Polyextremophilic Chitinolytic Activity by a Marine Strain (IG119) of Clonostachys rosea.

The investigation for novel unique extremozymes is a valuable business for which the marine environment has been overlooked. The marine fungus Clonostachys rosea IG119 was tested for growth and chitinolytic enzyme production at different combinations of salinity and pH using response surface methodology. RSM modelling predicted best growth in-between pH 3.0 and 9.0 and at salinity of 0-40‱, and maximum enzyme activity (411.137 IU/L) at pH 6.4 and salinity 0‱; however, quite high production (>390 IU/L) was still predicted at pH 4.5-8.5. The highest growth and activity were obtained, respectively, at pH 4.0 and 8.0, in absence of salt. The crude enzyme was tested at different salinities (0-120‱) and pHs (2.0-13.0). The best activity was achieved at pH 4.0, but it was still high (in-between 3.0 and 12.0) at pH 2.0 and 13.0. Salinity did not affect the activity in all tested conditions. Overall, C. rosea IG119 was able to grow and produce chitinolytic enzymes under polyextremophilic conditions, and its crude enzyme solution showed more evident polyextremophilic features. The promising chitinolytic activity of IG119 and the peculiar characteristics of its chitinolytic enzymes could be suitable for several biotechnological applications (i.e., degradation of salty chitin-rich materials and biocontrol of spoiling organisms, possibly solving some relevant environmental issues).

Assessment of the plasmidome of an extremophilic microbial community from the Diamante Lake, Argentina.

Diamante Lake located at 4589 m.a.s.l. in the Andean Puna constitutes an extreme environment. It is exposed to multiple extreme conditions such as an unusually high concentration of arsenic (over 300 mg L-1) and low oxygen pressure. Microorganisms thriving in the lake display specific genotypes that facilitate survival, which include at least a multitude of plasmid-encoded resistance traits. Hence, the genetic information provided by the plasmids essentially contributes to understand adaptation to different stressors. Though plasmids from cultivable organisms have already been analyzed to the sequence level, the impact of the entire plasmid-borne genetic information on such microbial ecosystem is not known. This study aims at assessing the plasmidome from Diamante Lake, which facilitates the identification of potential hosts and prediction of gene functions as well as the ecological impact of mobile genetic elements. The deep-sequencing analysis revealed a large fraction of previously unknown DNA sequences of which the majority encoded putative proteins of unknown function. Remarkably, functions related to the oxidative stress response, DNA repair, as well as arsenic- and antibiotic resistances were annotated. Additionally, all necessary capacities related to plasmid replication, mobilization and maintenance were detected. Sequences characteristic for megaplasmids and other already known plasmid-associated genes were identified as well. The study highlights the potential of the deep-sequencing approach specifically targeting plasmid populations as it allows to evaluate the ecological impact of plasmids from (cultivable and non-cultivable) microorganisms, thereby contributing to the understanding of the distribution of resistance factors within an extremophilic microbial community.

Isolation of ammonia gas-tolerant extremophilic bacteria and their application to the elimination of malodorous gas emitted from outdoor heat-treated toilets.

Ammonia gas-tolerant extremophilic bacteria capable of growing in atmospheres containing up to 4000 ppm of gaseous ammonia were isolated. These bacteria were capable of growing in nutrient broth containing high concentrations of ammonia water, with growth in medium augmented with 0.1-0.2% ammonia exceeding that in medium without ammonia. The minimal inhibitory concentration of ammonia in the medium was 0.5%. The isolated ammonia gas-tolerant bacterium was moderately alkaliphilic, with optimum growth was observed at pH 9. DNA sequence analysis of the 16SrRNA gene revealed that the isolated bacterium was Bacillus lentus. Furthermore, extremophilic bacteria cultured in a 1300 ppm ammonia gas atmosphere on agar medium containing no nitrogen sources were observed to use ammonia gas for growth. These bacteria were identified as Paenibacillus lentus and Bacillus altitudinis based on 16SrRNA gene sequence analysis. The deodorizing effect of ammonia odor by the isolated bacteria immobilized on sawdust was evaluated. The findings showed that forcing ammonia gas through a column containing B. altitudinis immobilized on sawdust reduced the concentration of ammonia gas by 30% compared to columns containing sawdust only. The isolated bacteria immobilized on wood sawdust lost the capacity deodorization after drying, but this function could be restored with increased moisture. The ammonia gas-tolerant extremophilic bacteria immobilized on sawdust show considerable potential for use in ameliorating malodors associated with outdoor heat-treated toilets.

The extremophile Anoxybacillus sp. PC2 isolated from Brazilian semiarid region (Caatinga) produces a thermostable keratinase.

The aim of this study was to select and identify thermophilic bacteria from Caatinga biome (Brazil) able to produce thermoactive keratinases and characterize the keratinase produced by the selected isolate. After enrichment in keratin culture media, an Anoxybacillus caldiproteolyticus PC2 was isolated. This thermotolerant isolate presents a remarkable feature producing a thermostable keratinase at 60°C. The partially purified keratinase, identified as a thermolysin-like peptidase, was active at a pH range of 5.0-10.0 with maximal activity at a temperature range of 50-80°C. The optimal activity was observed at pH 7.0 and 50-60°C. These characteristics are potentially useful for biotechnological purposes such as processing and bioconversion of keratin.

Microbiome and ecology of a hot spring-microbialite system on the Trans-Himalayan Plateau.

Little is known about life in the boron-rich hot springs of Trans-Himalayas. Here, we explore the geomicrobiology of a 4438-m-high spring which emanates ~70 °C-water from a boratic microbialite called Shivlinga. Due to low atmospheric pressure, the vent-water is close to boiling point so can entropically destabilize biomacromolecular systems. Starting from the vent, Shivlinga's geomicrobiology was revealed along the thermal gradients of an outflow-channel and a progressively-drying mineral matrix that has no running water; ecosystem constraints were then considered in relation to those of entropically comparable environments. The spring-water chemistry and sinter mineralogy were dominated by borates, sodium, thiosulfate, sulfate, sulfite, sulfide, bicarbonate, and other macromolecule-stabilizing (kosmotropic) substances. Microbial diversity was high along both of the hydrothermal gradients. Bacteria, Eukarya and Archaea constituted >98%, ~1% and <1% of Shivlinga's microbiome, respectively. Temperature constrained the biodiversity at ~50 °C and ~60 °C, but not below 46 °C. Along each thermal gradient, in the vent-to-apron trajectory, communities were dominated by Aquificae/Deinococcus-Thermus, then Chlorobi/Chloroflexi/Cyanobacteria, and finally Bacteroidetes/Proteobacteria/Firmicutes. Interestingly, sites of >45 °C were inhabited by phylogenetic relatives of taxa for which laboratory growth is not known at >45 °C. Shivlinga's geomicrobiology highlights the possibility that the system's kosmotrope-dominated chemistry mitigates against the biomacromolecule-disordering effects of its thermal water.

Serratia liquefaciens FG3 isolated from a metallophyte plant sheds light on the evolution and mechanisms of adaptive traits in extreme environments.

Serratia liquefaciens strain FG3 (SlFG3), isolated from the flower of Stachytarpheta glabra in the Brazilian ferruginous fields, has distinctive genomic, adaptive, and biotechnological potential. Herein, using a combination of genomics and molecular approaches, we unlocked the evolution of the adaptive traits acquired by S1FG3, which exhibits the second largest chromosome containing the largest conjugative plasmids described for Serratia. Comparative analysis revealed the presence of 18 genomic islands and 311 unique protein families involved in distinct adaptive features. S1FG3 has a diversified repertoire of genes associated with Nonribosomal peptides (NRPs/PKS), a complete and functional cluster related to cellulose synthesis, and an extensive and functional repertoire of oxidative metabolism genes. In addition, S1FG3 possesses a complete pathway related to protocatecuate and chloroaromatic degradation, and a complete repertoire of genes related to DNA repair and protection that includes mechanisms related to UV light tolerance, redox process resistance, and a laterally acquired capacity to protect DNA using phosphorothioation. These findings summarize that SlFG3 is well-adapted to different biotic and abiotic stress situations imposed by extreme conditions associated with ferruginous fields, unlocking the impact of the lateral gene transfer to adjust the genome for extreme environments, and providing insight into the evolution of prokaryotes.

The Intersection of Geology, Geochemistry, and Microbiology in Continental Hydrothermal Systems.

Decompressional boiling of ascending hydrothermal waters and separation into a vapor (gas) and a liquid phase drive extensive variation in the geochemical composition of hot spring waters. Yet little is known of how the process of phase separation influences the distribution of microbial metabolisms in springs. Here, we determined the variation in protein coding genes in 51 metagenomes from chemosynthetic hot spring communities that span geochemical gradients in Yellowstone National Park. The 51 metagenomes could be divided into 5 distinct groups that correspond to low and high temperatures and acidic and circumneutral/alkaline springs. A fifth group primarily comprised metagenomes from springs with moderate acidity and that are influenced by elevated volcanic gas input. Protein homologs putatively involved in the oxidation of sulfur compounds, a process that leads to acidification of spring waters, in addition to those involved in the reduction of sulfur compounds were enriched in metagenomes from acidic springs sourced by vapor phase gases. Metagenomes from springs with evidence for elevated volcanic gas input were enriched in protein homologs putatively involved in oxidation of those gases, including hydrogen and methane. Finally, metagenomes from circumneutral/alkaline springs sourced by liquid phase waters were enriched in protein homologs putatively involved in heterotrophy and respiration of oxidized nitrogen compounds and oxygen. These results indicate that the geological process of phase separation shapes the ecology of thermophilic communities through its influence on the availability of nutrients in the form of gases, solutes, and minerals. Microbial acidification of hot spring waters further influences the kinetic and thermodynamic stabilities of nutrients and their bioavailability. These data therefore provide an important framework to understand how geological processes have shaped the evolutionary history of chemosynthetic thermophiles and how these organisms, in turn, have shaped their geochemical environments.

Terrestrial Hot Spring Systems: Introduction.

This report reviews how terrestrial hot spring systems can sustain diverse and abundant microbial communities and preserve their fossil records. Hot springs are dependable water sources, even in arid environments. They deliver reduced chemical species and other solutes to more oxidized surface environments, thereby providing redox energy and nutrients. Spring waters have diverse chemical compositions, and their outflows create thermal gradients and chemical precipitates that sustain diverse microbial communities and entomb their remnants. These environments probably were important habitats for ancient benthic microbial ecosystems, and it has even been postulated that life arose in hydrothermal systems. Thermal spring communities are fossilized in deposits of travertine, siliceous sinter, and iron minerals (among others) that are found throughout the geological record back to the oldest known well-preserved rocks at 3.48 Ga. Very few are known before the Cenozoic, but it is likely that there are many more to be found. They preserve fossils ranging from microbes to trees and macroscopic animals. Features on Mars whose morphological and spectroscopic attributes resemble spring deposits on Earth have been detected in regions where geologic context is consistent with the presence of thermal springs. Such features represent targets in the search for evidence of past life on that planet.

Biomarker Profiling of Microbial Mats in the Geothermal Band of Cerro Caliente, Deception Island (Antarctica): Life at the Edge of Heat and Cold.

Substrate-atmosphere interfaces in Antarctic geothermal environments are hot-cold regions that constitute thin habitable niches for microorganisms with possible counterparts in ancient Mars. Cerro Caliente hill in Deception Island (active volcano in the South Shetland Islands) is affected by ascending hydrothermal fluids that form a band of warm substrates buffered by low air temperatures. We investigated the influence of temperature on the community structure and metabolism of three microbial mats collected along the geothermal band of Cerro Caliente registering 88°C, 8°C, and 2°C at the time of collection. High-throughput sequencing of small subunit ribosomal ribonucleic acid (SSU rRNA) genes and Life Detector Chip (LDChip) microarray immunoassays revealed different bacterial, archaeal, and eukaryotic composition in the three mats. The mat at 88°C showed the less diverse microbial community and a higher proportion of thermophiles (e.g., Thermales). In contrast, microbial communities in the mats at 2°C and 8°C showed relatively higher diversity and higher proportion of psychrophiles (e.g., Flavobacteriales). Despite this overall association, similar microbial structures at the phylum level (particularly the presence of Cyanobacteria) and certain hot- and cold-tolerant microorganisms were identified in the three mats. Daily thermal oscillations recorded in the substrate over the year (4.5-76°C) may explain the coexistence of microbial fingerprints with different thermal tolerances. Stable isotope composition also revealed metabolic differences among the microbial mats. Carbon isotopic ratios suggested the Calvin-Benson-Bassham cycle as the major pathway for carbon dioxide fixation in the mats at 2°C and 8°C, and the reductive tricarboxylic acid cycle and/or the 3-hydroxypropionate bicycle for the mat at 88°C, indicating different metabolisms as a function of the prevailing temperature of each mat. The comprehensive biomarker profile on the three microbial mats from Cerro Caliente contributes to unravel the diversity, composition, and metabolism in geothermal polar sites and highlights the relevance of geothermal-cold environments to create habitable niches with interest in other planetary environments.

Diversity and distribution of hidden cultivable fungi associated with marine animals of Antarctica.

In the present study, we surveyed the distribution and diversity of fungal assemblages associated with 10 species of marine animals from Antarctica. The collections yielded 83 taxa from 27 distinct genera, which were identified using molecular biology methods. The most abundant taxa were Cladosporium sp. 1, Debaryomyces hansenii, Glaciozyma martinii, Metschnikowia australis, Pseudogymnoascus destructans, Thelebolus cf. globosus, Pseudogymnoascus pannorum, Tolypocladium tundrense, Metschnikowia australis, and different Penicillium species. The diversity, richness, and dominance of fungal assemblages ranged among the host; however, in general, the fungal community, which was composed of endemic and cold-adapted cosmopolitan taxa distributed across the different sites of Antarctic Peninsula, displayed high diversity, richness, and dominance indices. Our results contribute to knowledge about fungal diversity in the marine environment across the Antarctic Peninsula and their phylogenetic relationships with species that occur in other cold, temperate, and tropical regions of the World. Additionally, despite their extreme habitats, marine Antarctic animals shelter cryptic and complex fungal assemblages represented by endemic and cosmopolitan cold-adapted taxa, which may represent interesting models to study different symbiotic associations between fungi and their animal hosts in the extreme conditions of Antarctica.

Ultra-small microorganisms in the polyextreme conditions of the Dallol volcano, Northern Afar, Ethiopia.

The Dallol geothermal area in the northern part of the Danakil Depression (up to 124-155 meter below sea level) is deemed one of the most extreme environments on Earth. The area is notable for being part of the Afar Depression, an incipient seafloor-spreading center located at the triple junction, between Nubian, Somali and Arabian plates, and for hosting environments at the very edge of natural physical-chemical extremities. The northern part of the Danakil Depression is dominated by the Assale salt plain (an accumulation of marine evaporite deposits) and hosts the Dallol volcano. Here, the interaction between the evaporitic deposit and the volcanisms have created the unique Dallol hot springs, which are highly acidic (pH ~ 0) and saline (saturation) with maximum temperatures ranging between 90 and 109 °C. Here we report for the first time evidence of life existing with these hot springs using a combination of morphological and molecular analyses. Ultra-small structures are shown to be entombed within mineral deposits, which are identified as members of the Order Nanohaloarchaea. The results from this study suggest the microorganisms can survive, and potential live, within this extreme environment, which has implications for understanding the limits of habitability on Earth and on (early) Mars.

Plastic Silica Conglomerate with an Extremophile Microbial Matrix in a Hot-Water Stream Paleoenvironment.

A new and unusual type of fossil, siliceous hot-spring deposit (sinter)-comprising monomictic, quartzose conglomerate encrusted with silicified microbial laminates-has been recognized in distal portions of Jurassic and Miocene paleo-geothermal fields of South and North America, respectively. The siliceous clasts are inferred to have originated as conduit-delivered hydrothermal silica gel, owing to their general plastic morphologies, which were then locally reworked and redistributed in geothermally influenced stream paleoenvironments. Today, hot-spring-fed streams and creeks, in places with silica-armored pavements, host microbial mats coating streambeds and/or growing over, and silicifying at, stream air-water interfaces, for example, in Yellowstone National Park (USA) and Waimangu Volcanic Valley (New Zealand). However, the modern deposits do not contain the plastically deformed silica cobbles evident in Mesozoic and Cenozoic examples described herein. Moreover, the fossil microbial laminates of this study are relatively dense and strongly coat the silica cobbles, suggesting the mats stabilized the clasts under fully submerged and hot, high-energy conditions. Thus, this new sinter facies, typically found a few kilometers from main spring-vent areas, is a perhaps unexpected extreme environment in which life took hold in hydrothermal-fluvial settings of the past, and may serve as an additional target in the search for fossil biosignatures of early Earth and possibly Mars.

Lipid Biomarker and Carbon Stable Isotope Survey on the Dallol Hydrothermal System in Ethiopia.

The remote Dallol Hot Springs, an active hydrothermal system in the volcanic region of Danakil (Ethiopia), is an interesting yet poorly studied polyextreme environment for investigating the limits of life. Here, we explored the presence of signs of life in five samples of sinter deposits at Dallol, by means of lipid biomarkers and stable isotope composition. The results reveal the existence of biological material with predominance of (presently or recently active) microbial sources, according to the relative abundance of low-over-high molecular weight moieties (n-alkanes, n-carboxylic acids, or n-alkanols), and the detection of diverse microbial-diagnostic compounds (i.e., monomethyl alkanes; C16:1 ω7, C18:1 ω9, C18:1 ω10, C18:2 ω6,9 and iso/anteiso C15 and C17 carboxylic acids; or short-chained dicarboxylic acids). The molecular lipid patterns at Dallol suggest a microbial community largely composed of thermophilic members of the Aquificae, Thermotogae, Chroroflexi, or Proteobacteria phyla, as well as microbial consortia of phototrophs (e.g., Cyanobacteria-Chloroflexi) in lower-temperature and higher-pH niches. Autotrophic sources most likely using the Calvin cycle, together with the acetyl coenzyme A (CoA) pathway, were inferred from the depleted bulk δ13C ratios (-25.9/-22.6‰), while sulfate-reducing bacteria were considered according to enriched sulfate (7.3/11.7‰) and total sulfur (20.5/8.2‰) δ34S ratios. The abundance of functionalized hydrocarbons (i.e., n-carboxylic acids and n-alkanols) and the distinct even-over-odd predominance/preference on the typically odd n-alkanes (CPIalkanes ≤ 1) pointed to active or recent microbial metabolisms. This study documents the detection of biosignatures in the polyextreme environment of Dallol and raises the possibility of finding life or its remnants in other remote locations on Earth, where the harsh environmental conditions would lead to expect otherwise. These findings are relevant for understanding the limits of life and have implications for searching for hypothetical life vestiges in extreme environments beyond Earth.

Physiology, Metabolism, and Fossilization of Hot-Spring Filamentous Microbial Mats.

The evolutionarily ancient Aquificales bacterium Sulfurihydrogenibium spp. dominates filamentous microbial mat communities in shallow, fast-flowing, and dysoxic hot-spring drainage systems around the world. In the present study, field observations of these fettuccini-like microbial mats at Mammoth Hot Springs in Yellowstone National Park are integrated with geology, geochemistry, hydrology, microscopy, and multi-omic molecular biology analyses. Strategic sampling of living filamentous mats along with the hot-spring CaCO3 (travertine) in which they are actively being entombed and fossilized has permitted the first direct linkage of Sulfurihydrogenibium spp. physiology and metabolism with the formation of distinct travertine streamer microbial biomarkers. Results indicate that, during chemoautotrophy and CO2 carbon fixation, the 87-98% Sulfurihydrogenibium-dominated mats utilize chaperons to facilitate enzyme stability and function. High-abundance transcripts and proteins for type IV pili and extracellular polymeric substances (EPSs) are consistent with their strong mucus-rich filaments tens of centimeters long that withstand hydrodynamic shear as they become encrusted by more than 5 mm of travertine per day. Their primary energy source is the oxidation of reduced sulfur (e.g., sulfide, sulfur, or thiosulfate) and the simultaneous uptake of extremely low concentrations of dissolved O2 facilitated by bd-type cytochromes. The formation of elevated travertine ridges permits the Sulfurihydrogenibium-dominated mats to create a shallow platform from which to access low levels of dissolved oxygen at the virtual exclusion of other microorganisms. These ridged travertine streamer microbial biomarkers are well preserved and create a robust fossil record of microbial physiological and metabolic activities in modern and ancient hot-spring ecosystems.

The Deep-Sea Polyextremophile Halobacteroides lacunaris TB21 Rough-Type LPS: Structure and Inhibitory Activity towards Toxic LPS.

The structural characterization of the lipopolysaccharide (LPS) from extremophiles has important implications in several biomedical and therapeutic applications. The polyextremophile Gram-negative bacterium Halobacteroideslacunaris TB21, isolated from one of the most extreme habitats on our planet, the deep-sea hypersaline anoxic basin Thetis, represents a fascinating microorganism to investigate in terms of its LPS component. Here we report the elucidation of the full structure of the R-type LPS isolated from H. lacunaris TB21 that was attained through a multi-technique approach comprising chemical analyses, NMR spectroscopy, and Matrix-Assisted Laser Desorption Ionization (MALDI) mass spectrometry. Furthermore, cellular immunology studies were executed on the pure R-LPS revealing a very interesting effect on human innate immunity as an inhibitor of the toxic Escherichia coli LPS.

An ecological approach to discover new bioactive extracts and products: the case of extremophile plants.

OBJECTIVES: Eight extremophile plants from Tunisia were screened to find natural products with benefits in human health. METHODS: These plants were collected in different areas in Tunisia. Their methanolic extracts were evaluated for their total phenolic content and for their antiradical (DPPH), antimicrobial (on 35 bacteria and one yeast), antiviral (hepatitis C virus, HCV) and cytotoxic activity (against WI38 and J774 cell lines). The most active species were subjected to a bioguided fractionation. KEY FINDINGS: The screening revealed promising activity for four plants, but two species have both antiradical and antimicrobial activity: Juncus maritimus and Limonium virgatum. The rhizomes extract of J. maritimus showed the highest activity against HCV, a selective antibacterial activity against Streptococcus dysgalactiae, and a moderate antiradical activity which is due to luteolin isolated in one step by centrifugal partition chromatography. The stems' and leaves' extracts of L. virgatum were rich in polyphenols responsible for the antiradical activity. Also, Limonium extracts showed an antibacterial activity with a broad spectrum. CONCLUSIONS: Extremophile plants have proven to be a promising source for bioactive metabolites. They have a powerful antioxidant system highly influenced by biotic and abiotic factors and the ability to produce secondary metabolites with antimicrobial activity.

Resilient microorganisms in dust samples of the International Space Station-survival of the adaptation specialists.

BACKGROUND: The International Space Station (ISS) represents a unique biotope for the human crew but also for introduced microorganisms. Microbes experience selective pressures such as microgravity, desiccation, poor nutrient-availability due to cleaning, and an increased radiation level. We hypothesized that the microbial community inside the ISS is modified by adapting to these stresses. For this reason, we analyzed 8-12 years old dust samples from Russian ISS modules with major focus on the long-time surviving portion of the microbial community. We consequently assessed the cultivable microbiota of these samples in order to analyze their extremotolerant potential against desiccation, heat-shock, and clinically relevant antibiotics. In addition, we studied the bacterial and archaeal communities from the stored Russian dust samples via molecular methods (next-generation sequencing, NGS) and compared our new data with previously derived information from the US American ISS dust microbiome. RESULTS: We cultivated and identified in total 85 bacterial, non-pathogenic isolates (17 different species) and 1 fungal isolate from the 8-12 year old dust samples collected in the Russian segment of the ISS. Most of these isolates exhibited robust resistance against heat-shock and clinically relevant antibiotics. Microbial 16S rRNA gene and archaeal 16S rRNA gene targeting Next Generation Sequencing showed signatures of human-associated microorganisms (Corynebacterium, Staphylococcus, Coprococcus etc.), but also specifically adapted extremotolerant microorganisms. Besides bacteria, the detection of archaeal signatures in higher abundance was striking. CONCLUSIONS: Our findings reveal (i) the occurrence of living, hardy microorganisms in archived Russian ISS dust samples, (ii) a profound resistance capacity of ISS microorganisms against environmental stresses, and (iii) the presence of archaeal signatures on board. In addition, we found indications that the microbial community in the Russian segment dust samples was different to recently reported US American ISS microbiota.