Environmental Microbiology (EM) | Microbial Ecology and Diversity
Microbiol. Biotechnol. Lett. 2024; 52(1): 76-87
https://doi.org/10.48022/mbl.2401.01010
Ahmed Mohamed Ali1, Tahany M.A. Abdel-Rahman1, and Mohamed G. Farahat1,2*
1Botany and Microbiology Department, Faculty of Science, Cairo University, Giza 12613, Egypt
2Biotechnology Department, Faculty of Nanotechnology for Postgraduate Studies, Sheikh Zayed Branch Campus, Cairo University, Sheikh Zayed City, Giza 12588, Egypt
Correspondence to :
Mohamed G. Farahat, farahat@cu.edu.eg
Halophilic bacteria are promising reservoirs for halotolerant enzymes that have gained much attention in biotechnological applications due to their remarkable activity and stability. In this study, 62 halophilic bacterial strains isolated from a solar saltern were screened for the production of various extracellular enzymes. The results revealed that 31 strains (50%) were positive for amylase production while 26 strains (41.9%) were positive for protease. Further, 22 strains (35.48%) exhibited β-glucosidase activity and only 17 (27.41%) demonstrated lipase activity. Of the investigated halophiles, ten strains growing in the presence of ≥15% NaCl (w/v) were selected and identified based on their 16S rRNA gene sequences as Halomonas meridiana, Salinivibrio costicola, Virgibacillus oceani, Virgibacillus marismortui, Marinobacter lipolyticus, Halobacillus karajensis, Salicola salis, Pseudoalteromonas shioyasakiensis, Salinicoccus amylolyticus, and Paracoccus salipaludis. Therefore, the present study highlights the diversity of the culturable halophilic bacteria in a Mediterranean solar saltern, harboring various valuable halotolerant enzymes.
Keywords: Halophilic bacteria, solar salterns, hydrolytic enzymes, bacterial diversity
Microorganisms have various adaptive strategies to tolerate environmental stresses, especially extremophiles which can survive under adverse conditions, such as high temperature, pH, and salinity [1]. This can be achieved by many mechanisms including the ability to produce particular secondary metabolites, which have many applications in various industrial fields and biotechnological research [2]. Extremophilic microorganisms that are capable of surviving in high salinity can grow in habitats such as the Dead Sea, solar salterns, and salt lakes. These organisms can produce extracellular enzymes with potential interest in several biotechnological and industrial applications due to their high activity and stability at low water levels [1, 3, 4]. It was reported that halophiles-derived enzymes were active under extreme conditions because their surfaces carry a high number of amino acid residues of negative charges [5]. Hydrolytic enzymes such as proteases, lipases, β-glucosidase, inulinases, cellulases, and amylases received increased attention due to their significant industrial roles in wastewater treatment, biosynthetic processes, agriculture, medicine, foods, detergent, textile, paper, and biofuel industries [6−10]. Also, hydrolytic enzymes have crucial roles in bioremediation processes and biodegradation of pollutants [11−14]. In this context, various bacteria strains isolated from marine habitats were reported as promising sources for a wide range of significant commercial hydrolytic enzymes including cellulases, nucleases, inulinases, xylanases, amylases, galactosidases, glucosidases, lipases, chitinases, dextranases, proteases, etc. [15−20]. Amylases are classified as hydrolyzing enzymes that hydrolyze starch into monosaccharide units by acting on its α-1,4-glucosidic linkages [21, 22]. Various amylases were applied in industrial sectors for starch liquefication, biomasses saccharification, fabrics desizing, beverage fermentation, and syrups production [23, 24]. In this regard, α-amylase from the marine bacterium
Furthermore, the halophilic
Sediments and brine samples were collected from a solar saltern, in Egypt (30°59'24.9"N 29°37'00.4"E) in sterile plastic bags and transferred to the laboratory through a short time after sampling and surveyed immediately for bacterial isolation.
Isolation of halophilic bacteria was performed using tryptic soy agar medium (TSA, Condalab, Spain) supplemented with 10% (w/v) NaCl. In brief, 100 μl of each collected sample were plated and the inoculated plates were incubated at 37℃ and checked daily for up to 7 days. The recovered colonies were transferred on new agar plates and sub-cultured many times until pure cultures were obtained. For the preservation of bacterial isolates, stocks of 20% glycerol of pure cultures were prepared and stored at -80℃.
Amylase activity. The potential of halophilic isolates for amylase production was investigated using starch agar medium containing (g/l): yeast extract, 1; peptone, 5; soluble starch, 2; agar, 20; supplemented with 10% NaCl. After incubation at 37℃ for 3−5 days, plates were flooded with iodine-potassium iodide solution. The formation of a clear zone around the bacterial colonies was recorded as starch hydrolysis [57].
Protease activity. The proteolytic activity was assessed by the method described by [57] with some modifications. Each strain was inoculated on skim milk agar medium containing (g/l): skim milk, 20; glucose, 1; tryptone, 5; yeast extract, 2.5; agar, 20; supplemented with 10% NaCl. After incubation at 37℃ for 5 days, the plates were checked. The appearance of a clear zone around indicates protease activity.
Lipase activity. The investigation of lipase activity was conducted using a screening medium containing (g/l): peptone, 10; CaCl2·2H2O, 0.1; agar, 20, supplemented with 10% NaCl (w/v). After autoclaving, sterile tween 80 was added to a final concentration of 1% (v/v). Each bacterial strain was inoculated and the plates were incubated at 37℃ for 4−5 days. Subsequently, the appearance of white precipitate around the bacterial growth indicated the lipase activity [58].
β-Glucosidase activity. The β-glucosidase activity was inspected using TSA medium containing (g/l): beef extract, 3; peptone, 5; agar, 20, supplemented with 10% NaCl, 0.05% esculin and 0.01% ferric citrate. The formation of a brown-black zone around the bacterial colonies after incubation at 37℃ for 5 days indicated β-glucosidase activity [51].
All isolated strains were inoculated in tryptic soy broth supplemented with different concentrations of NaCl (10, 15, 20 and 25%) and incubated at 37℃ in a shaking incubator (140 rpm). The cultures were checked daily for bacterial growth up to 15 days.
Extraction of genomic DNA from bacterial isolates was performed by using GeneJET™ Genomic DNA Purification Kit according to the manufacturer protocol. Amplification of nearly full-length 16S rRNA gene was conducted by PCR using universal primers, 27F (AGAGTTTGATCMTGGCTCAG) and 1492R (TACGGYTACCTTGTTACGACTT) [59]. Afterward, the amplicons were purified and sequenced using ABI 3730xI sequence analyzer (Applied Biosystems, USA), and the sequences were analyzed using the Basic Local Alignment Search Tool (http://www.ncbi.nlm.nih.gov/blast). Construction of the phylogenetic tree was performed using the Neighborjoining method based on bootstrap values (1000 replications with MEGAX software). The sequences of the 16S rRNA gene were deposited in the GenBank.
In the present study, 62 halophilic bacterial strains were isolated on TSA medium supplemented with 10% NaCl, from a solar saltern, in Egypt. After that, all isolated halophiles were screened for the production of various extracellular hydrolytic enzymes.
All isolated bacterial halophiles were screened for their ability to produce four extracellular hydrolytic enzymes (amylase, protease, lipase, and β-glucosidase) in the presence of 10% NaCl. Of the investigated strains, 54/62 (87.09%) were positive for at least one extracellular enzyme. On the other hand, eight strains (12.91%) did not produce any of the investigated extracellular enzymes. Results showed that 31 strains (50%) had amylase activity (Fig. 1A). Regarding the protease activity, 26 strains (41.9%) exhibited proteolytic activity on skimmed milk agar plates (Fig. 1B). Seventeen strains (27.41%) showed lipase activity (Fig. 1C) and 22 halophilic strains (35.48%) were found to produce dark brown zones around their colonies, indicating β-glucosidase activity (Fig. 1D). Results revealed that only three bacterial strains were positive for all investigated enzymes and seven strains produced three different enzymes. Moreover, 19 bacterial strains exhibited positive activity for two investigated enzymes, and 25 strains exhibited activity for only one enzyme (Fig. 2).
Bacterial growth in different concentrations of NaCl (10−25%, w/v) was investigated. All the investigated isolates exhibited significant growth at 10% NaCl. Results revealed that three strains (SHB1, SHB21 and SHB59) were able to grow at NaCl concentrations up to 25%, while SHB7, SHB26 and SHB37 exhibited growth up to 20% NaCl. Three strains (SHB13, SHB22, SHB55 and SHB60) showed growth up to 15% NaCl (Table 1). Among 62 halophilic strains, 10 exhibiting the ability to grow in high salt concentrations (≥15%) were selected while 52 strains that did not grow on more than 10% NaCl were maintained at -80℃ for future investigations. The selected strains were subjected to molecular identification based on 16S rRNA analysis.
Table 1 . Salinity tolerance of halophilic strains isolated from a Mediterranean solar saltern located at the northern coast of Egypt.
Strain | Growth with NaCl (%, w/v) | |||
---|---|---|---|---|
10 | 15 | 20 | 25 | |
SHB1 | + | + | + | + |
SHB7 | + | + | + | - |
SHB13 | + | + | - | - |
SHB21 | + | + | + | + |
SHB22 | + | + | - | - |
SHB26 | + | + | + | - |
SHB37 | + | + | + | - |
SHB55 | + | + | - | - |
SHB59 | + | + | + | + |
SHB60 | + | + | - | - |
‘+’ positive for the test, ‘-’ negative for the test.
Based on 16S rRNA gene analysis, the selected halophilic strains were identified as
Table 2 . Identification of extreme halophilic bacterial strains based on 16S rRNA gene sequence analysis.
Strain | Accession Number | Closest Species | Similarity (%) |
---|---|---|---|
SHB1 | PP035898 | 99.93 | |
SHB7 | OR616804 | 99.30 | |
SHB13 | OR616805 | 99.79 | |
SHB21 | OR616806 | 99.87 | |
SHB22 | OR616807 | 99.86 | |
SHB26 | OR616808 | 99.79 | |
SHB37 | OR616809 | 99.64 | |
SHB55 | OR616810 | 99.78 | |
SHB59 | OR616811 | 99.55 | |
SHB60 | OR616812 | 99.77 |
Concerning the selected bacterial strains, results revealed that ten identified strains activity of possess at least one hydrolytic enzyme (Table 3). The most active strain that exhibited activity of all tested enzymes was
Table 3 . Extracellular hydrolytic enzymes of extreme halophilic bacteria.
Strain | Extracellular Enzyme | |||
---|---|---|---|---|
Amylase | Protease | Lipase | β-Glucosidase | |
+ | - | + | - | |
+ | - | + | - | |
- | + | + | + | |
- | + | - | + | |
- | - | + | - | |
+ | + | + | + | |
+ | + | + | - | |
+ | + | - | + | |
+ | - | - | + | |
- | + | + | - |
‘+’ positive for the test, ‘-’ negative for the test.
Recently, halophilic bacteria have been regarded as prolific sources of novel economically important stable enzymes that are functional under extreme conditions. Due to their unique properties, halophilic-derived enzymes are extensively used in many industrial fields. In the present work, 62 halophilic bacteria were isolated from a solar saltern in Egypt among them ten halophilic strains that can grow at NaCl concentrations equal to or more than 15% belonging to nine genera were selected. Because of operating at high salt concentrations with the reducing water activity, enzymes from the extreme halophilic microorganisms exhibit extraordinary stability and are thought to be robust biocatalysts in aqueous/ organic and nonaqueous media with superior activity in various biotechnological applications [60, 61]. Solar salterns are one of the natural habitats that are characterized by the presence of high salt concentrations and have been reported as a rich source for the isolation of halophilic microorganisms. In a similar study, various halophilic bacteria have been isolated from solar salterns in Tunisia, including
In the present study, amylase was the most prevalent enzyme followed by protease, while lipase was the least prevalent enzyme. The ten extreme halophilic identified strains showed at least one activity of the tested enzymes. The most active strain that showed activity of the four enzymes was
In this investigation, we identified seven promising lipase-producing halophilic bacterial strains:
In conclusion, this investigation shed light on the diversity of halophilic bacterial strains from the extreme ecosystem, Mediterranean solar saltern in Egypt. The majority of the recovered halophilic strains were able to produce salt-tolerant hydrolytic enzymes. We found that amylase was the most prevalent enzyme, while lipase was uncommon. Furthermore, ten extreme halophilic bacterial strains belonging to nine genera were identified. Of these strains,
The authors have no financial conflicts of interest to declare.
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