Psychrophiles : Ecological significance and potential industrial application

The bulk of earth’s biosphere is cold 5 °C that sustains a broad diversity of microbial life by triggering physiological response(s) to ensure survival in cold and frozen ecosystems. The strategy of adaptation to cold environments includes changes in membrane composition and induction of a set of specific cold-active proteins, polyunsaturated lipids and exopolysaccharides. These adaptive features provide an enormous natural reservoir of enzymes that function effectively in cold environments, and these coldactive enzymes have been targeted for innovative applications useful to humankind. This review provides an overview of the existence, distribution and adaptation strategies of psychrophilic microorganisms worldwide with great emphasis on their recently emerged industrial applications in the textile industry, food and dairy industry, brewing and wine industry, laundry detergent industry, and others. 
 Significance: 
 • The outcome of these studies may also help in the exploration of the possibility of life in distant frozen planets.



Introduction
A great proportion of the earth's biosphere (>85%) permanently experiences temperatures below 5 °C. 1 The largest coverage of these cold environments is successfully colonised by a wide diversity of extremophilic microorganisms, including bacteria, archaea, yeasts, filamentous fungi and algae. 2,3In 1887, Forster first reported the existence of bacteria capable of growth at 0 °C, especially in sea water and ocean fish. 4Cold-adapted or coldloving microbes are termed 'psychrophiles', and have cardinal growth temperatures (minimum, optimum and maximum) at or below 0 °C, 15 °C and 20 °C, respectively, while microorganisms that withstand cold temperatures with a higher growth optimum and maximum (above 25 °C) are called 'psychrotolerant'. 5 The ability to thrive at such low temperatures requires a vast array of adaptations to maintain the metabolic rates and sustain growth compatible with life in these severe environmental conditions. 6Because of their attractive properties, extensive research on psychrophilic microorganisms has been conducted to understand their survival strategies that include genetic and acclimation processes and adaptation mechanisms. 7e purpose of this review is to summarise the tremendous significance of this group of microorganisms in fundamental research, pharmaceuticals, medicine and recent biotechnological applications.

Psychrophiles: Historical background
In 1887, Forster early investigated the isolation of microorganisms from fish preserved by cold temperatures that were bioluminescent. 8Later in 1892, Forster reported other bacterial isolates from various environments (natural water, food, surface and intestines of freshwater and seawater fish) that were able to grow at 0 °C. 4chmidt-Nielsen (1902) first mentioned the term 'psychrophile' to describe bacteria capable of growing at 0 °C. 9owever, Müller (1903) criticised this term and demonstrated that while these organisms were able to grow at low temperature they actually grew more rapidly at elevated temperatures. 10Thus, Kruse (1910) suggested that these bacteria might be better called 'psychrotolerant', but this suggestion or others of similar nature did not gain acceptance and the term 'psychrophile' has retained. 11This confusion was ended in 1975, when Morita proposed a definition to the term 'psychrophile' for cold-adapted or cold-loving microbes, having minimum, optimum and maximum growth temperatures at or below 0 °C, 15 °C and 20 °C, respectively. 12

Habitats and biodiversity
Cold habitats dominate the vast majority of our planet, covering three-quarters of the earth's surface, and span from the Arctic to the Antarctic and from high-mountain regions to the deep ocean. 13,14The major fraction of this low temperature environment is represented by the deep sea (90% of the ocean volume), followed by snow (35% of land surface), permafrost (24% of land surface), sea ice (13% of the earth's surface) and finally glaciers (10% of land surface).Other cold environments are cold-water lakes, cold soils, cold deserts and caves. 15These earth dominant environments are successfully colonised by enormously diverse communities of psychrophilic bacteria, archaea, algae, yeast [16][17][18][19][20] , insects 21 and fish 22 , that are able to thrive and even maintain metabolic activity at subzero temperatures.
Bacteria represent very important members of the sea ice habitat, including many unique taxa. 23Heterotrophic gasvacuolate bacteria, not reported in other marine habitats, have been discovered in and near sea ice. 24Among those cold-adapted bacteria, the genus Colwellia provides an unusual case.Members of this genus produce extracellular enzymes capable of degrading high-molecular-weight organic compounds.These traits make Colwellia species important to carbon and nutrient cycling wherever they occur in the cold marine environment, from contaminated sediments to ice formations as analogs for possible habitats on other planets and moons (e.g.Mars and Europa). 13presentatives of the family Vibrionaceae are among the most commonly reported bacteria to populate almost all extreme environments. 25Nevertheless, a wide range of phylogenetic diversity within the genera

Existence and metabolic adaptation
Survival of psychrophiles in harsh and extremely cold environments is interesting and requires a vast array of unique adaptive features from all their cellular components. 34,35Chintalapati et al. 36 categorised three phases of cold-shock response.Phase I, Acclimation Phase, immediately follows cold exposure, leading to reduced growth rate as a result of reduced membrane fluidity, and several cold-shock proteins are produced.During Phase II, Recovery Phase, cells are considered 'cold-adapted' and resume growth and bulk protein synthesis restarts.During Phase III, Stationary Phase, non-cold-shock proteins are synthesised, allowing cells to proceed to slow-rate growth at low temperature. 37 general, bacteria owe the ability to cope with such challenges to complex strategies.One important strategy is directed towards their extremely efficient DNA repair mechanism sustained under frozen conditions. 38his evidence for active DNA repair mechanism in icy environments is a true reflection of their physiological potential and survival in frozen substrates for extended time frames of up to 600 000 years. 39

Regulation of membrane fluidity
Another important and the most frequent adaptive strategy relates to the ability of the cell to regulate or modulate the fluidity of the membrane in freezing environments.The membrane is the first barrier that can sense environmental changes and it acts as an interface between external and internal environments, so as to overcome the deleterious effects of harsh conditions. 36Shivaji and Prakash 35 reported that membranes become more rigid at cold temperatures, which activates a membrane-associated sensor resulting in upregulation of genes involved in membrane fluidity modulation for exchange of metabolites from and to the cell.Consequently, changes in the membrane lipid composition facilitate this process.This is achieved by modifications in the lipids' fatty acyl chains that serve to maintain optimum membrane fluidity. 40In general, lower growth temperatures govern the activation of a group of cold-shockactivated enzymes called 'desaturases' which convert saturated acyl fatty acids to unsaturated ones and aid the increase in the proportion of unsaturated acyl chains, reduction in the acyl chain length and increased methyl-branched fatty acids. 36Fungal cell membranes show evidence of similar changes at cold ecosystems to maintain their fluid state.The degree of unsaturated fatty acids increases at low temperatures in Candida, Leucosporidium and Torulopsis as reported by Kerekes and Nagy 41 .

Carotenoid pigments
Predominance of carotenoid pigments, in several bacteria isolated from Antarctic sea ice, has been reported to play an important role in the maintenance of membrane structure, fluidity and protection from UV radiation. 42Accumulation of C-50 carotenoid, Bacterioruberin, observed in the psychrotrophic strain, Arthrobacter agilis, was postulated to play a crucial role in the regulation of membrane fluidity at low temperatures. 43urthermore, several polar and non-polar carotenoid pigments, synthesised by Micrococcus roseus and Sphingobacterium antarcticus Antarctic strains, were found to bind vesicles, made of both synthetic and natural lipids, and to rigidify them. 44

Anti-freeze proteins
Anti-freeze proteins (AFPs) are ice-binding proteins that have the ability to decrease the freezing point of water and show extracellular ice recrystallisation inhibition activity during latter stages of the warming cycle. 45,46Furthermore, AFPs have been well known in promoting super cooling of the body fluids at subzero temperatures to prevent freezing of blood in polar fish. 47In addition, those proteins have been reported in insects and plants. 48,49Duman and Olsen 50 first demonstrated the presence of AFPs in cold-adapted bacteria and the bacterium Moraxella sp. was the first reported Antarctic strain to produce an AFP. 51Some psychrophilic AFPs have been purified from cell extracts of Micrococcus cryophilus, Pseudomonas putida and Rhodococcus erythropolis. 52

Cryoprotectants
Cryoprotectants are exopolymeric substances (e.g.sugars, alcohols and amino acids), produced in high amounts and believed to prevent cold-induced aggregation of proteins and maintain optimum membrane fluidity under unfavorable low temperature. 53,54In 1994, Ko et al. 55 demonstrated the growth-enhancing effect of glycine betaine on Listeria monocytogenes at low temperature.Furthermore, trehalose showed evidence in preventing protein denaturation and aggregation in psychrophilic bacteria. 56In fungi, trehalose is an important stress protectant and stabiliser of membranes, accumulated in fungal hyphae in large quantities at low temperatures. 57

Cold-shock proteins
Cold-shock proteins have been extensively characterised and are considered the most prominent response of cells to cold shock in order to counteract the detrimental effect of temperature downshift; they play a critical role in cold adaptation. 58Cold-shock proteins have been functionally linked to the regulation of cellular protein synthesis, particularly at the level of transcription and initiation of translation.They also act as chaperones by preventing the formation of mRNA secondary structures (mRNA 'folding') and maintenance of chromosome structure. 59,60These stress proteins, together with cold-acclimation proteins, have been detected and exclusively overexpressed during the entire growth of several Antarctic bacteria. 56Furthermore, Kawahara et al. 61 believed that the cold acclimation protein (Hsc 25) produced in an ice-nucleating bacterium Pantoea ananas KUIN-3, was capable of refolding cold-denatured enzymes that sustain biological activities following an abrupt temperature downshift.

Cold-active enzymes
Cold-active or 'cold-adapted' enzymes are those enzymes that display high catalytic efficiency at low temperature compared to their mesophilic counterparts. 62Furthermore, Struvay and Feller 63 reported multiple adaptive features developed by constrained psychrophiles to design enzymes perfectly compatible to the given environment.In the first attempt, psychrophiles produce enzymes that are highly flexible in structure, having an up to 10-fold higher specific activity (k cat ) than their mesophilic homologues, thus providing better access to the active site of substrates at lower temperatures. 64These cold-active enzymes offset the inhibitory effect of low temperatures on reaction rates and maintain adequate metabolic fluxes to the growing organism.Secondly, the apparent maximal activity of cold-active enzymes is shifted towards low temperatures to cope with the increased viscosity of the aqueous environment induced under cold conditions.In this context, these enzymes are heat labile and frequently inactivated at temperatures that are not detrimental for their mesophilic counterparts. 65Finally, apart from their high catalytic efficiency, the adaptation to cold is not usually perfect, as the specific activity exhibited by most psychrophilic enzymes around 0 °C remains generally lower than that of their mesophilic counterparts at 37 °C.
Pioneering studies compared cold-active enzymes to their conventional mesophilic forms and observed significant differences in amino acid composition, mostly in the active site domain, responsible for their activity under cold conditions. 66The criteria for structural alterations include increased clusters of glycine residues (providing local mobility) http://www.sajs.co.za May/June 2018 and reduction in proline and arginine residues in loops, which provides enhanced chain flexibility between secondary structures and the capability of forming multiple salt bridges and hydrogen bonds, respectively. 6However, surprisingly, Aghajari et al. 67 reported great similarity in the three-dimensional structure of a cold-adapted α-amylase enzyme of a psychrophilic bacterium, Alteromonas hulopfuncris, to those of mammalian α-amylases.

Biotechnological applications
Over recent years, considerable attention has been focused on psychrophilic microorganisms that proliferate in extremely cold niches and the fascinating biotechnological potential of their cold adaptation.Although Witter 11 reported the significant opportunity of psychrophilic bacteria in the dairy industry and in keeping milk fresh under refrigeration facilities for longer holding times as far back as 1961, studies on psychrophiles have been accentuated by the advancements and applications in avoiding potentially disastrous situations in various industries, including those concerned with food production, waste processing, mining, environmental bioremediations, agriculture, and medicine and molecular diagnostics. 75,76

Bioremediation
Because of increasing human activities and demand for fossil fuel energy, spillage of petroleum products is a growing menace to the environment.Psychrophiles hold tremendous potential as 'environmental cleaners' to successfully degrade pollutants petroleum hydrocarbons in extreme cold conditions. 77,78Whyte et al. 79 reported that the microbial catabolic pathways responsible for the degradation of petroleum hydrocarbons, including n-alkanes and polycyclic aromatic hydrocarbons, are widespread in cold regions.Furthermore, several indigenous, cold-adapted microbial populations that use petroleum hydrocarbons have been detected, including Rhodococcus 80 , Pseudomonas 81 and Pseudoalteromomas 82 .

Food industry
It has indeed emerged that cold-active enzymes represent an extremely powerful tool in the food industry.There is an increasing industrial trend to treat foodstuffs under low temperature conditions in order to avoid detrimental effects on taste, texture and nutritional value, and also to save energy.Cold-active β-galactosidase, which hydrolyses lactose to glucose and galactose at refrigerating temperature, is a potentially important enzyme in the dairy industry.It can be used to produce lactosefree milk-derived foods for lactose-intolerant people, who represent approximately 30% of the world population. 83Furthermore, it can be used to convert lactose in whey to D-tagatose -a natural high-addedvalue sweetener with low caloric and glycemic index. 63Hoyoux et al. 84 patented a cold-active β-galactosidase from an Antarctic psychrophile, Pseudoalteromonas haloplanktis, for its capacity to hydrolyse lactose during milk storage at low temperatures.
In this context, application of cold-active pectinases in the fruit juice industry is highly demanding to retain the quality and nutritional properties of the fruit juice and facilitate different processing stepsliquefaction, clarification and juice extraction -at low temperatures.Furthermore, residual enzyme activity could be easily eliminated by enzyme inactivation by moderate heat input after treatment. 85In wine industries, cold-active pectinases, isolated from yeasts and fungi, are believed to increase the production and retention of volatile compounds, thereby improving the aromatic profile of wines. 86so, it is worth mentioning that cold-active xylanases, hydrolysing β-1,4-xylan present in all flours, are one of the key ingredients of industrial dough conditioners used at cool temperatures required for dough resting to improve bread quality.Following careful baking trials, xylanase from the Antarctic bacterium Pseudoalteromonas haloplanktis, effectively improved the mechanical dough properties and final bread quality with a positive effect on loaf volume. 63

Medical and pharmaceutical applications
There is a growing interest in studying psychrophilic bacteria as new tools in pharmaceutical and cosmetic applications.Consequently, several promising psychrophilic strains were detected as a valuable source of new active antimicrobial compounds at low temperatures, as reported by Tomova et al. 87 Recently, compounds produced by the halophilic Antarctic actinomycete Nocardioides sp.strain A-1, with antimicrobial activity against Xanthomonas oryzae which causes bacterial blight disease in rice-producing countries, exhibited promising application in agriculture for plant protection. 88In addition, it was found that Antarticine-NF3, an antifreeze glycoprotein produced by the Antarctic bacterium Pseudoalteromonas, is effective for scar treatment and has been included in cosmetic regeneration creams. 89rthermore, polyunsaturated fatty acids, produced to improve membrane permeability and nutrient transport in psychrophilic bacteria, may constitute an economic alimentary source for aquaculture industries with favorable activities on cholesterol and triglyceride transport for optimal nervous system and cardiovascular health. 90 the industrial level, the polar yeast Candida antarctica is used to produce two cold-active lipases, A and B, sold as Novozym 435 by Novozymes (Bagsvaerd, Denmark) involved in a very large number of applications related to pharmaceuticals and cosmetics. 91

Detergent and fabric industry
Considering the versatile properties of cold-active enzymes -such as high catalytic efficiencies at low temperature, lower thermal stability and novel substrate specificities -they offer a large reservoir of potentially novel biotechnological exploitation. 75Nowadays, the detergent field, which represents 30-40% of all enzymes produced worldwide, requires more enzymes that are capable of working at low temperatures in the context of energy saving.Cold-adapted enzymes actively used in detergent formulations -lipases, proteases and α-amylases -are systematically introduced to improve the efficiency of detergents and reduce the amount of chemicals used in order to protect the texture and colours of fabrics and reduce wear and tear during washing. 6,64,92urrently, cold-active subtilisins, isolated from Antarctic Bacillus species, are incorporated in cold-active detergents that combine storage alkaline stability and cold activity required for optimal washing results. 63rthermore, the application of cold-adapted cellulases in fabric production and denim finishing could increase the smoothness and softness of tissues, decolourisation of textile effluents and textile bleaching and allows the development of environmentally friendly technologies in fibre processing. 93

Conclusion
Over recent years, a wealth of knowledge has been accumulated on psychrophilic microorganisms and their cold-shock response catalysts.Because of their unique biological features, psychrophiles can be significantly exploited in biotechnological industries such as the pharmaceutical, enzyme production, bioremediation, biosensor, cosmetic, agriculture, domestic purposes and textile industries.The outcome of these studies may also help to explore the possibility of life in distant frozen planets and their satellites.