User:Ku Itoi/Brine pool

Metagenomics Analysis
Metagenomics is a powerful genomic analysis to identify the microbiome communities in a variety of environments. Previous gene analysis requires culturing microorganisms that is problematic since most microorganisms present in nature are not cultivable. Metagenomics overcomes this problems by allowing researchers to directly sample and analyze the microbe community from the desired environment. Despite the harsh environment for living organisms, metagenomics has revealed the presence of previously unknown microbiome communities in multiple brine pools. Common procedures of marine microbe metagenomics include sampling, filtration and extraction, DNA sequencing, and database analysis. Shotgun sequencing and 454 pyrosequencing are typically used to sequence genes, and 16S rRNA is used for the identification of species.

Main clades
The taxonomy of the main microbe community found in Atlantis II and Discovery without minor or unknown species to avoid ambiguity. . It is important to note that the list is based on the data provided from the primary articles. Because identification of microbes and construction of phylogeny is controversial, phylogeny has been reconstructed indicating some taxonomic rank might not be up-to-date.

Animals are have also been found living in these anaerobic brine pools, such as the first ever described metazoan by Danovaro et. al., in 2010, even though much research still has to be done about their entire life cycle. Many novel undefined taxa that live in these extreme environments are still unknown.

Environmental challenges and adaptations
The lack of mixing of the water column in combination with the extreme conditions of salinity, anoxia, temperature and hydrostatic pressure, has resulted in an environment which has been separated from the upper water column. These environmental characteristics promote unique extreme challenges and selective pressures on the organisms that live in these ecosystems, which has overtime resulted in the selection of novel microbial structural components, allowing them to survive and thrive in brine pool habitats.

Challenges
Due to high levels of salinity of the environment, cell membranes cannot avoid the rapid loss of intracellular water, severely impairing cell turgor and functioning. Thus, organisms that live in this ecosystem had the evolutionary need to develop novel strategies to decrease cell metabolic activity as much as possible to be able to survive. Another big challenge that these organisms had to face to survive in these extreme environments face, in combination with the multiple stressors, is the significant ionic, kosmotropic and chaotropic effects on the cells caused by the brine pool environment. Lastly, the lack of oxygen in brine pools also increase the difficulty of organisms to yield energy and to adapt to these extreme environments, as oxygen is usually extensively used as the most energy-yielding electron acceptor.

Adaptations
In order to solve the challenges imposed by these extreme environments, organisms successfully developed different evolutionary adaptations to survive under the new selective pressures, such as the "salt-in" and the "compatible-solute" strategy to decrease the risk of the chaotropic effects on the cells developed by halophilic archaea. This novel strategy increases intracellular ionic concentration (mostly K+) to decrease the osmotic pressure, and thus forcing these organisms to adapt their entire metabolic machinery to increase salt concentration inside of their cells to ensure their survival.

Another important adaptation example is the novel synthesis of thermoprotective molecules, such as hydroxyketone molecules by piezophilic microorganisms, as it allowed organisms to survive in extreme water temperatures and hydrostatic pressures. Thus, these novel features were evolutionary selected for during generations, and ultimately successfully avoiding denaturation of proteins due to the extreme selective pressures, intrinsically increasing their fitness in extreme hydrostatic pressures and decreasing the risk of desiccation imposed by the environment.

Lastly, another important adaptation is the usage of alternative electron acceptors molecules to yield energy, such as iron, manganese, sulfate, elemental sulfur , carbon dioxide, nitrite and nitrate , which are abundantly available in these deep water layers.

Chemical composition and metabolic significance
As the name suggests, Brine pools, or Deep Hypersaline Anoxic Basins (DHABs), are characterized by a very high salt concentration and anoxic conditions. Sodium, chloride, magnesium, potassium, and calcium ion concentration are all extremely high in brine pools. And, due to low mixing rates between the above seawater and the brine water, brine pool water becomes anoxic within the first ten centimeters or so. While there are large variations in the geochemical composition of individual pools, as well as extreme chemical stratification within the same pool, conserved chemical trends are present. Deeper layers of DHABs will be saltier, hotter, more acidic and more anaerobic than the preceding layers. The concentration of heavy metals (Fe, Mn, Si, Cu) and certain nutrients (NO2-, NH4+, NO3-, and PO4-) will tend to increase with depth, while the concentration of SO4- and both organic and inorganic carbon decrease with depth. While these trends are all observed to some capacity in DHABs, the intensity and distance over which these trends take effect can vary in depth from one meter, to tens of meters.

The heavy stratification within DHABs has led to increased microbial metabolic diversity and varying cell concentrations between layers. The majority of cell biomass has been found at the interfaces between the distinct chemical layers (with the highest concentrations of cells located at the brine-surface interface). Microbes exploit the sharp chemical gradients between the layers to make their metabolisms more thermodynamically favorable.

Four heavily studied DHABs are Urania, Bannock, L’Atalante, and Discovery. All four of these Brine pools are located in the Mediterranean sea, yet they all exhibit distinct chemical properties: Urania has the highest concentration of Sulfuric Acid observed (at ~16mM)--compared to normal sea water (2.6 x 10^-6mM) or the next highest [HS-] in the Bannock basin (~3mM). Discovery has an extremely low concentration of Na+ (68mM) and an extremely high concentration of Mg2+ (4995mM)--compared to the surrounding seawater with concentrations of 528mM and 60mM respectively. The L’Atalante basin has a high [SO4 2-] compared to the other pools.

Carbon Cycling
While it was initially thought that particulate organic matter (POM) was an important source of carbon for DHABs, due to their depth the concentration of POM reaching the pools was not significant as originally thought. The majority of fixed carbon is now thought to come from autotrophy, specifically methanogenesis. Direct measurements of methane production in DHABs have provided extensive molecular evidence of methanogenesis in these environments. Proteomic analysis further support the presence of methanogenesis by identifying the enzyme RuBisCo in various DHABs. Interestingly, it has been suggested that instead of CO2 or acetoclastic methanogenesis, prokaryotes in DHABs use methylotrophic methanogenesis as it allows for a higher energy yield and the intermediates can be used for osmoprotectants.

Nitrogen Cycling
One of the key metabolic features of DHABs is the dissimilatory reduction of nitrogen. In Bannock basin and L’Atalante basin anammox and denitrification pathways have been identified using a combination of transcriptomics and direct isotope tracking. Other DHABs have been analyzed for anammox pathways using metatranscriptomic techniques with little positive results, which may be due to the limitations of transcriptomic sensitivity. In deeper DHAB layers, nitrogen fixation and ammonium assimilation has been observed. These reductive pathways require a lot of energy and are mainly performed by methanogens to synthesize osmoprotectants.

Sulfur Cycling
Due to the high concentration of sulfate (especially in the Uranian Basin), sulfate reduction is extremely important in the biogeochemical cycling of DHABs. The highest rates of sulfate reduction tend to be found in the deepest DHAB layers, where redox potential is lowest. Sulfate reducing bacteria have been found in the brines of Kebrit deep, Nereus Deep, Erba deep, Atlantis II deep, and Discovery Deep. Oxidative sulfur pathways help close the biogeochemical sulfur loops within the DHABs. There are three main sulfur oxidizing pathways which are likely found in DHABs: 1) sulfur-oxidizing multienzyme complex which can oxidize sulfide or thiosulfate to sulfate (w/ elemental sulfur or sulfite as an intermediate), 2) a sulfide/quinone complex which oxidizes hydrogen sulfide to elemental sulfur, 3) polysulfide reductase, which reduces precipitate sulfur to sulfide. A combination between the second and third pathway would allow for increased energetic yield. In addition, some novel groups have been isolated from saline lakes which can anaerobically respire sulfur using acetate, pyruvate, formate, or hydrogen as a sole electron donors.

Microbial Symbiosis
There is a high concentration of bacteria present in Brine Pools that serve essential roles for the ecosystem, such as being part of Symbiotic Relationships or acting as a food source for several organisms in this habitat. Examples include tubeworms and clams having a symbiotic relationship with many of these bacteria. Tube worms and clams have a symbiotic relationship with many of these bacteria where both species (clams and tube worms) use bacteria to convert chemical energy from hydrogen sulfide, obtaining their food source from a symbiotic bacteria that is inhabiting in an organ inside their body called the trophosome. In exchange for the bacteria making food used to develop and reproduce, the tube worm provides a safe habitat for the bacteria to live in.

Mussels are another example of organisms that are abundant and also provide safe habitat for bacteria that feeds on methane to produce sugars. Even though there is an absence of primary producers that are light-driven, mussels are able to thrive due to the chemosynthetic, carbon-fixing bacterial symbionts that inhabit their gill tissues. This symbiosis is very commonly seen, as the shores of brine pools can be surrounded or lined with mussels with bacterial mats covering them.

Bacteria can also act as epibiotic symbiont, which were found to play an important role in the adaptations of microorganisms to these environments, such as organisms from the flagellated group Euglenozoa that have been thriving in brine pools due to this relationship.