Living organisms that have colonised planet Earth for billions of years have evolved mechanisms and strategies that allow them to inhabit environments that are potentially harmful or lethal from an anthropocentric point of view. One of the groups of organisms that have had the longest evolution time to adapt to harsh habitats is bacteria. These inhabit the most deadly and unfriendly environments for humans and are in general called extremophiles. Depending on the factor that limits the growth of the microbes, they are categorised as thermophiles (elevated temperature), psychrophiles (low temperature), acidophiles (low pH), alkaliphiles (high pH), radiophiles (high radiation resistance), piezophiles and barophiles (high pressure), xerophiles (low water activity) and polyextremophiles (tolerating or preferring more than one extreme factor) (1-3). Another interesting group of extremophiles are the halophiles. The bacteria grow and proliferate in environments with high salinity, e.g. salt mines, ponds and springs or salt deposits on rocks or buildings (4, 5). Colonization and the ability to proliferate in environments with extreme osmotic pressure is a major challenge for bacteria. Microorganisms have a low cell volume to surface ratio, which can lead to easier loss of intracellular water and consequently make it difficult for them to effectively control the osmotic balance (6).

To survive, halophiles have developed specific intracellular mechanisms that allow them to retain intracellular water. In general, they have two main mechanisms of action. The first is called the "salt-in strategy" and is based on the accumulation of salts within the cell, the concentration of which balances external osmotic pressure and prevents leakage of water. This is common in archaea and certain Halobacteria (7). The second method, which is more common among procaryotes, is much more complicated and is based on the synthesis of small organic molecules within the cell called compatible solutes or osmoprotectants. These molecules are produced in the cells in high concentrations without affecting central metabolism and they belong mainly to the polyols and carbohydrate derivatives. These compounds are commonly found in bacterial and plant kingdoms, including glycerol, myo-inositol, glucosylglycerol and trehalose (8).

In terms of potential commercial application, two hydroxyproline derivatives, osmoprotectants – ectoine and its hydroxylated derivative – hydroxyectoine, are worth mentioning. Both bind water molecules strongly and are found in bacterial cells. Their concentration depends on the type of bacteria and the environmental conditions. Ectoine concentration can increase from a few millimoles per litre to up to 100 mM in response to stress like high salt, drought or temperature changes (1).

Apart from their osmoprotective properties, they also act as antifreezing agents, UV filters, soothing and anti-inflammatory molecules (9). These properties make both molecules incredibly attractive components for the cosmetic and food industries. Due to their low irritability, they are also widely used in pharmaceutical products, including high-purity drugs like eye or nose drops (10).

The primary aims of the research were to isolate a wild-type bacterial strain that synthesises ectoine at low or moderate salt concentrations and to carry out preliminary work to find cost-effective and simple growth media.

We isolated 56 bacterial strains that can grow in the presence of a 10 % salt mixture. Fifteen originated from the vicinity of Tyrawa spring (BHTA), 30 from Złockie in the vicinity of Na Mokradłach spring (BHMM) and 11 from Rajcza (BHEGO).

Most bacterial strains were Gram-positive and rod-shaped. BHTA20 and BHTA24 were the only strains classified as cocci. Nevertheless, microscopic observations confirmed that we obtained a diversified set of strains. The morphological differences of the isolates are shown in Fig. 1.

The more diverse the collection of microorganisms obtained before the screening, the higher the probability of effective selection. Significant morphological differences between the isolated strains suggested positive screening results, i.e. the isolation of ectoine producer. The isolated bacteria were grown in a nutrient-rich medium containing 10 % seawater. The presence of ectoine and its derivative hydroxyectoine was then determined semiquantitatively using the LC-MS method. The common logarithm of the number of mass detector counts was calculated for easier presentation of the results (Fig. 2). Moderately halophilic bacteria, which were the subject of the search, grow well in a salt range between 5 and 20 %. Therefore, the absorbance was compared with the estimated amount of ectoine (Fig. 2).

Most bacteria did not synthesise ectoine and hydroxyectoine or produced them in minimal quantities. Nevertheless, based on the number of mass detector counts, we identified 10 strains that were able to biosynthesise significant amounts of desirable products. Strains BHTA2, BHTA18, BHTA19, BHTA21, BHTA22, BHTA24, BHTA25 and BHTA30 synthesised mainly ectoine and negligible amounts of hydroxyectoine. Strain BHTA20 synthesised only ectoine, while BHTA8 synthesised mainly hydroxyectoine. Most of the selected microorganisms grew well under aerobic conditions in the medium containing a total of 10 % seawater. Only BHTA22 showed a low biomass concentration, so it was included in further studies. ANOVA analysis was carried out to validate the statistical differences observed between the strains. In all cases, the statistical significance of the results was confirmed and the p-value was less than 0.001.

All strains that produced significant amounts of ectoine and its derivative hydroxyectoine were isolated from the vicinity of Tyrawa spring. We identified them based on the 16S rDNA sequence and basic biochemical characteristics (Table 1).

The results confirmed the differentiation of the isolated strains. Among the microorganisms, we identified six genera of bacteria. Four strains were classified as Halobacillus spp., two as Virgibacillus spp. and one each as Bacillus sp., Pseudalkalibacillus sp., Salimicrobium sp. and Thalassobacillus sp. In addition to the genetic identification, basic biochemical characteristics were also determined (Table 2).

According to current knowledge, Halobacillus halophilus is the only ectoine producer previously described in detail (18). However, the final amount of ectoine synthesised by this species is low. Therefore, H. halophilus has not been industrially utilised for ectoine biosynthesis (18, 19). There is a lack of knowledge about the cultivation conditions and the biosynthesis of ectoine by the other strains. Therefore, we decided to analyse the influence of temperature, pH and different salt amount on the bacteria (Table 2).

All bacteria showed catalase activity and lacked l-alanine aminopeptidase. Only Pseudalkalibacillus hwajinpoensis BHTA18 and Thalassobacillus devorans BHTA24 did not show oxidase activity. All strains grew in the entire analysed temperature range. However, the optimal value of this parameter varied depending on the strain. Most bacteria preferred temperatures around 30 °C, but B. velezensis BHTA8, P. hwajinpoensis BHTA18, Halobacillus sp. BHTA2 and H. sediminis BHTA30 had their optimum at 37 °C (Table 2). The pH range was strain dependent. Nevertheless, most of the tested microorganisms preferred slightly alkaline or neutral pH. This is not surprising since most halophiles normally live in such environments (20).

The growth of most halophilic microorganisms requires the presence of inorganic salt. High-salt medium is mandatory to stimulate ectoine biosynthesis. On the other hand, cell growth and excretion of the product can be limited by a high salt amount (21). Moreover, specific ions can affect ectoine metabolism in bacterial cells (22). There is no information about the media used for ectoine biosynthesis with the isolated strains. Therefore, to select the inorganic components of the media and determine their content range, the growth of the bacteria was tested in the presence of: NaCl, MgSO₄, KCl and (NH₄)₂SO₄ (Table 2).

Halophilic bacteria require the presence of sodium ions for their growth and metabolism, some are not able to grow in the absence of salt. Halophiles can be categorised into three primary groups based on their optimal salt preferences: mild (1–3 %), moderate (3–15 %) and extreme (15–30 %) (23). NaCl content, particularly sodium ions, is a known factor responsible for the regulation of ectoine biosynthesis. Ectoine production tends to show a positive correlation with increasing NaCl amount (24). The effect of NaCl is also observed at the molecular level. It influences the cell transport system and the expression of genes involved in the biosynthesis of ectoine (ectABC) and hydroxyectoine (ectD) (25). On the other hand, a high value of this factor limits the growth of bacteria.

In the case of KCl, it has been shown that some of the halophilic bacteria, e.g. Halanaerobiales sp. or Salinibacter sp., accumulate KCl to ensure osmotic balance in the cells and to stabilise the acid proteome. It is known that diaminobutyrate-2-oxoglutarate transaminase (EctB) is a pyridoxal phosphate (PLP)-dependent enzyme and K⁺ ions are necessary for its stability and activity (26, 27). Ono et al. (26) reported that the presence of 0.01–0.5 M KCl increases the activity of EctB more than the presence of NaCl. Moreover, K⁺ is obligatory for the activity of many enzymes in the cells of extreme halophiles, which show a salt-dependent osmoregulation (28).

The ammonium ions can increase the expression of the ectABC gene cluster, which is involved in the synthesis of ectoine (29). It has also been assumed that ammonium sulphate increases the amount of l-aspartate-β-semialdehyde, which is the precursor of ectoine, and glutamate, which is responsible for the supply of co-substrates for EctB (30). These actions improve the production of ectoine by increasing the flow of substrate through the ectoine pathway.

Another factor that can influence the metabolism of halophilic bacteria is the presence of Mg²⁺. It has been shown that halophilic bacteria have a higher demand for this ion during their growth than non-halophilic microorganisms (31). It has also been suggested that high amounts of Mg²⁺ protect and stabilise macromolecules during dormancy. Moreover, the ratio between Mg²⁺ and Na⁺ varies depending on the physiological state and the presence of nutrients (32).

The results presented in Table 2 show that only B. velezensis BHTA8, P. hwajinpoensis BHTA18, Virgibacillus salarius BHTA19 and T. devorans BHTA24 were able to grow in a medium without added salt. The optimal NaCl content range is different for each strain. However, according to the nomenclature proposed by Kushner (23), most of the isolated strains can be classified as moderately halophilic bacteria, because they have an optimum in the range of 3–15 %. In the case of Halobacillus sediminis BHTA25 and BHTA30, there are some doubts and lower salt amounts should also be tested.

We observed no growth of Halobacillus sp. BHTA2, H. sediminis BHTA25 and BHTA30 in the presence of KCl only. Interestingly, the optimal value of this parameter reached 20 % for S. halophilum BHTA20. Growth in the presence of KCl depended significantly on the strain (Table 2). A similar effect was observed in the case of (NH₄)₂SO₄. Halobacillus sp. BHTA2, H. sediminis BHTA25 and BHTA30 as well as V. litoralis BHTA22 did not grow. In other cases, the optimal amount of salt did not exceed 10 %. Most of the tested strains grew well in the presence of MgSO₄·7H₂O and had their optimum in the range of 5-10 % of the salt. The obtained results do not allow a simple determination of content limits. Future studies should consider synergistic and antagonistic effects between the ions. Therefore, artificial seawater (Instant Ocean) was used for further analysis. Artificial seawater is a mixture of different salts that mimics the natural environment, and it is easy to use in growth media. The isolated bacteria grow well when the seawater amount is up to 15 %. Additionally, P. hwajinpoensis BHTA18, V. salarius BHTA19, H. sediminis BHTA25 and BHTA30 were able to proliferate even in a 25 % solution (Table 2).

According to the presented results, we carried out biosynthesis of ectoine at optimal temperature and pH for each of the strains. We calculated the mean value for the optimal pH range, if the optimal value was defined as a range. The fermentation media contained 15 % Instant Ocean (seawater). Absorbance values were measured during cultivation (Fig. 3). Ectoine and hydroxyectoine were extracted after 48 h of fermentation and their concentration was quantified using HPLC (Table 3).

All isolated strains grow well under the described conditions. The highest increase in absorbance was observed in the cultures B. velezensis BHTA8 and P. hwajinpoensis BHTA18, while the lowest value was observed in Halobacillus sp. BHTA2 and V. litoralis BHTA22. Ectoine concentration reached 274, 4.5, 1.5 and 5.3 mg/L in V. salarius BHTA19, S. halophilum BHTA20, V. litoralis BHTA22 and H. sediminis BHTA25, respectively. Hydroxyectoine was not detected. ANOVA analysis confirmed the statistical significance of the results and the p-value was less than 0.001.

Ectoine is produced on an industrial scale by Halomonas elongata DSM142 through a “bacterial milking” process. Using this microorganism Kunte et al. (8) obtained 7.4 g/L of ectoine, with a volumetric productivity of 0.22 g/L/h. The main disadvantage of the process was high salinity, up to 2.57 M, which can lead to corrosion of the equipment, increase production costs and inhibit the growth of the microorganisms. In addition, bacterial milking is effective only for Gram-negative bacteria. Therefore, other microorganisms were also considered as ectoine producers. At a much lower salt concentration, the biosynthesis of ectoine is carried out by Brevibacterium epidermis DSM20659. The strain produced 1.42 g/L of ectoine, with a productivity of 0.08 g/L/h (33). Halomonas salina BCRC17875 is another wild-type strain used for the biosynthesis of ectoine. In this case, ectoine production reached a concentration of 13.96 g/L after 44 h of cultivation and a NaCl concentration of 2 M (34). High concentrations of ectoine can be achieved with the use of genetically modified microorganisms. Escherichia coli and Corynebacterium glutamicum are commonly used for this purpose. The genetically modified E. coli BL21 strain yielded 60.7 g/L with a volumetric productivity of 1.08 g/L/h using glucose as the sole carbon source under low salt concentration conditions (35). The metabolically engineered C. glutamicum produced significant amounts of ectoine in low-salt environments. C. glutamicum Ect10 showed the ability to synthesise 115.87 g/L of ectoine, making it one of the most efficient ectoine producers documented so far (36).

The biosynthesis of hydroxyectoine is closely related to the presence of ectD gene, which encodes ectoine hydroxylase. Hydroxyectoine impedes the purification of ectoine and reduces its biosynthesis yield, so strains with high ectoine hydroxylase activity require a genetic modification to inhibit this function (37, 38). The presence of the ectD gene in V. salarius has been confirmed (39). V. salarius BHTA19 synthesised hydroxyectoine, as shown by LC-MS analysis (Fig. 2), but it was not detected by HPLC (Table 3). This suggests that the expression of the ectD gene and the activity of ectoine hydrolase depend on the culture conditions and require further investigation.

Among the isolated strains, V. salarius BHTA19 showed the most promising properties. The strain not only produced the highest concentration of ectoine, but it also showed broad tolerance to the type of salt and its amount as well as temperature and pH. There is a lack of knowledge about the cultivation conditions and the biosynthesis of ectoine by V. salarius. However, Virgibacillus sp. is already known as an ectoine producer. To our knowledge, only a few genera have been described so far. Virgibacillus pantothenticus, Virgibacillus halodenitrificans and Virgibacillus salarius 19.PP.SC1.6 are worth mentioning (25, 39, 40). Unfortunately, their production efficiency is not impressive. Considering that V. salarius BHTA19 was cultivated in a flask culture, the obtained amount of ectoine seems to be promising. For this reason, further studies are required to determine the potential of the strain for industrial application.

Ectoine is one of the most marketable biotechnological products. There is a consistent increase in the number of its applications, leading to a growing demand for this metabolite. Although ectoine is produced on an industrial scale, the process still needs to be improved. The high salt amounts used in current methods require the use of the equipment that is suitably resistant. Furthermore, managing production waste that contains significant quantities of salt is a challenge. Ongoing research efforts should focus not only on the improvement of efficiency and the reduction of production costs, but also on the identification of natural ectoine-producing strains that can perform biosynthesis at low or moderate salt amounts.

As a result of the presented research, a moderate halophile Virgibacillus salarius BHTA19 has been identified as a new potential producer of ectoine. This study showed that after 24 h of shake flask culture, the concentration of ectoine reached 274 mg/L. The concentration of ectoine is lower than currently reported for other microorganisms, but BHTA19 was not optimised, so the results cannot be directly compared. Nevertheless, the presented research provides a solid basis to encourage the development of an innovative technology.