Microalgae from Cold Environments and Their Possible Biotechnological Applications

Cold environments include deep ocean, alpine, and polar areas. Even if the cold conditions are harsh and extreme for certain habitats, various species have been adapted to survive in them. Microalgae are among the most abundant microbial communities which have adapted to live in low light, low temperature, and ice coverage conditions typical of cold environments by activating different stress-responsive strategies. These species have been shown to have bioactivities with possible exploitation capabilities for human applications. Even if they are less explored compared to species living in more accessible sites, various activities have been highlighted, such as antioxidant and anticancer activities. This review is focused on summarizing these bioactivities and discussing the possible exploitation of cold-adapted microalgae. Thanks to the possibility of mass cultivating algae in controlled photobioreactors, eco-sustainable exploitation is in fact possible by sampling a few microalgal cells without impacting the environment.


Introduction
Oceans cover more than 70% of the Earth's surface, and are characterized by different environment typologies to which several organisms, sometimes specific ones, are adapted. Cold ecosystems, with a stable temperature below and/or at 0 • C, include deep ocean, alpine, and polar environments, areas where environmental conditions can become extreme. As the most abundant cold-adapted life-forms on earth, microorganisms dominate these environments in terms of species diversity and biomass [1]. Cold-adapted microorganisms include "psychrophilic" and "psychrotrophic" organisms; the former enjoy optimal growth at temperature ≤15 • C, while the latter are able to grow at temperatures below 15 • C but show a maximum optimal growth rate at >18 • C [2, 3]. Thanks to their high-efficiency and energy-saving catalytic properties, cold-adapted microorganisms have become valuable natural resources with potential in various biological fields. The Arctic and Antarctic ecosystems are characterized by a high level of biological and genetic diversity, but are less studied compared to more accessible sites and are largely unexplored from the bioactivity point of view. This is mainly due to difficulties in sampling in such extreme habitats, e.g., ice coverage, as well as costs.
Environmental adaptations underlying physiological and molecular strategies are mechanisms allowing survival among cold-adapted microorganisms [4,5]. Adaptations to extreme, even hostile, temperatures can allow organisms to not only survive but to thrive and tolerate the extreme conditions of these natural environments. One of the major adaptations of metabolic function is the maintenance of membrane fluidity, which influences growth and photosynthesis at low temperatures [6][7][8]. Other general strategies include the ability to regulate the expression of structural enzymes, the expression of heat shock proteins, and the presence of compatible solutes.
Considering that temperature influences all the biochemical reactions of cells, the study of adaptations to temperature conditions is widespread. Recently, Lauritano et al. [4] reviewed physiological and molecular responses to the main environmental stressors of microalgae in polar marine environments. As reported in their review, low temperatures induced the maintenance of membrane fluidity in microalgae thanks to unsaturated fatty acids and the expression of cold shock/antifreeze proteins. In addition, depending on the concomitant variation of salinity and light intensity, variations in various lipid and pigment concentrations have been reported [4,9]. In particular, in the presence of high levels of ultraviolet radiation, an increase in photoprotective pigments, chlorophylls, and carotenoids has been found [10].
Furthermore, in the past decade cold-adapted microorganisms have attracted attention for their potential applications in biotechnology. Due to their metabolic activity at colder temperatures, these organisms or their derived products can be used in manufacturing or processing steps that require low temperatures [11,12]. Cold marine environments, including high latitude oceans, are dynamic across multiple spatial and temporal scales due to the complex interactions of ice formation and melting, ocean circulation and mixing, and terrestrial and atmospheric inputs.
The primary production of phytoplankton and sea ice algae in high latitude oceans is very high [13,14]. Microalgae are between the most abundant sea ice microbial communities that have adapted to live in extreme conditions and thrive within the distinct microhabitats created when sea ice forms and develops [15][16][17][18][19][20]. After becoming embedded in the ice, the algae must survive in extreme conditions of little or no light, high salinity, and cold temperatures within the sea ice brine. Their capability to live in different environments results in a portfolio of different taxonomic species and in the production of very different chemical mediators [21]. Microalgae have in fact been shown to be excellent producers of several bioactive molecules, and many studies have demonstrated that microalgal raw extracts, fractions, and/or pure molecules have biological activities with possible applications in the treatment of human pathologies, including antioxidant, anticancer, anti-microbial, and immunomodulatory activities [21].
In addition to studies on cold adapted species, experiments in the laboratory have been performed to mimic natural conditions in order to study both their growth and physiological responses as well as to stress microalgae and induce the activation of metabolic pathways to obtain new activities and products. For instance, various researchers have applied the OSMAC ("one strain, multiple compounds") approach, which means that different culturing conditions are used in order to stimulate the production of a high number of metabolites. Stressful culturing conditions may include variations in medium salinity, pH, temperature, nutrient concentrations, bubbling, and exposure to predators. Previous reviews have focused on bacterial and microalgal physiological and molecular responses in polar marine environments [4], omics approaches applied to polar microalgae linking the results with biochemical and physiological data ( [22] and chapter by [23]), and extremophile microalgae, including various acidophilic/metallotolerant, alkaliphilic, endolithic/endophytic, halophilic, halotolerant, and cryophilic/psychrophilic microalgae, as well as their potential exploitation in the context of astrobiology [24]. The aim of the current review is to collect bioactivity information on microalgae living in cold environments, mainly polar ones, or exposed to cold stress in laboratory conditions, in order to discuss their activity properties and possible eco-sustainable exploitation for different applications (Figure 1).

Figure 1.
This figure summarizes how microalgal biodiversity from cold environments, as well as microalgae exposed to cold stress, may be sources of valuable products with different industrial applications, including ingredients for cosmetics, drugs, and biodiesel production. Chemical structures of (a) eicosapentaenoic acid (EPA) and (b) docosahexaenoic acid (DHA) were retrieved from the public database PubChem [25-27].

Cold-Living Microalgae Bioactivities
The adaptation mechanisms of polar microalgae have evolved by developing ecological, physiological, and molecular defensive and adaptive strategies to withstand the harsh polar environment characterized by low temperatures, freeze-thaw cycles, desiccation, salinity, and high and variable photosynthetically active and ultraviolet radiation [28,29]. These adaptive strategies include the synthesis of a huge diversity of compounds originating from different metabolic pathways that protect them from stress caused by extreme environmental conditions [30]. Different compounds such as PUFAs [31,32], carotenoids [33,34], phenolic [34], UV-absorbing compounds [33], and exopolysaccharides [35] are overproduced by extreme environment microalgae [29], and have attracted biotechnological interest in various sectors [36].
In addition to studies analyzing single species, Ingebrigtsen et al. [40] investigated fifteen bulk plankton samples from three environmentally different areas (three samples from the coast of Finnmark, seven from the East of Hopen Island, and five from North West of Svalbard) for their antibacterial, anticancer, anti-diabetes, and antioxidant activities. After a freeze-drying step, the biomass composition present in each area was evaluated in terms of species characterization. Samples collected in the areas of Hopen East and northwest of Svalbard were completely or almost completely made up of phytoplankton. Each sample was fractionated using a gradient of MilliQ water, methanol, and acetone. Eight fractions per sample were obtained by this fractionation procedure and their bioactivity was further evaluated. Several of the fractions (from Fraction 4 to Fraction 7) showed cytotoxic activity against human melanoma A2058 cells. Only one of the fractions (Fraction 6 of an extract from northwest of Svalbard) was active in the cellular antioxidant assay (CAA), while in the cellular lipid peroxidation assay (CLPAA) there was detectable bioactivity in all fractions (1 to 8) in all areas with the greatest presence of phytoplankton, namely, Hopen East and northwest of Svalbard. Ingebrigtsen et al. compared the observed bioactivity with that of a mass-cultivated diatom, Porosira glacialis, identifying higher bioactivity in the field phytoplankton samples compared to the cultivated species P. glacialis [40]. Active marine microalgae are summarized in Table 1. Table 1. This table reports bioactivities reported for microalgae living in cold environments, with the sampling location, performed assays, and tested concentration. DPPH stands for 1,1-diphenyl-2picrylhydrazyl; ELISA for Enzyme-linked immunosorbent assay; FITC for fluorescein isothiocyanate; MTS for 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; MTT for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; and ROS for reactive oxygen species.

Cold Stress Exposure Experiments
Shukla et al. [46] evaluated the growth characteristics of the Chlorella mirabilis strain L-10 (isolated from deglaciated soil on King George Island, South Shetland, maritime Antarctic) in micro-scale mass cultivation systems under different nitrogen and carbon sources, and performed analyses of fatty acid contents. The biomass cultivation was performed in stable (indoor) and variable (outdoor) conditions. The indoor cultivation was performed in a closed cultivation chamber with an irradiation of 75 µmol photons m −2 s −1 at 15 • C. The outdoor condition was performed in a temperate region of the Czech Republic, Central Europe, selected as late winter or early spring (February-April), in order to mimic the temperature environments of the polar summer. The authors manipulated the growth conditions by treating the algae with various culture media rich in different nutrients. The fatty acid analyses in Chlorella mirabilis in the indoor experiment revealed increased production of linoleic acid in glycerol treatments (5% v/v). The authors concluded that Chlorella mirabilis could be considered a possible strain for linoleic acid production at low temperatures [46]. Linoleic acid is the most highly consumed polyunsaturated fatty acid (PUFA) in the human diet; it is an essential nutrient and is typically provided in enteral, parenteral, and infant clinical formulas [47].
In the same year, Boelen et al. [48] investigated the intraspecific variability in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) content in response to irradiance and temperature in the diatoms Chaetoceros brevis and Thalassiosira weissflogii, along with the interspecific variability of the same compounds in two polar diatoms (Chaetoceros brevis, Thalassiosira weissflogii) and three temperate microalgae (the green alga Pyramimonas sp., the haptophyta Emiliania huxleyi, and the raphidophyta Fibrocapsa japonica). Boelen et al. noted that higher relative and absolute levels of DHA were found in Emiliania huxleyi and Pyramimonas sp. cultivated at 3 • C and standard irradiation of 75 µmol photons m −2 s −1 , while EPA levels were higher in Fibrocapsa japonica, Chaetoceros brevis, and Emiliania huxleyi cultivated at 16 • C and standard irradiation of 75 µmol photons m −2 s −1 [48]. EPA and DHA are well-known omega-3 fatty acids, and have been reported to play an important role for the immune system [49]. They have been shown to have anticancer [50], antibacterial, and antioxidant activities [51]. In particular, EPA has shown antibacterial activity against Bacillus cereus and Staphylococcus aureus [52] as well as anticancer properties against human A549 lung cancer cells and a human esophageal cancer cell line [53,54]. DHA has previously shown anticancer properties against breast (MDA-MB-231, MCF-7), pancreatic (MiaPaca-2), and colorectal (CaCo-2, SW-620) cancer cell lines [55]. Recently, Cheregi et al. [56] showed that the use of microalgal strains adapted to the local conditions in Nordic countries may be advantageous for biofuel and high-value compound production, especially for their high content of EPA and DHA. For instance, the diatom Phaeodactylum tricornutum (M28) from Norwegian fjords (at 10-15 • C) was able to accumulate 43% (dry weight) of lipids, with 7.33% EPA and 0.6% DHA. The diatom Attheya septentrionalis (M21; at 10-15 • C) from the Arctic region was able to accumulate 24% EPA.
In 2017, El-Sheekh et al. [58] studied the effects of different temperatures (15,20,25,30,35, and 40 • C) on the growth, lipid productivity, and fatty acid profiles of the green alga Scenedesmus acutus as a possible feedstock for biodiesel production. The highest fatty acid content was observed at low temperature (15 • C). With increased temperatures, the fatty acid content decreased; hence, the authors suggested culturing S. acutus at low temperatures for biodiesel production purposes.
In the same year, Demirel et al. [59] determined the effects of three different culture media: Bold's Basal Medium (BBM), Blue-Green medium (BG-11), and RD medium, along with two regimes of temperature values (22 and 28 • C), on the growth rate of Desmodesmus protuberans, and investigated the antioxidant and antimicrobial bioactivity of the crude extracts. The crude extract of Desmodesmus protuberans grown with BBM medium at 28 • C showed major antibacterial activities, inhibiting the growth of Candida albicans, while with the RD medium at 22 • C it showed major antioxidant activity [59]. Menegol et al. [60] studied the effects of temperatures of 22, 27, and 32 • C along with different sodium nitrate concentrations of 12, 24, 36, 48, or 60 mg L −1 on the chlorophyta Heterochlorella luteoviridis. H. luteoviridis was shown to be a great source of xanthophylls, carotenes, and polyunsaturated fatty acids. In particular, low temperatures improved the biosynthesis of ω3-type fatty acids by lowering the ω6:ω3 ratio [60].
Similarly, Stirk et al. [61], exposed six green microalgae, Chlorococcum ellipsoideum, Gyoerffyana humicola, Nautococcus mamillatus, Acutodesmus acuminatus, Protococcus viridis and Chlorella vulgaris, to salt and low temperature stress by the addition of 36 g L −1 NaCl and transfer from 25 • C to 15 • C. They analyzed and quantified changes in endogenous brassinosteroid content. Exposure for 30 min at 36 g L −1 NaCl defined an increase of brassinosteroid content in all six species compared with the corresponding untreated cultures. Only three species of microalgae, Chlorococcum ellipsoideum, Acutodesmus acuminatus, and Protococcus viridis, showed an increase in cell division when the temperature was lowered from 25 to 15 • C; on the contrary, salt stress had a negative effect on cell division [61]. As the six microalgae strains analyzed in this study were part of a previous study in which endogenous auxins, cytokinins, gibberellins, and brassinosterides were detected and quantified in four-day cultures [62,63], the authors suggested that it is likely that the rapid increase in endogenous brassinosterides in response to salt stress would have been accompanied by changes in the concentrations of other hormones as well.
Recently, Zaho et al. demonstrated lipid accumulation in the diatom Phaeodactylum tricornutum, the green alga Chlorella vulgaris, and the brown alga Nannochloropsis sp. grown in low temperature (15 • C) conditions. In this work, Zaho et al. determined the relative lipids and unsaturated fatty acid content with Fourier transform infrared (FTIR) spectroscopy [64]. In 2004, Jiang et al. showed that the production of eicosapentaenoic acid (EPA) and polyunsaturated fatty acids (PUFA) from the marine diatom Pheodactylum tricornutum increased following the induction of temperature stress (from 25 to 10 • C). The highest yield per dry mass unit was 4.9% for PUFA (dry mass: 12.4 mg L −1 ) and 2.6% for EPA (dry mass: 6.6 mg L −1 ) when the temperature was shifted from 25 to 10 mid-hour [65]. In 2022, Fierli et al. [66] further studied lipogenesis and carotenogenesis in the diatom Pheodactylum tricornutum exposed to a cold temperature shock (from 20 to 10 • C) with addition of NaCl salt (5 mg mL −1 ) and the phytohormone abscisic acid (4 mg L −1 ). At 24 h and 5 days from stress induction with low temperature, salt, and the phytohormone abscisic acid, Fierli et al. analyzed the expression of key genes such as diacylglycerol O-acyltransferase 1 (DAGT), zeaxanthin epoxidase (ZEP3), and coding enzymes involved in the synthesis of lipids and carotenoids, demonstrating that cold temperature influenced lipid production, increasing it by 22%. At the same time, they noted an increase in EPA of 22% and fucoxanthin of 33% [66]. Lipids can have applications in the nutraceutical and cosmeceutical fields, while fucoxanthin has been shown to have anticancer [67] and anti-obesity activities [68]. Active marine microalgae are summarized in Table 2. Table 2. This table shows bioactivities reported for microalgae exposed to cold stress along with the experiments, performed assays, and tested concentrations. BBM stands for Bold's Basal Medium; BG-11 for Blue-Green medium; DHA for docosahexaenoic acid; ELISA for Enzyme-linked immunosorbent assay; EPA for eicosapentaenoic acid; FRAP for Ferric Reducing Antioxidant Power; FTIR for Fouriertransform infrared spectroscopy; MTS for 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PTP1B for Phosphatase1B Inhibition Assay; and TBARS for thiobarbituric acid reactive substances.

Conclusions
Many microalgae have been reported to live in cold extreme environments. While certain algal cells can form resting spores/cysts, many polar algae appear to remain in a vegetative state during winter [69]. This review shows that cold-adapted species are able to exert bioactivities for different human applications. In particular, anticancer (mainly melanoma cells), immunomodulatory, and antioxidant activities have been reported.
Microalgae are photosynthetic unicellular organisms that, in certain cases, can be massively cultivated under controlled conditions in photobioreactors in an eco-friendly and eco-sustainable way, thereby reducing destructive collection practices applied to sampling huge numbers of individuals of certain macro-organisms. This means that a few sampled cells are sufficient, and that culturing and successive studies can be performed in laboratory conditions without impacting natural environments. In addition, recent advances in -omics technologies, such as genomics, metagenomics, transcriptomics, metatranscriptomics, proteomics, and metabolomics, can be applied to microalgae to perform in silico analysis of metabolites of interest (e.g., [70]). In particular, these approaches have helped to shed light on biosynthetic pathways that can be involved in the synthesis of compounds with ecological functions (e.g., chemical mediators, communication, and defense), as well as possible biotechnological applications (e.g., pharmaceutical, nutraceutical, cosmeceutical, and biofuel applications).
Among the most active species, for anticancer activity there is Botrydiopsidaceae sp. (ETBO; active at 6.25 µg/mL); for immunomodulatory activity Chloromonas reticulata (ETCH) and Micractium sp. (ETMI) (active at 5 µg/mL); and for antioxidant activity, Micractinium sp. (P9-1), with an IC 50 of 0.52 mg mL −1 , Desmodesmus opoliensis SQ-2, with an IC 50 of 0.58 mg mL −1 , and Scenedesmus sp. B2-2, with an IC 50 of 0.59 mg mL −1 . Considering that these are raw extracts, not pure compounds, successive purification of bioactive metabolites is necessary in order to compare them to the marine pharmaceuticals already on the market, many of which are active at the level of 0.6-7 ng/mL (see Table 3 in Martinez et al. [71]).
Algae have been found to be excellent sources of various compound categories, such as fatty acids, peptides, carbohydrates, vitamins, polyphenols, and pigments [21,72]. Among the most active reported compounds in cold microalgal species are the omega-3 fatty acids EPA and DHA. Humans produce very low rates of EPA and DHA, while these are fundamental nutrients in our diet. EPA and DHA are hormonal precursors, play an important role in the immune system [49], and have been shown to have anticancer [50] and antioxidant activities [51], which is why they are of great economic interest. It has been hypothesized that microalgae living in polar regions have higher levels of PUFAs, as in addition to playing a role in the functioning of the thylakoid membrane, they serve to contain the fluidity of the cell membrane [48,73]. For this reason, polar microalgae adapted to cold and showing high growth rates in low-temperature and low-irradiance conditions could be used as key species for mass production of EPA and DHA.
In addition to production of bioactive compounds, cold-adapted microalgal species have shown promising bioremediation properties [74,75]. For example, Gojkovic and collaborators tested eight microalgal species from Northern Sweden and the culture collection strain Scenedesmus obliquus RISE (UTEX 417) to remove nineteen pharmaceuticals from growth medium in bioreactors. Lipophilic compounds such as Biperiden, Trihexyphenidyl, Clomipramine, and Amitriptyline were removed efficiently from the culture medium (>70% in average within 2 days). Interestingly, Chlorella vulgaris 13-1 and Chlorella saccharophila RNY were highly efficient in removing all nineteen pharmaceuticals from the growth medium within 12 days.
Warming oceans, changing ocean circulation, decreasing ice cover, and increasing freshwater inputs are all expected to impact polar ecosystems in the future. To date, while many efforts have been made to measure changes in phytoplankton biomass and in community composition in cold environments, less attention has been paid to the physiological responses and metabolites they may produce, which require further studies. Physiological changes in microalgae influence their growth rate, fitness, nutritional quality, and compound/secondary metabolite production [76], and further investigations can shed light on new details regarding the influence on primary production rates, community composition, cold environment ecology, and possible biotechnological exploitation.