Thermoacidophil bacteria: general concepts – Culture; Molecular identification (PCR and sequencing) 

Information 11-07-2017. 

A thermoacidophil bacteria is an extremophile microorganism that is both thermophilic and acidophilic, meaning that it can grow under conditions of high temperatures, and at low pH. Most thermoacidophil bacteria are archaea (mainly Crenarchaeota and Euryarchaeota) or bacteria, although some Eukaryotes have occasionally been reported (e.g., red algae such as Galdieria sulphuraria that has acquired genes by gene transfer from Archaea and bacteria). Thermocidophils can be found in thermal or sulphurous waters (e.g., Sulfolobus), in deep sea or in other geothermal environments. They can also be found in acid mining drains.

There are few examples that are tolerant to both media (pH < 2, and > 80°C). Many archaea have aerobic or microaerophilic metabolism, although obligate anaerobes (e.g., Acidilobales) have been described.

Archaea are unicellular microorganisms with a cellular structure similar to bacteria, but with some differences, so they were previously considered as ancient bacteria (Aqueobacteria). Currently considered a group of independent living beings (domain Archeae), independent of the other two groups of living beings the bacteria (domain Bacteria) and eukaryotes (domain Eukaria). In this last domain, animals, plants, fungi and protozoa are included. 

Despite being similar in size and shape to bacteria, some have very different shapes, with flat and square cells. Sometimes their genes and metabolic pathways are more similar to those of eukaryotes, as is the case with enzymes for transcription and translation. They also have different molecules in their structure (lipid ethers in their cell membranes), and use many energy sources (organic compounds, ammonia, metal ions or hydrogen). They tolerate high salinity, use sunlight as a source of energy, and can fix carbon. They are divided by binary fission, fragmentation or budding, unlike other living beings.

They are considered extremophile microorganisms, that is to say that they can develop under "extreme" conditions, as it happens in thermal water of high temperature, salty waters, but they can also be found in the soil, the oceans, the marshes and in the human intestine or of the ruminants, in which it helps to digest food.

Through the analysis of ribosomal RNA, the existence of five evolutionary groups is admitted: Euryarchaeota, Crenarchaeota, Korarchaeota, Nanoarchaeota and Thaumarchaeota, of which the first two are more frequent. The Euryarchaeota include very varied microorganisms such as methanogens, thermoacidophils and hiperhalophiles. Crenarchaeota include hyperthermophils, acidophiles, sulfur reducers and/or oxidants and chemolithoheterotrophs. The Korarchaeota are very rare and are found in hot springs. Nanoarchaeota are very small hyperthermophiles or acidophiles, with a size of 300 nm in diameter that would make them the smallest prokaryotes. The Thaumarchaeota are nitrifying quimiolitoautotrophs of marine and terrestrial environments.

The size of the archaea can be from 0.1 μm to more than 15 μm, with round, elongated, filamentous, spiral, lobed or flat shapes. Some form aggregates or filaments up to 200 μm, or can even form macroscopic filamentous colonies. Its structure is similar to that of bacteria, without internal structures, its cell membrane is surrounded by a cell wall and may have flagella. They resemble gram-positive bacteria with the cytoplasmic membrane and the cell wall, without periplasmic space, with some exception in which there is a periplasmic space in which vesicles with a membrane can be found.

However, the lipids of the archaea membranes are very different from those of other life forms, such as bacteria or eukaryotes, and are probably more resistant to extreme conditions. In bacteria and in eukaryotes, there are phospholipids, which are composed of a non-polar hydrophobic part (fatty acid without branches, or rings), a hydrophilic polar part linked to glycerol (D-glycerol) and phosphate, through a type bond ester, forming layered structures (lipid bilayer). The most common esters are formed by the union of an acid and an alcohol (-COOH and -OH groups), so that the hydroxyl radical -OH, is replaced by the -COO group of the fatty acid. The hydrogen atom (H) of the acid group joins with the hydroxyl radical of the alcohol to form water (H2O).

In archaea, phospholipids are also composed of a hydrophobic part (long and branched isoprenoid structure, sometimes with cyclohexane or cyclopropane rings) and a polar part of glycerol (L-glycerol) and phosphate, but linked by an ether-type bond, whose resistance is much higher, for example, at high temperatures. Isoprenoids, or terpenes, are hydrocarbons of 5 carbon atoms derived from 2-methylbutane-1,3-diene. The ether type bond is a group of type R-O-R', where R and R' are alkyl groups, for example of two alcohols (ROH + HOR' à R-O-R'+ H2O). These groups are very hydrophobic and do not tend to be hydrolyzed. Some Archaea, instead of having a lipid bilayer, have a monolayer resulting from the fusion of two hydrophobic chains, forming a single molecule with two hydrophilic polar groups that could confer greater rigidity to extreme conditions, such as acidity.

Most archaea have a cell wall composed of proteins that form a rigid grouping (S layer) that covers the outside of the cells forming a mesh that protects the cell membrane chemical and physical. They lack peptidoglucan, except for methanogenic archaea that have a pseudopeptidoglycan that lacks N-acetylmuramic acid and amino acids

Archaea can obtain their energy from inorganic compounds such as sulfur or ammonia (lithotropic archaea, such as nitrifiers, methanogens and anaerobic methane oxidants) and as a carbon source inorganic compounds or nitrogen fixation. Others use sunlight as a source of energy (phototrophic archaea, but without photosynthesis) and as a carbon source they use organic compounds. Finally, others obtain the energy of organic compounds (organotrophic archaea) and the carbon source is obtained from organic compounds or carbon fixation.

Archaea can live in many habitats and could make up 20% of the Earth's biomass. Many are extremophiles and it was thought that this was their only ecological niche. For example, some of them live at very high temperatures (thermophilic archaea - > 45ºC- and hyperthermophilic - > 80ºC-), even higher than 100ºC; others are found in very cold habitats, in very saline waters (halophilic archaea), acidic (acidophilic archaea, even at pH 0) or alkaline (alkalophilic archeae). Others develop at milder temperatures (mesophilic archaea) and live in soft, humid conditions such as oceans and soils.

Relationship of archaea with other living beings

The archaea are found in free life in the wild in soils, waters, extremophile environments such as hot water springs and acid or alkaline waters. They have also been found in relation to living beings in situations of mutualism or commensalism. In relation to situations of pathogenicity, its implication in oral infections has only been suggested.

In situations of mutualism methanogenic archaea have been found in the digestive system of animals that digest cellulose such as ruminants and termites, associated with protozoa. Protozoa would digest plant cellulose for energy, releasing hydrogen, but this would reduce the energy released. However, the presence of archaea would convert hydrogen into methane (CH4) by using CO2 as the final electron acceptor (4H2 + CO2 à CH4 + 2H2O), benefiting the protozoa. Some archaea would live inside protozoa consuming the hydrogen produced by them in the hydrogenosomes, and the same would happen in some marine sponges. In the human intestinal flora there is the methanogen Methanobrevibacter smithii, which, as in termites, could be mutualists interacting with other microbes to contribute to the digestion of food. They have also been associated with corals and with the roots of plants.

Interest in technology and industry

The extreme conditions in which these microorganisms can develop are possible because they have enzymes that can exert their function under these conditions. For this reason some of its enzymes are being used to perform reactions in extreme conditions. This is the case of some thermostable DNA polymerases, such as the Pfu DNA polymerase (Pyrococcus furiosus) that is used in Molecular Biology. The same occurs with amylases, galactosidases, etc., of other Pyrococcus species that perform their function at more than 100 °C, with which food can be processed at high temperatures (milk low in lactose or whey). Methanogenic archaea are being used to treat wastewater by performing the anaerobic digestion of waste producing biogas. Acidophilic archaea are used in mining to obtain metals such as gold, cobalt and copper. The obtaining of antibiotics of these microorganisms is being considered.

Tests carried out in IVAMI:

  • Cultivation and molecular identification of archaea isolated in culture.

As it is possible that different species of archaea can coexist in any of the environmental conditions in which they are found, we must cultivate the sample instead of going directly to its molecular detection. For example, hypersaline environments include Haloarcula spp., Haloterrigena spp., Haloferax spp., Halorubrum spp., Halobacterium spp., Halonotius spp., Haloquadratum spp., etc.). In some environments halophilic archaea may coexist along with methanogenic archaea.

The client must indicate the conditions in which the sample was to guide the cultivation in the laboratory: salinity and its concentration if known; temperature; acidity; alkalinity, intestinal; ...

  • Molecular detection only: when you only want to know if archaea exist, molecular detection could be carried out.

Recommended sample:

  • The sample will depend on the place where you want to detect its presence: water, soil, intestine of animals, food, ...: Approximately 100 grams or 100 mL are required.

Conservation and shipment of the sample:

  • Environmental conditions in which the sample is usually found, except in the case of samples that are at high temperatures that can be remitted at room temperature.

Delivery of results:

  • 10 to 15 days depending on whether we have to identify or not. The culture is prolonged since an enrichment must be carried out with subcultures at 48 hours and 7 days, with the molecular identification if they are present.

Cost of the test:

  • Molecular identification: Consult to (must be decided how many identifications they want to be made if more than one type of different colonies are isolated by phenotypic characteristics or if they only want the majority, both more abundant, or all).