ISOLATION AND CHARACTERIZATION OF MODERATELY HALOPHILIC BACTERIA IN SALINE ENVIRONMENTS OF ARAL SEA REGION

ABSTRACT

The purpose of research is to isolate and characterize exopolysaccharide producing halophilic bacteria strain from saline environments of the Aral Sea region, Karakalpakstan. The bacterial isolates grew optimally at 35ºC, in pH 7.0-7.5, and in the presence of 11.7-15.6% w/v NaCl. On the basis of the taxonomic evidence presented in this study, it was concluded that strain UzAS3 should be classified as a species of Halovibrio, for which the name Halovibrio sp. is proposed, with the type strain UzAS3.

АННОТАЦИЯ

Цель исследования - выделение и характеристика экзополисахарид продуцирующего штамма галофильных бактерий из засоленных сред Приаралья, Каракалпакстана. Бактериальные изоляты оптимально росли при 35ºC, pH 7,0-7,5 и в присутствии 11,7-15,6% NaCl. На основании морфологических, биохимических и таксономических данных, полученных в результате исследований, установлено, что галофильный бактериальный изолят UzAS3 следует классифицировать как род Halovibrio, а Halovibrio sp. UzAS3 был предложен в качестве штамма.

Keywords: hypersaline environments, Aral Sea region, halophilic bacteria, phenotypic and biochemical characteristics.

Ключевые слова: гиперсоленые среды, Приаралье, галофильные бактерии, фенотипические и биохимические характеристики.

Halophilic microorganisms are widespread in such hypersaline environments as saline soils, saline lakes, and salterns. Halophiles are found in all three domains of life. Based on their response to NaCl, they can be classified into three groups: slight halophiles grow optimally at 1-3%; moderate halophiles grow optimally at 3-15%; and extreme halophiles grow optimally above 15-20% [1, 2]. To survive in high-salt conditions, two principal mechanisms have been evolved by halophilic microorganisms: (i) salt-in-cytoplasm, and (ii) compatible solute strategies [3]. In the second mechanism, accumulate organic compounds such as sugars, amino acids or amino acid derivatives (ectoine, hydroxyectoine and glycine betaine) in response to an osmotic stress [3]. The production of compatible solutes and other compounds such as bacteriorhodopsins, exopolysaccharides, hydrolases and biosurfactants has remarkable potentials in industry [4].

The genus Halovibrio, which belongs to the family Halomonadaceae, was proposed by Fendrich [5] for Gram-negative, strictly aerobic, heterotrophic, salt-requiring, mesophilic and non-sporeforming rods, which were motile by means of single polar flagella. There are only two species with validly published names in the genus Halovibrio: Halovibrio variabilis [6] and Halovibrio denitrificans [7].

The purpose of research is to isolate and characterize exopolysaccharide producing halophilic bacteria from saline environments of the Aral Sea region, Karakalpakstan.

Materials and methods

Sample Collection and Their Physicochemical Analyses. Water samples (water and its sediments) were collected from hypersaline lake of the Aral Sea region, Kungrad, in May, 2019 (N 43º 07 77", E 58º 79’ 48"). These water and sediment samples were taken in a sterile 250 ml bottles and 50 ml falcon tubes, respectively.

Isolation and culture conditions. The samples were diluted in 5% (w/v) sea salts solution, transferred to plates containing HM agar medium supplemented with different concentrations of sea salts solution (5, 10, 20, 25) [8] and incubated at 35 ºC for 7 days. Cultures were purified and identified using morphological characteristics. Selected strains were transferred to new plates of the same medium and stored aerobically at 4 ºC.

One of the isolates, designated UzAS3, corresponded with Halovibrio variabilis DSM 3050 included as a validly published bacterial name, and has been characterized using a phenotypic approach. Halovibrio variabilis DSM 3050 was used as the reference strain [7].

Morphological and biochemical characteristics. Characterization of the physiological and biochemical properties of the bacterial strain included Gram-staining, motility test, catalase and oxidase activities, degradation of casein, starch, gelatin, Tween 80 and urea, endospore formation and nitrate reduction [9]. All results were systematically analyzed according to the Bergey’s Manual of Determinative for Bacteriology [10]. The various salinities (0.5-20% w/v) contained in HM were adjusted by addition of NaCl. Inoculated media were incubated at 35 ºC for 8-10 days. The temperature range for growth was determined by incubating cultures at 15-50 ºC for 8-10 days in HM agar. Growth at pH 5-9 was determined in HM agar. Motility was assessed in a hanging-drop preparation at × 1000 magnification from 72-h cultures in HM. The cell size and morphology, flagellation pattern were determined using fluorescence microscopy (Leica 1000) of negatively stained cells grown on HM at 35 ºC for 3 day [9].

Results and discussion

Hypersaline lakes in the Aral Sea region featuring high mineralization (50-280 g/l) decline their level due to evaporation or completely dry up during summer time and spread salt to the surround territories. The temperature and salinity of the water at the time of sampling were 28.9ºC and 127.8 g/L, respectively. The physicochemical parameters measured at the time of sampling (may 2019) are presented in Table 1.

Table 1.

Physicochemical analyses of water sample and its deposit from the Aral Sea region (Kungrad region)

*Measurable quantities of trace elements lower than <0.2 mg/L considered as Trace.

Morphological, physiological and biochemical characteristics. UzAS3 was a short rod-shaped (0.4-0.7 x 1.0-3.0 μm in size), strictly aerobic, salt-requiring bacterium with single, polar flagella. Growth did not occur in a medium without Na⁺ or sea salts. The cultural, physiological and biochemical characteristics of strain UzAS3 are given in the species description (Fig. 1).

Figure 1. Colony and microscopy of negatively stained cells of UzAS3

For most phenotypic characteristics, strain UzAS3 had properties that are typical for its phylogenetically related species in the genera Halovibrio variabilis and Halovibrio sp. as described by Fendrich, 1989 and Sorokin et al., 2006, such as the presence of Gram-negative, strictly aerobic, rod-shaped cells with polar flagella, which were isolated from the marine environment. Positive for catalase, oxidase and urease, but not H₂S and indole production.It could also be differentiated from phylogenetically related Halovibrio sp. by different reactions for caseinase and utilization of lactose and mannose [7].

Table 2.

Phenotypic and biochemical characteristics distinguishing UzAS3 from its phylogenetically related species

A summary of the major differential properties between UzAS3 and phylogenetically related species is given in Table 2. Thus, based on morphological, physiological and biochemical properties, strain UzAS3 was identified to be a Halovibrio-related.

Description of Halovibrio sp. UzAS3. Cells are Gram-negative, catalase- and oxidase-positive, and strictly aerobic short rods (0.4-0.7 x 1.0-3.0 mm) with single, polar flagella. No spores are observed. Forms circular, convex, pale red-coloured, colonies with entire margins and a diameter of 2-5 mm on HM after 3-5 days of incubation at 33-35 ºC. Growth occurs at 25 and 43 ºC, but not at 4 and 50 ºC. Grows well at 28-37 ºC (optimum – 35 ºC). It is capable of growing in 11.7-15.6% (w/v) NaCl (optimum = 15.0%). The pH range for growth is 6.5-8.5 (optimum 7.5). Positive responses are recorded for the amylase, lipase, urease and utilization of Tween 80, D-glucose, sucrose. Negative for Voges-Proskauer, indole and H₂S production, nitrate reduction, caseinase. Acids are produced from sucrose and glucose.

Consequently, based on our findings, we conclude that the identified halophilic bacterium isolated from the saline lake in Kungrad, Karakalpakstan, were mostly from genus Halovibrio, which possess variant species and strains with different degrees of tolerance to salinity. The isolation and characterization of Halovibrio sp. UzAS3 was carried out for the first time in Uzbekistan saline ecosystems.

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