Extremophilic microbial metabolism and radioactive waste disposal.

Decades of nuclear activities have left a legacy of hazardous radioactive waste, which must be isolated from the biosphere for over 100,000 years. The preferred option for safe waste disposal is a deep subsurface geological disposal facility (GDF). Due to the very long geological timescales required, and the complexity of materials to be disposed of (including a wide range of nutrients and electron donors/acceptors) microbial activity will likely play a pivotal role in the safe operation of these mega-facilities. A GDF environment provides many metabolic challenges to microbes that may inhabit the facility, including high temperature, pressure, radiation, alkalinity, and salinity, depending on the specific disposal concept employed. However, as our understanding of the boundaries of life is continuously challenged and expanded by the discovery of novel extremophiles in Earth's most inhospitable environments, it is becoming clear that microorganisms must be considered in GDF safety cases to ensure accurate predictions of long-term performance. This review explores extremophilic adaptations and how this knowledge can be applied to challenge our current assumptions on microbial activity in GDF environments. We conclude that regardless of concept, a GDF will consist of multiple extremes and it is of high importance to understand the limits of polyextremophiles under realistic environmental conditions.

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).

Extremophile enzyme optimization for low temperature and high salinity are fundamentally incompatible.

The evolutionary mechanisms behind cold and high-saline co-adaptation of proteins are not thoroughly understood. To explore how enzymes evolve in response to multiple environmental pressures we developed a novel in silico method to model the directed evolution of proteins, the Protein Evolution Parameter Calculator (PEPC). PEPC carries out single amino acid substitutions that lead to improvements in the selected user-defined parameters. To investigate the evolutionary relationship between increased flexibility and decreased isoelectric point, which are presumed indicators of cold and saline adaptation in proteins, we applied PEPC to a subset of core haloarchaea orthologous group (cHOG) proteins from the mesophilic Halobacterium salinarum NRC-1 and cold-tolerant Halorubrum lacusprofundi strain ATCC 49239. The results suggest that mutations that increase flexibility will also generally increase isoelectric point. These findings suggest that enzyme adaptation to low temperature and high salinity might be evolutionarily counterposed based on the structural characteristics of probable amino acid mutations. This may help to explain the apparent lack of truly psychrophilic halophiles in nature, and why microbes adapted to polar hypersaline environments typically have mesophilic temperature optima. A better understanding of protein evolution to extremely cold and salty conditions will aid in our understanding of where and how life is distributed on Earth and in our solar system.

Deep Hypersaline Anoxic Basins as Untapped Reservoir of Polyextremophilic Prokaryotes of Biotechnological Interest.

Deep-sea hypersaline anoxic basins (DHABs) are considered to be among the most extreme ecosystems on our planet, allowing only the life of polyextremophilic organisms. DHABs' prokaryotes exhibit extraordinary metabolic capabilities, representing a hot topic for microbiologists and biotechnologists. These are a source of enzymes and new secondary metabolites with valuable applications in different biotechnological fields. Here, we review the current knowledge on prokaryotic diversity in DHABs, highlighting the biotechnological applications of identified taxa and isolated species. The discovery of new species and molecules from these ecosystems is expanding our understanding of life limits and is expected to have a strong impact on biotechnological applications.

The Polyextremophilic Bacterium Clostridium paradoxum Attains Piezophilic Traits by Modulating Its Energy Metabolism and Cell Membrane Composition.

In polyextremophiles, i.e., microorganisms growing preferentially under multiple extremes, synergistic effects may allow growth when application of the same extremes alone would not. High hydrostatic pressure (HP) is rarely considered in studies of polyextremophiles, and its role in potentially enhancing tolerance to other extremes remains unclear. Here, we investigated the HP-temperature response in Clostridium paradoxum, a haloalkaliphilic moderately thermophilic endospore-forming bacterium, in the range of 50 to 70°C and 0.1 to 30 MPa. At ambient pressure, growth limits were extended from the previously reported 63°C to 70°C, defining C. paradoxum as an actual thermophile. Concomitant application of high HP and temperature compared to standard conditions (i.e., ambient pressure and 50°C) remarkably enhanced growth, with an optimum growth rate observed at 22 MPa and 60°C. HP distinctively defined C. paradoxum physiology, as at 22 MPa biomass, production increased by 75% and the release of fermentation products per cell decreased by >50% compared to ambient pressure. This metabolic modulation was apparently linked to an energy-preserving mechanism triggered by HP, involving a shift toward pyruvate as the preferred energy and carbon source. High HPs decreased cell damage, as determined by Syto9 and propidium iodide staining, despite no organic solute being accumulated intracellularly. A distinct reduction in carbon chain length of phospholipid fatty acids (PLFAs) and an increase in the amount of branched-chain PLFAs occurred at high HP. Our results describe a multifaceted, cause-and-effect relationship between HP and cell metabolism, stressing the importance of applying HP to define the boundaries for life under polyextreme conditions.IMPORTANCE Hydrostatic pressure (HP) is a fundamental parameter influencing biochemical reactions and cell physiology; however, it is less frequently applied than other factors, such as pH, temperature, and salinity, when studying polyextremophilic microorganisms. In particular, how HP affects microbial tolerance to other and multiple extremes remains unclear. Here, we show that under polyextreme conditions of high pH and temperature, Clostridium paradoxum demonstrates a moderately piezophilic nature as cultures grow to highest cell densities and most efficiently at a specific combination of temperature and HP. Our results highlight the importance of considering HP when exploring microbial physiology under extreme conditions and thus have implications for defining the limits for microbial life in nature and for optimizing industrial bioprocesses occurring under multiple extremes.

Culture-dependent diversity of bacteria from Laohugou glacier, Qilian Mts., China and their resistance against metals.

In the current study, psychrophilic, endolithic, and epilithic bacterial strains were isolated and characterized from the nonpolar Laohugou glacier (LHG) no. 12, the largest valley glacier in the western Qilian Mts. located on the northeastern edge of the Tibetan Plateau. Five different types of samples, rocks, soil, glacial water, ice/snow, and cryoconite, were collected. A total of 48 bacterial strains were isolated by using the R2A bacterial cultural medium. The findings revealed that the Gram-positive bacteria 41 (85.4%) dominated the Gram-negative bacteria 7 (14.6%) in this extremely harsh environment. Molecular characterization based on 16S ribosomal RNA gene sequencing exhibited that the obtained isolates belong to four phyla, among which the diversity of Firmicutes (58.33%) was higher followed by Actinobacteria (23.0%), Proteobacteria (14.6%), and least diversity was reported in Euryarchaeota (4.2%). The bacterial communities were most dominant in soil samples followed by cryoconite sample and least dominant in the ice and snow samples. Moreover, the obtained bacterial isolates were found resistant to high concentrations of heavy metals (Cr3+ , Cd2+ , Hg2+ , and Ar3+ ) and sodium chloride, and, therefore, exhibited polyextremophilic characteristics. LHG no. 12 is rich in bacterial and archaeal diversities and provides a potentially curious site for further in-depth exploration of microbial diversity and their biotechnological applications.

Extremophiles as sources of inorganic bio-nanoparticles.

Industrial use of nanotechnology in daily life has produced an emphasis on the safe and efficient production of nanoparticles (NPs). Traditional chemical oxidation and reduction methods are seen as inefficient, environmentally unsound, and often dangerous to those exposed and involved in NP manufacturing. However, utilizing microorganisms for biosynthesis of NPs allows efficient green production of a range of inorganic NPs, while maintaining specific size, shape, stability, and dispersity. Microorganisms living under harsh environmental conditions, called "Extremophiles," are one group of microorganisms being utilized for this biosynthesis. Extremophiles' unique living conditions have endowed them with various processes that enable NP biosynthesis. This includes a range of extremophiles: thermophiles, acidophilus, halophiles, psychrophiles, anaerobes, and some others. Fungi, bacteria, yeasts, and archaea, i.e. Ureibacillus thermosphaericus, and Geobacillus stearothermophilus, among others, have been established for NP biosynthesis. This article highlights the extremophiles and methods found to be viable candidates for the production of varying types of NPs, as well as interpreting selective methods used by the organisms to synthesize NPs.

Engineered ketocarotenoid biosynthesis in the polyextremophilic red microalga Cyanidioschyzon merolae 10D.

The polyextremophilic Cyanidiophyceae are eukaryotic red microalgae with promising biotechnological properties arising from their low pH and elevated temperature requirements which can minimize culture contamination at scale. Cyanidioschyzon merolae 10D is a cell wall deficient species with a fully sequenced genome that is amenable to nuclear transgene integration by targeted homologous recombination. C. merolae maintains a minimal carotenoid profile and here, we sought to determine its capacity for ketocarotenoid accumulation mediated by heterologous expression of a green algal ß-carotene ketolase (BKT) and hydroxylase (CHYB). To achieve this, a synthetic transgene expression cassette system was built to integrate and express Chlamydomonas reinhardtii (Cr) sourced enzymes by fusing native C. merolae transcription, translation and chloroplast targeting signals to codon-optimized coding sequences. Chloramphenicol resistance was used to select for the integration of synthetic linear DNAs into a neutral site within the host genome. CrBKT expression caused accumulation of canthaxanthin and adonirubin as major carotenoids while co-expression of CrBKT with CrCHYB generated astaxanthin as the major carotenoid in C. merolae. Unlike green algae and plants, ketocarotenoid accumulation in C. merolae did not reduce total carotenoid contents, but chlorophyll a reduction was observed. Light intensity affected global ratios of all pigments but not individual pigment compositions and phycocyanin contents were not markedly different between parental strain and transformants. Continuous illumination was found to encourage biomass accumulation and all strains could be cultivated in simulated summer conditions from two different extreme desert environments. Our findings present the first example of carotenoid metabolic engineering in a red eukaryotic microalga and open the possibility for use of C. merolae 10D for simultaneous production of phycocyanin and ketocarotenoid pigments.

Extremophiles: the species that evolve and survive under hostile conditions.

Extremophiles possess unique cellular and molecular mechanisms to assist, tolerate, and sustain their lives in extreme habitats. These habitats are dominated by one or more extreme physical or chemical parameters that shape existing microbial communities and their cellular and genomic features. The diversity of extremophiles reflects a long list of adaptations over millions of years. Growing research on extremophiles has considerably uncovered and increased our understanding of life and its limits on our planet. Many extremophiles have been greatly explored for their application in various industrial processes. In this review, we focused on the characteristics that microorganisms have acquired to optimally thrive in extreme environments. We have discussed cellular and molecular mechanisms involved in stability at respective extreme conditions like thermophiles, psychrophiles, acidophiles, barophiles, etc., which highlight evolutionary aspects and the significance of extremophiles for the benefit of mankind.

Genome-guided prediction of acid resistance mechanisms in acidophilic methanotrophs of phylogenetically deep-rooted Verrucomicrobia isolated from geothermal environments.

Verrucomicrobia are a group of microorganisms that have been proposed to be deeply rooted in the Tree of Life. Some are methanotrophs that oxidize the potent greenhouse gas methane and are thus important in decreasing atmospheric concentrations of the gas, potentially ameliorating climate change. They are widespread in various environments including soil and fresh or marine waters. Recently, a clade of extremely acidophilic Verrucomicrobia, flourishing at pH < 3, were described from high-temperature geothermal ecosystems. This novel group could be of interest for studies about the emergence of life on Earth and to astrobiologists as homologs for possible extraterrestrial life. In this paper, we describe predicted mechanisms for survival of this clade at low pH and suggest its possible evolutionary trajectory from an inferred neutrophilic ancestor. Extreme acidophiles are defined as organisms that thrive in extremely low pH environments (≤ pH 3). Many are polyextremophiles facing high temperatures and high salt as well as low pH. They are important to study for both providing fundamental insights into biological mechanisms of survival and evolution in such extreme environments and for understanding their roles in biotechnological applications such as industrial mineral recovery (bioleaching) and mitigation of acid mine drainage. They are also, potentially, a rich source of novel genes and pathways for the genetic engineering of microbial strains. Acidophiles of the Verrucomicrobia phylum are unique as they are the only known aerobic methanotrophs that can grow optimally under acidic (pH 2-3) and moderately thermophilic conditions (50-60°C). Three moderately thermophilic genera, namely Methylacidiphilum, Methylacidimicrobium, and Ca. Methylacidithermus, have been described in geothermal environments. Most of the investigations of these organisms have focused on their methane oxidizing capabilities (methanotrophy) and use of lanthanides as a protein cofactor, with no extensive study that sheds light on the mechanisms that they use to flourish at extremely low pH. In this paper, we extend the phylogenetic description of this group of acidophiles using whole genome information and we identify several mechanisms, potentially involved in acid resistance, including "first line of defense" mechanisms that impede the entry of protons into the cell. These include the presence of membrane-associated hopanoids, multiple copies of the outer membrane protein (Slp), and inner membrane potassium channels (kup, kdp) that generate a reversed membrane potential repelling the intrusion of protons. Acidophilic Verrucomicrobia also display a wide array of proteins potentially involved in the "second line of defense" where protons that evaded the first line of defense and entered the cell are expelled or neutralized, such as the glutamate decarboxylation (gadAB) and phosphate-uptake systems. An exclusive N-type ATPase F0-F1 was identified only in acidophiles of Verrucomicrobia and is predicted to be a specific adaptation in these organisms. Phylogenetic analyses suggest that many predicted mechanisms are evolutionarily conserved and most likely entered the acidophilic lineage of Verrucomicrobia by vertical descent from a common ancestor. However, it is likely that some defense mechanisms such as gadA and kup entered the acidophilic Verrucomicrobia lineage by horizontal gene transfer.

Exobiology of the Venusian Clouds: New Insights into Habitability through Terrestrial Models and Methods of Detection.

The search for life beyond Earth has focused on Mars and the icy moons Europa and Enceladus, all of which are considered a safe haven for life due to evidence of current or past water. The surface of Venus, on the other hand, has extreme conditions that make it a nonhabitable environment to life as we know it. This is in contrast, however, to its cloud layer, which, while still an extreme environment, may prove to be a safe haven for some extreme forms of life similar to extremophiles on Earth. We consider the venusian clouds a habitable environment based on the presence of (1) a solvent for biochemical reactions, (2) appropriate physicochemical conditions, (3) available energy, and (4) biologically relevant elements. The diversity of extreme microbial ecosystems on Earth has allowed us to identify terrestrial chemolithoautotrophic microorganisms that may be analogs to putative venusian organisms. Here, we hypothesize and describe biological processes that may be performed by such organisms in the venusian clouds. To detect putative venusian organisms, we describe potential biosignature detection methods, which include metal-microbial interactions and optical methods. Finally, we describe currently available technology that can potentially be used for modeling and simulation experiments.

Forced Biomineralization: A Review.

Biologically induced and controlled mineralization of metals promotes the development of protective structures to shield cells from thermal, chemical, and ultraviolet stresses. Metal biomineralization is widely considered to have been relevant for the survival of life in the environmental conditions of ancient terrestrial oceans. Similar behavior is seen among extremophilic biomineralizers today, which have evolved to inhabit a variety of industrial aqueous environments with elevated metal concentrations. As an example of extreme biomineralization, we introduce the category of "forced biomineralization", which we use to refer to the biologically mediated sequestration of dissolved metals and metalloids into minerals. We discuss forced mineralization as it is known to be carried out by a variety of organisms, including polyextremophiles in a range of psychrophilic, thermophilic, anaerobic, alkaliphilic, acidophilic, and halophilic conditions, as well as in environments with very high or toxic metal ion concentrations. While much additional work lies ahead to characterize the various pathways by which these biominerals form, forced biomineralization has been shown to provide insights for the progression of extreme biomimetics, allowing for promising new forays into creating the next generation of composites using organic-templating approaches under biologically extreme laboratory conditions relevant to a wide range of industrial conditions.

Metagenomic Investigation of a Low Diversity, High Salinity Offshore Oil Reservoir.

Oil reservoirs can represent extreme environments for microbial life due to low water availability, high salinity, high pressure and naturally occurring radionuclides. This study investigated the microbiome of saline formation water samples from a Gulf of Mexico oil reservoir. Metagenomic analysis and associated anaerobic enrichment cultures enabled investigations into metabolic potential for microbial activity and persistence in this environment given its high salinity (4.5%) and low nutrient availability. Preliminary 16S rRNA gene amplicon sequencing revealed very low microbial diversity. Accordingly, deep shotgun sequencing resulted in nine metagenome-assembled genomes (MAGs), including members of novel lineages QPJE01 (genus level) within the Halanaerobiaceae, and BM520 (family level) within the Bacteroidales. Genomes of the nine organisms included respiratory pathways such as nitrate reduction (in Arhodomonas, Flexistipes, Geotoga and Marinobacter MAGs) and thiosulfate reduction (in Arhodomonas, Flexistipes and Geotoga MAGs). Genomic evidence for adaptation to high salinity, withstanding radioactivity, and metal acquisition was also observed in different MAGs, possibly explaining their occurrence in this extreme habitat. Other metabolic features included the potential for quorum sensing and biofilm formation, and genes for forming endospores in some cases. Understanding the microbiomes of deep biosphere environments sheds light on the capabilities of uncultivated subsurface microorganisms and their potential roles in subsurface settings, including during oil recovery operations.

Understanding High-Salt and Cold Adaptation of a Polyextremophilic Enzyme.

The haloarchaeon Halorubrum lacusprofundi is among the few polyextremophilic organisms capable of surviving in one of the most extreme aquatic environments on Earth, the Deep Lake of Antarctica (-18 °C to +11.5 °C and 21-28%, w/v salt content). Hence, H. lacusprofundi has been proposed as a model for biotechnology and astrobiology to investigate potential life beyond Earth. To understand the mechanisms that allow proteins to adapt to both salinity and cold, we structurally (including X-ray crystallography and molecular dynamics simulations) and functionally characterized the ß-galactosidase from H. lacusprofundi (hla_bga). Recombinant hla_bga (produced in Haloferax volcanii) revealed exceptional stability, tolerating up to 4 M NaCl and up to 20% (v/v) of organic solvents. Despite being cold-adapted, hla_bga was also stable up to 60 °C. Structural analysis showed that hla_bga combined increased surface acidity (associated with halophily) with increased structural flexibility, fine-tuned on a residue level, for sustaining activity at low temperatures. The resulting blend enhanced structural flexibility at low temperatures but also limited protein movements at higher temperatures relative to mesophilic homologs. Collectively, these observations help in understanding the molecular basis of a dual psychrophilic and halophilic adaptation and suggest that such enzymes may be intrinsically stable and functional over an exceptionally large temperature range.

Corrigendum: Living at the Extremes: Extremophiles and the Limits of Life in a Planetary Context.

[This corrects the article DOI: 10.3389/fmicb.2019.00780.].

The Dallol Geothermal Area, Northern Afar (Ethiopia)-An Exceptional Planetary Field Analog on Earth.

The Dallol volcano and its associated hydrothermal field are located in a remote area of the northern Danakil Depression in Ethiopia, a region only recently appraised after decades of inaccessibility due to severe political instability and the absence of infrastructure. The region is notable for hosting environments at the very edge of natural physical-chemical extremities. It is surrounded by a wide, hyperarid salt plain and is one of the hottest (average annual temperatureDallol: 36-38°C) and most acidic natural systems (pHDallol ≈0) on Earth. Spectacular geomorphologies and mineral deposits produced by supersaturated hydrothermal waters and brines are the result of complex interactions between active and inactive hydrothermal alteration of the bedrock, sulfuric hot springs and pools, fumaroles and geysers, and recrystallization processes driven by hydrothermal waters, degassing, and rapid evaporation. The study of planetary field analog environments plays a crucial role in characterizing the physical and chemical boundaries within which life can exist on Earth and other planets. It is essential for the definition and assessment of the conditions of habitability on other planets, including the possibility for biosignature preservation and in situ testing of technologies for life detection. The Dallol area represents an excellent Mars analog environment given that the active volcanic environment, the associated diffuse hydrothermalism and hydrothermal alteration, and the vast acidic sulfate deposits are reminiscent of past hydrothermal activity on Mars. The work presented in this paper is an overview of the Dallol volcanic area and its hydrothermal field that integrates previous literature with observations and results obtained from field surveys and monitoring coupled with sample characterization. In so doing, we highlight its exceptional potential as a planetary field analog as well as a site for future astrobiological and exploration programs.

Living at the Extremes: Extremophiles and the Limits of Life in a Planetary Context.

Prokaryotic life has dominated most of the evolutionary history of our planet, evolving to occupy virtually all available environmental niches. Extremophiles, especially those thriving under multiple extremes, represent a key area of research for multiple disciplines, spanning from the study of adaptations to harsh conditions, to the biogeochemical cycling of elements. Extremophile research also has implications for origin of life studies and the search for life on other planetary and celestial bodies. In this article, we will review the current state of knowledge for the biospace in which life operates on Earth and will discuss it in a planetary context, highlighting knowledge gaps and areas of opportunity.

Thermophilic and alkaliphilic Actinobacteria: biology and potential applications.

Microbes belonging to the phylum Actinobacteria are prolific sources of antibiotics, clinically useful bioactive compounds and industrially important enzymes. The focus of the current review is on the diversity and potential applications of thermophilic and alkaliphilic actinobacteria, which are highly diverse in their taxonomy and morphology with a variety of adaptations for surviving and thriving in hostile environments. The specific metabolic pathways in these actinobacteria are activated for elaborating pharmaceutically, agriculturally, and biotechnologically relevant biomolecules/bioactive compounds, which find multifarious applications.