-altitude wetland holds freshwater springs, evaporitic ponds and lagoon with variable salinity and nutrients, potentially influencing the ecology of nitrifying communities. In this study, nitrifying microorganisms in Salar de Huasco (Chile) were surveyed to determine bacterial and archaeal contribution to ammonium (AO), nitrite oxidation (NO), ammonium uptake (AU) during wet and dry seasons. The activity signals from these groups were assessed by specific amoA-qPCR transcription, 15N tracer studies and addition of group specific inhibitor experiments for nitrifying microorganisms (N1-guanyl-1, 7-diaminoheptane [GC7]-archaeal specific and allylthiourea [ATU]-bacterial specific). Nitrifying communities, i.e. Nitrosopumilus, Nitrosospira, Nitrosomonas, Kuenenia and Nitrospira, were more frequent (∼0.25% of 16S rRNA sequences) at low salinity sites. Bacterial amoA-qPCR transcripts also increased at low salinity and along in situ ammonium increase observed between wet/dry seasons. Nutrient changes through time and 15N tracer experiments results showed that AO and NO were detected and peaked mainly at low salinity-high ammonium sites (<37 000 µS cm-1 and >0.3 µM), whereas AU was predominant at evaporitic sites. Our results indicate that salinity and ammonium affect the nitrifying communities that are potentially more active at low-salinity sites but persistent at saltier evaporitic areas of the wetland when ammonium is available.

Bacterial Active Community Cycling in Response to Solar Radiation and Their Influence on Nutrient Changes in a High-Altitude Wetland.

Microbial communities inhabiting high-altitude spring ecosystems are subjected to extreme changes in solar irradiance and temperature throughout the diel cycle. Here, using 16S rRNA gene tag pyrosequencing (cDNA) we determined the composition of actively transcribing bacteria from spring waters experimentally exposed through the day (morning, noon, and afternoon) to variable levels of solar radiation and light quality, and evaluated their influence on nutrient recycling. Solar irradiance, temperature, and changes in nutrient dynamics were associated with changes in the active bacterial community structure, predominantly by Cyanobacteria, Verrucomicrobia, Proteobacteria, and 35 other Phyla, including the recently described Candidate Phyla Radiation (e.g., Parcubacteria, Gracilibacteria, OP3, TM6, SR1). Diversity increased at noon, when the highest irradiances were measured (3.3-3.9 H', 1125 W m-2) compared to morning and afternoon (0.6-2.8 H'). This shift was associated with a decrease in the contribution to pyrolibraries by Cyanobacteria and an increase of Proteobacteria and other initially low frequently and rare bacteria phyla (< 0.5%) in the pyrolibraries. A potential increase in the activity of Cyanobacteria and other phototrophic groups, e.g., Rhodobacterales, was observed and associated with UVR, suggesting the presence of photo-activated repair mechanisms to resist high levels of solar radiation. In addition, the percentage contribution of cyanobacterial sequences in the afternoon was similar to those recorded in the morning. The shifts in the contribution by Cyanobacteria also influenced the rate of change in nitrate, nitrite, and phosphate, highlighted by a high level of nitrate accumulation during hours of high radiation and temperature associated with nitrifying bacteria activity. We did not detect ammonia or nitrite oxidizing bacteria in situ, but both functional groups (Nitrosomona and Nitrospira) appeared mainly in pyrolibraries generated from dark incubations. In total, our results reveal that both the structure and the diversity of the active bacteria community was extremely dynamic through the day, and showed marked shifts in composition that influenced nutrient recycling, highlighting how abiotic variation affects potential ecosystem functioning.

Microbial Activity Response to Solar Radiation across Contrasting Environmental Conditions in Salar de Huasco, Northern Chilean Altiplano.

In high altitude environments, extreme levels of solar radiation and important differences of ionic concentrations over narrow spatial scales may modulate microbial activity. In Salar de Huasco, a high-altitude wetland in the Andean mountains, the high diversity of microbial communities has been characterized and associated with strong environmental variability. Communities that differed in light history and environmental conditions, such as nutrient concentrations and salinity from different spatial locations, were assessed for bacterial secondary production (BSP, 3H-leucine incorporation) response from short-term exposures to solar radiation. We sampled during austral spring seven stations categorized as: (a) source stations, with recently emerged groundwater (no-previous solar exposure); (b) stream running water stations; (c) stations connected to source waters but far downstream from source points; and (d) isolated ponds disconnected from ground sources or streams with a longer isolation and solar exposure history. Very high values of 0.25 µE m-2 s-1, 72 W m-2 and 12 W m-2 were measured for PAR, UVA, and UVB incident solar radiation, respectively. The environmental factors measured formed two groups of stations reflected by principal component analyses (near to groundwater sources and isolated systems) where isolated ponds had the highest BSP and microbial abundance (35 microalgae taxa, picoeukaryotes, nanoflagellates, and bacteria) plus higher salinities and PO43- concentrations. BSP short-term response (4 h) to solar radiation was measured by 3H-leucine incorporation under four different solar conditions: full sun, no UVB, PAR, and dark. Microbial communities established in waters with the longest surface exposure (e.g., isolated ponds) had the lowest BSP response to solar radiation treatments, and thus were likely best adapted to solar radiation exposure contrary to ground source waters. These results support our light history (solar exposure) hypothesis where the more isolated the community is from ground water sources, the better adapted it is to solar radiation. We suggest that factors other than solar radiation (e.g., salinity, PO43-, NO3-) are also important in determining microbial productivity in heterogeneous environments such as the Salar de Huasco.