Extremophilic (salt rock). These sites are of particular interest

Extremophilic organisms are ‘extreme-loving’, meaning that
they inhabit some of the harshest conditions known to man, including extreme
temperatures, ion concentrations and salinity. Merely a few decades ago it was thought
that it was near impossible to find life in such harsh environments, but It has
since been revealed that extremophilic microorganisms have evolved to thrive in
these extreme conditions.

Halophiles are extremophilic microorganisms, such as
bacteria, archaea and some eukaryote, which thrive is high salt (NaCl)
environments. Halophiles can be classified as slight (<5% salinity), moderate (5% - 20%), or extreme (20% - 30%) depending on the degree of halotolerance. These microbes have adapted to thrive in hypersaline (high salinity) environment which would kill most microorganisms via plasmolysis. This study focuses on halophiles in the archaea and bacteria domain. These halophiles utilize two key methods to maintain an osmotic balance across their cellular membrane, therefore preventing plasmolysis. The most widely used method is the incorporation of osmoprotectants (compatible solutes) into the cells cytoplasm, these molecules act as osmolytes, negating the osmotic stress of the high salt environment by behaving like salt molecules. An alternative method induces an influx of NaCl into the cells cytoplasm, this causes the NaCl concentration across the cellular membrane to be balanced. This method has a notable floor, NaCl dehydrates and denatures proteins such as enzymes, ultimately causing cell death. The halophiles which utilize this method develop negatively charged amino acid residues attached to the cytoplasmic exposed exterior of the enzyme, these negative residuals attract water molecules to the enzyme keeping the protein hydrated, therefore preventing denaturing of the enzyme. These accumulating factors add to the resilience of halophiles making them key research subjects for biotechnological applications. 2.2. Hypersaline Biotopes Halophiles inhabit a significant proportion of the earth due to a rich array of hypersaline (high salinity) environments, partly due to 96.5% of the water on earth containing salt, as well as the abundance of non-liquid salt environments. A range of hypersaline have been selected for study, including: Anderton Boat lift (brine springs), INOVYN (artificial brine), and Winsford Salt Mine (salt rock). These sites are of particular interest due to the environments containing a range of salinities allowing a diverse microbiome of halophiles to evolve filling specific niches. Additionally, minimal research has previously been undertaken on all sites, therefore increasing the probability of novel discoveries for biotechnological applications with the added potential of finding new and innovative species. 2.3. Biotechnology Biotechnology is an area of biology which focuses on harnessing the natural processors of living systems and organisms for use in technological and industrial processes. Microorganisms are widely used in biotechnology due to their unique properties, such as, bioplastic production, bioremediation, pharmacology and Food processing. These versatile uses all stem from the ability of microbes to produce enzymes (biocatalysts). Despite recent developments and successes in biotechnology, wider use is frequently hindered by a combination of several factors. Among these, the mismatch between resilience of used microbial strains (and their biomolecules) and the harsh conditions associated with industrial processes, as well the high economic costs of maintaining a sterile environment, and extracting bioproducts, are seen as major limitations.  Many of these can be overcome by the use of halophiles, which explains the current boom in biotechnological research associated with these organisms. 2.2.1. Hydrolase Hydrolase are a collection of enzymes which degrade molecules by break intermolecular bonds during a hydrolysis reaction. The most common hydrolase enzymes include, protease, lipase and amylase, which degrade proteins, lipids, and starch, respectively. All these enzymes are known to be secreted by select microbes. This is due to many microbes encountering these substrates in their environment, and therefore utilize the materials they encounter for cellular functions. The application of these enzymes are restricted due to a reduced stability in exstream pH, ion concentrations and salinity. Hydrolase have been used in biotechnology for century's and have undergone new uses as time has progressed. In particular, the hydrolase enzymes, protease, lipase and amylase have seen wide use in biotechnology, they each have specific uses but also share a wider application. For example, all three have been used in extensively in the food industry during cheese fermentation and the production of bread products. Additionally, they have seen a more recent use in detergents, pharmaceuticals, cosmetics and medical applications, such as Pancreatic Enzyme Replacement Therapy. This illustrates the significants of discovering novel enzyme producing microbes which can produce a greater yield of produces and are more efficient at the diversity of roles they are applied to. 2.2.1.1. Protease Proteins are created in all organism via condensation reactions forming covalent peptide bonds between amino acids. Many microbes rely on proteins in their environment as a source of supplementary amino acids. Hence why microbes can produce protease which hydrolyses peptide bonds, causing large polymer proteins to be broken into amino acid peptides and monomers that can then be metabolized by the microbe. This process has specific biotechnological applications in agriculture due to the recycling of proteins being essential to the global nitrogen and carbon cycles. 2.2.1.2. Lipase Lipids are biomolecules which are not soluble in non-polar solvents due to them primarily consisting of fatty acids terminating in a carboxylic acid group; this structure results in a molecule with a polar (hydrophilic) end, and a non-polar (hydrophobic) end. These lipids constitute a large quantity of organic products such as fats and oils. Many of the molecules these substances consist of are frequently used in biotechnology, therefore lipase has been introduced as an effective biocatalyst. For example, the growing demand for biofuels has rendered a requirement of an increased yield and more successful purification process (Andualema et al., 2012). Lipase has been successfully used to convert oils to fatty acid methyl esters (FAME), FAME molecules are the main components of biofuel and can be acquired using lipase more readily than traditional methods (M. A. Naidu, P. Saranraj 2013). 2.2.1.3. Amylase Carbohydrates such as starch are a key source of energy for many microbes. Starch molecules consist of a chain of glucose molecules, these glucose molecules can be obtained and used for energy via hydrolysis of the starch bonds using the enzyme amylase.  Much like other hydrolase enzymes amylase can be extracted from animals, plants and microbes. But the resilience of microbial enzymes extracted from halophiles and generally faster growth rate than other organisms have allowed halophiles to meet the industrial standards. This has happened to such an extent that amylase has almost completely replaced standard chemical hydrolysis of starch in industry (M. A. Naidu, P. Saranraj 2013). Due to Amylase producing glucose sugars it has found a wide us in the food industry for the production of syrups such as fructose corn syrup and maltose syrups, as well as used in paper and textiles industry's. 2.2.2 Bioplastics With an ever-increasing amount of plastic waste being produced, there is a growing interest for alternative material with the same diverse application of plastics derived from crude oil, but without the dangerous environmental impacts. Microbes could be the optimum producer such a material. Bioplastics are polymers which are not formed via the traditional methods of plastic production, but rather produced as a by-product of microbial metabolism. Unlike conventional plastics, bioplastics are eco-friendly due to their biodegradability but still maintain many of the useful characteristics as standard plastics. Polyhydroxyalkanoates (PHA) is one such plastic, known to be produced by halophiles which express the enzyme PHA Synthase. PHAs are polyesters which are produced by microorganisms primarily through the fermentation of sugars and lipids. The variability of this bioplastic is amplified by its potential to bind with over 150 different monomers changing its structure and therefore its biotechnological uses, for example PHAs can be both thermoplastic or elastomeric, meaning that they can have both strong molecular bonds (hard plastics), and weak molecular bonds (weak plastics), therefore have a range of uses from surgical mesh to nails. Additionly, what makes PHAs a sustainable plastic in biotechnology is the yield with some microbes 80% of their dried weight being PHAs. Halophiles have been used for this pourpose partily due to the possibility of using sea water insead of fresh water which as well as reduced contaminations is far more abundant than fresh water. Also, halophiles are able to contiunulously produce PHA allowing for a continuous fermentation process instead of a batch one, further increasing the yield. Another advantage is that PHA can be made from wast productis such as kitchen waste. 2.2.4 Anti-Microbial Potential Due to increased use of anti-microbial drugs in the past 200 years microbes have evolved to accumulate an array of resistances to the widely used drugs. This has initiated an "arms race" between drug development and antibiotic resistance in microbes. It is of upmost importance to discover new antimicrobial compounds to aid those whose immune systems cannot fend of microbial attacks. Many microbes produce anti-microbial compounds in order to out compete the many microbes in their natural environment. Many habitats have been screened for anti-microbial producing microbes, one such habitat that has undergone limited screening for anti-microbial producing microbes is hypersaline biotopes. The few studies which focus on this area have proven that there is great promise on finding novel anti-moicrpbial compounds, in particular there appears to be an increased rate of anti-microbial compounds which can disrupt the growth of Gram-positive bacteria opposed to Gram-negative bacteria (Lei Chen et al., 2010). Discovory of such compounds could potentially lead to the development of new antibiotics. 2.3 Hypersaline Biotopes Hypersaline biotopes are a form of extreme environment which surpass the standard salinity of sea water (3.5%). Salt is an ionic compound formed from sodium cations (Na+), and chloride anions (Cl-) which are abundant in hypersaline environments in the form of sodium chloride (NaCl). Hypersaline environments are found across the earth in an array of terrestrial environments such as salt mines and flats. Additionally, salt can dissolve in polar aqueous (water) environments to form brine springs, pools and lakes. Cheshire salt district located in North West United Kingdom (UK) is home to a range of hypersaline environments both terrestrial and aquatic. This district consists of Upper Triassic Mercia Mudstone containing large salt deposits which lay the foundations This diverse salt driven area has a large salt vein running under it which has been utilized as a source of income for the districts over the past few centuries. Very limited research has been done on these sites when looking for halophiles, furthermore virtually no research has been done of the potential of biotechnologically relevant microbes on these sites. There are three key sites that have been screened for biotechnological halophiles. 1) Anderton Boat Lift, this nature reserve is home to a selection of brine springs and their resulting brine pool. Natural water springs reside beneath the site, while being pushed to the surface the water passes through layers of salt rock which dissolve into the water giving it the high salt concentrations associated with brine. In the past companies utilized these brine springs for the production of sodium carbonate (Na2CO3), the by-products of this reaction include calcium chloride (CaCl2). The waste products where then dumped on the location of the brine springs giving the site an unusual composition giving rise to rare flora, hence why the site is now considered a Site of Biological Interest (SBI). 2) INOVYN, this chemical company produces artificial brine which is utilized in industrial processes. They acquire this brine by pumping high presser water into the salt veins which dissolved the salt into the water and then they extract the water. The caverns grow larger as more salt is extracted giving a larger surface area, therefore the older the brine the high its salt concentration, two brines where sampled an old and a new. 3) Winsford salt mine, this is the largest salt mine is the UK as well as being old, being mines since the 17th century. This mine consists of copious quantities of near pure rock salt which is home to numerous halophiles. Additionally, this mine also has a brine pool formed via condensation during the mining process. All three of these locations have unique qualities making them key sites for the discovery of novel halophiels.

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