Exploring halo enzymes and halo biosurfactants production from halophilic and halotolerant bacteria isolated from Lake Qarun solar salterns

Exploring halo enzymes and halo biosurfactants production from halophilic and halotolerant bacteria isolated from Lake Qarun solar salterns | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Exploring halo enzymes and halo biosurfactants production from halophilic and halotolerant bacteria isolated from Lake Qarun solar salterns Ahmed Ahmed Abdelmonaem Mousa, Basma Mahmoud Ebraheim Elkot, Adel Elsayed Elbeltagy This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7456751/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Halophilic and halotolerant bacteria are microorganisms that thrive in hypersaline environments. They have much potential in several fields in life, such as Industry and agriculture. In this study, five isolates were obtained from four saline ponds of Qarun Lake using Nutrient Agar (NA) medium prepared with water from the ponds. Among them, QSLA17 was considered a halotolerant bacterium, while QSLA1, QSLA7, QSLA14, and QSLA16 were considered halophilic bacteria. The 16S rRNA analysis demonstrated that the isolates were affiliated with three groups: Firmicutes, Gammaproteobacteria, and Alphaproteobacteria. The ability of isolates to produce hydrolytic enzymes and biosurfactants was tested. After initial screening, all isolates were found to have positive lipase activity using Tween 80 as a substrate, while QSLA1 and QSLA7 isolates were able to produce lipase using olive oil. QSLA14, QSLA16, and QSLA17 isolates demonstrated the ability for esterase production, while QSLA1, QSLA7, and QSLA14 isolates succeeded in producing casease. Gelatinase (protease) was excreted by isolates QSLA1, QSLA7, QSLA14, and QSLA17, while chitinase was produced by isolates QSLA1, QSLA7, QSLA14, and QSLA16. Moreover, all isolates demonstrated their ability to produce biosurfactants while growing in Modified Landy Medium with different salt concentrations, and in this context, the QSLA1 isolate demonstrated the best results compared to the other isolates. Biological sciences/Biochemistry Biological sciences/Biotechnology Biological sciences/Microbiology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 Figure 17 Figure 18 Figure 19 Introduction Hypersaline ecosystems are widely distributed habitats, including a variety of terrestrial lakes and deep-sea basins with salt concentrations exceeding three times seawater up to saturation. Hypersaline habitats can be divided into two main types, thalassohaline and athalassohaline waters‎ [ 49 ] . Thalassohaline waters or brines are of marine origin and have ionic composition similar to that of seawater. Athalassohaline waters or brines, such as the Dead Sea and soda lakes, are often found inland and therefore not directly connected to marine waters. Hypersaline environments are spreading all over the world, in arid, coastal, and deep-sea locations, underground salt mines, and artificial salterns ‎[ 43 ] . Solar salterns are widely distributed and can be found at sea level in arid and semiarid regions, both as naturally occurring or human-made salterns ‎ [ 73 ] . Halophiles are salt-loving microorganisms present in saline habitats as normal inhabitants ‎ [ 74 ] and can be found in all three domains of life: Archaea, Bacteria, and Eukarya ‎ [ 21 ] . They are an excellent source of a great variety of extracellular hydrolytic enzymes such as protease, lipase, esterase, gelatinase, and chitinase. Such enzymes are not only salt-tolerant but also can withstand and carry out reactions efficiently under extreme conditions ‎ [ 35 ] . The isolation of moderate and extreme halophiles able to produce hydrolases will provide the possibility to have optimal activities at different salt concentrations that could be useful in some industrial processes ‎ [ 41 ] . The halophilic members in the genera Bacillus , Halomonas , Salinovibrio , Chromohalobacter , and Salinicoccus have been identified with potential in the production of a variety of exo-enzymes under hypersaline habitats ‎ [ 62 ] . Also, they considered a highly promising source for discovering novel biosurfactant molecules that are used currently for pharmacological, food, and cosmetics applications ‎[ 33 ] . Qarun Lake is located in the north of Fayoum Governorate and is currently saline, turbid, and without surface outflow ‎ [ 21 ] . Solar salterns of Qarun Lake typically consist of several ponds interconnected to form the so-called multipond system. These ponds are used by a local company for salt and minerals production. These different ponds have different salt concentrations, therefore have different microbial communities. This study was undertaken with the aim of isolating and identifying halophilic/halotolerant bacterial communities that can reside in the solar saltern ponds of Qarun Lake and test their ability to produce salt-tolerant hydrolytic enzymes and biosurfactants. Materials and Methods Bacterial isolates During September 2019, water samples were collected from solar saltern ponds. The collected water samples were filtered to remove impurities, analyzed for salt concentration by refractometer and pH by pH meter, before use. Ten liters of water were collected from each pond in sterile plastic jars, placed in ice packs, and transferred to the Biotechnology Laboratory, Botany Department, Faculty of Agriculture, Menoufia University. A sterilized Nutrient agar medium (NA) was used for bacterial isolation by streaking 100 µl of the water sample on NA plates. The plates were incubated at 30°C for 7–30 days ‎ [ 9 ] . A subsequent streaking and sub-culturing were performed on NA medium prepared by pond water for purification, and the purified isolates were kept in 30% glycerol in the freezer till further use. Morphological and physiological characterization of the isolates Colonies developed on the plates were morphologically examined for their shape, pigmentation, elevation, and optical properties. In addition, cell shape, Gram staining, and endospore formation procedure ‎ [ 30 ] , motility by hanging drop method ‎[ 25 ] , and catalase activity method ‎ [ 76 ] , were also evaluated. Salinity tolerance of the isolates Bacterial isolates were screened for salt tolerance using NA medium supplemented with various levels of NaCl (0, 7, 12.5, 20, and 22%). The plates were incubated for 7 days at 30°C, and the growth was recorded ‎[ 53 ]‎ . Molecular Identification of the isolates by 16S rDNA DNA Extraction and Polymerase Chain Reaction (PCR) Genomic DNA was extracted from bacterial cells grown aerobically in nutrient broth‎ [ 12 ] . The extracted DNA was purified and visualized under UV by staining with ethidium bromide ‎ [ 61 ] and kept at -20°C until use ‎ [ 48 ] . Amplification of the 16S rRNA gene sequence, using bacterial primers 27F and 1492R ( Table 1 ), was performed using a model PTC-100 thermal cycler (MJ Research Inc., USA) ‎ [ 59 ] . Presence of amplified products was confirmed by applying 7 µl of PCR product on 1% agarose gel in 1X TAE buffer containing ethidium bromide stain, then visualized by Gel documentation system (Bio-Rad Laboratories) ‎ [ 61 ] . The PCR products obtained were purified using the QIAquick PCR purification Kit protocol (Qiagen) and sent for sequencing. Table (1) primers used for 16S rRNA sequencing analysis (PCR) ‎ [ 72 ] . Primer name Orientation Priming site Sequence (5´- 3´) 27F Forward 8–27 AGAGTTTGATCCTGGCTCAG 1492R Reverse 1492–1513 GGTTACCTTGTTACGACTT 16S rRNA sequencing and evolutionary relationships of taxa The sequence was carried out by Colors Laboratories (El-Etihad Square, Maadi, Cairo, Egypt) according to the laboratory instructions. The sequences were edited by eliminating all gaps (Complete deletion option) using the CHROMAS PRO software version 1.5. The 16S rRNA gene sequences were then compared with those published in GenBank databases using Basic Local Alignment Search Tool (BLAST) in the National Centre for Biotechnology Information (NCBI) website (htt// www.ncbi.nih.gov ). Alignment was done using CLUSTAL W 1.6 software ‎ [ 4 ] . The evolutionary history was inferred using the Neighbor-Joining method ‎ [ 60 ] . Evolutionary analyses were conducted in MEGA X ‎ [ 36 ] . The sequences were aligned using the embedded muscle algorithm, and the output was used to build a phylogenetic tree by calculating distance matrices for Neighbor-Joining (NJ) analysis. Detection of hydrolytic enzymes Isolates were examined for lipase, esterase, casease, gelatinase (protease), and chitinase production. Bacterial inoculum used for the enzymatic screening was prepared by growing the isolates in 10 ml NB medium with 12.5% total salt under shaking at 120 rpm for about 72 h. Tests were done by the disc diffusion method ‎ [ 41 ] . Plates were incubated for seven days at 30°C. All assays were carried out in triplicate. An enzymatic rate determination was used, with the following formula ‎ [ 37 ] : Lipolytic enzyme assay on phenol red agar medium supplemented with olive oil Bacterial isolates were plated on phenol red agar plates containing 1% olive oil ‎ [ 54 ] . Phenol red has an endpoint at pH 7.3–7.4, where a slight decrease in pH will turn its color from pink to yellow. Lipase-producing bacteria will turn the dye into yellow ‎[ 38 ] . Lipolytic enzyme assay using Tween 80 agar medium Bacterial isolates were plated on Tween 80 agar plates and incubated as described above ‎ [ 3 ] . Tween 80 (fatty acid esters of polyoxyethylene sorbitan) is mostly hydrolyzed by the lipase as it contains esters of oleic acid and rarely by esterases ‎ [ 35 ] . Detection of esterase For the detection of esterase-producing halophilic bacteria, Tween 20 (fatty acid esters of polyoxyethylene sorbitan) has been used as a substrate in Tween 20 agar medium ‎ [ 3 ] . Tween20 is easily hydrolyzed by esterase as it contains esters of lower-chain fatty acids ‎ [ 35 ] . Screening using tweens (Tween 20 and Tween 80) agar plates demonstrated precipitation around the esterase/lipase-producing bacteria. This is based on the precipitation of the calcium salt of the fatty acids released by the hydrolysis of the Tweens. Detection of casease To detect casease enzyme, the isolates were plated on modified skim milk agar medium using skim milk agar disc diffusion test ‎[ 11 ] . For this purpose, a solution of defatted skim milk powder was autoclaved separately for five minutes at 115°C, then well mixed with NA medium and poured onto petri dishes ‎ [ 13 ] . Positive results for casease production were indicated by clear zones around the isolates on the plates, whereas for the negative results, the isolates lacked zones of clearance ‎ [ 34 ] . Detection of gelatinase (protease) Gelatin liquefaction detects the breakdown of gelatin to polypeptides and amino acids by enzyme gelatinase ‎ [ 26 ] . Gelatin is a solid at room temperature, but above 25°C it liquefies. When gelatinase hydrolyses this protein into amino acids, it remains liquefied even at the low temperatures of an ice bath ‎ [ 14 ] . The method described by Ingroff et al. (1988) was used for testing gelatinase (protease) activity was tested by preparing nutrient gelatin medium which was sterilized at 100°C for 20 min, then cooled to 40°C and inoculated with 1ml of each isolate, then incubated at 30°C for 7 days. Proteolytic activity was detected after being placed in a refrigerator at 4°C for 60 min (until the non-inoculated gelatin solidified) ‎ [78] . Chitinolytic activity The detection was carried out using Luria Bertani (LB) colloidal chitin agar medium. Colloidal chitin suspension was prepared ‎ [ 56 ] , mixed with LB medium, and autoclaved at 15 lbs. and 121°C for 20 minutes ‎ [ 2 ] and inoculated with isolates using the disc diffusion method ‎ [ 41 ] . Plates were incubated at 30°C for 7 days. Clear zone diameter was measured in (cm) at the average of three-dimensional measures and used as an indicator of chitinase activity ‎ [ 22 ] . Biosurfactant production Growth of isolated bacteria on spent engine oil Fifty milliliter of modified Landy broth medium in 250 ml Erlenmeyer flask was amended with 1% of spent engine oil ‎ [ 15 ] , supplemented with salt water in different concentrations, was inoculated with 1/2 ml (1%) of bacterial inoculum and incubated at 30°C for 7 days to determine the ability of bacterial isolates to grow on spent engine oil which enhance biosurfactant production. Bacterial cultures were centrifuged at 3000 rpm for 20 min. to use the supernatant for determining the ability of selected strains for biosurfactant production ‎ [ 66 ] . The halophilic isolates QSLA1, QSLA7, QSLA14, and QSLA16 were examined for biosurfactant production on Landy medium with 3.5 and 7% salt concentrations, while isolate QSLA17 was examined for biosurfactant production on Landy medium with 0, 3.5, and 7% salt concentrations, as it was a halotolerant bacterium. Detection and quantitative screening for biosurfactant production The quantitative screening for biosurfactant production from the isolates was carried out by oil displacement test, emulsification index, and measurement of surface tension ‎ [ 68 ] . Oil displacement activity A modification was carried out on the oil spreading technique ‎ [ 45 ] . Ten µl of spent engine oil was previously put onto the surface of 20 ml of distilled water in a petri dish (70 mm in diameter) to form a thin membrane. The culture of selected isolates was centrifuged at 3000 rpm, then ten µl of bacterial culture supernatant was added on top of the spent engine oil layer. A zone of displacement in the oil was observed, and the diameter of oil displacement (cm) was measured as the activity of biosurfactants. The method was effective for measuring biosurfactant activity, especially when the activity and the quantity of biosurfactant were not high enough. Emulsification index (E24%) Using the method described by Selim et al. (2019), two mL of cell-free supernatant were added to 2 mL of spent engine oil in a screw capped tube and mixed vigorously by vortex at high speed for 2 min. The tubes were kept standing at room temperature for 24 hrs. ‎ [ 66 ] . The emulsification index (E24%) was calculated as the ratio of emulsion zone height to the total height of the three phases using the equation described below ‎ [ 67 ] . Surface tension measurement using a stalagometer The surface tension of supernatant broth containing biosurfactant was measured at 25°C using a stalagometer (Fig. 1) (Stalagometer Rohr B Abgew) by the drop method ‎ [ 69 ] . In this assay, a Traube stalagometer was used to measure the surface tension of a liquid ‎ [ 18 ] . A Traube stalagometer is an instrument that allows for the consistent formation of uniform volumes of liquid droplets and subjects them to gravitational force within a modified capillary tube. The number of drops that fall per unit of volume is compared to a control liquid (i.e., water) of known surface tension. This information is then used to calculate the surface tension of the sample according to the following equation: γ0 = γ (n/n0) Where γ0 and γ are the surface tensions of the reference solvent (for water, γ0 = 72 dyne/cm2) and biosurfactant-containing broth solution, and n0 and n are the drop numbers of the reference solvent and biosurfactant-containing solution, respectively. The reference solution was taken as uninoculated Modified Landy broth medium ‎ [ 69 ] . Statistical analysis The data were further subjected to statistical analysis using analysis of variance (ANOVA), and the differences between Means were compared by a high-range statistical domain using Tukey’s test and Tukey's post hoc analysis in order to distinguish homogeneous–heterogeneous groups among different variables. The multiple comparisons of Means were considered at a probability level p = 0.05. The results are presented as the average means ± Standard Errors (SE) of triplicate. Results and discussion Isolation and purification of halophilic bacteria from solar salterns' water Bacterial isolates This study included five halo-bacterial isolates; QSLA7, that was isolated from the first pond with 4% salinity, QSLA1 and QSLA17 which were obtained from the second pond with 8.2% salt concentration, while QSLA14 was isolated from the third pond with 17.2% salt concentration and QSLA16 that obtained from the fourth pond with 24.2% salinity on nutrient agar (NA) medium that prepared by dissolving their component in saline water taken from the pond water samples instead of distilled water. Results were shown in Table 4. Cultural, morphological, and biochemical characterization of the isolates Colonial, morphological and biochemical characterizations, which aids in partial identification of microorganism ‎ [ 69 ] , were checked and it was observed that QSLA1, QSLA7, and QSLA16 isolates were gram negative and non-spore forming bacteria, while QSLA17 was gram positive and QSLA14 was gram variable, also both of them were spore forming. All isolates were catalase producers and motile. Moreover, QSLA1, QSLA14, QSLA16, and QSLA17 were rod-shaped, while QSLA7 was curved rod-shaped, as shown in Table 2. Colony morphology All colonies of the isolates were translucent, flat, shiny, and round. The colonies' edge of QSLA7, QSLA14, QSLA16, and QSLA17 isolates were ragged, while the colony edge of QSLA1 was lobate. Concerning colony size, QSLA1 and QSLA14 were pinpoint-sized, while the others were small. Moreover, QSLA1, QSLA7, and QSLA17 have a creamy color, while QSLA14 and QSLA16 were pink and yellow colored, respectively, as described in Table 3. Salinity tolerance assay For the halo-tolerance assay, the isolates demonstrated varied tolerance to different NaCl concentrations, 0, 7, 12.5, 20, and 22% NaCl. All isolates appeared in NA medium with salt concentrations of 12.5% NaCl and did not appear using 22% NaCl in the same culture medium, while only the QSLA1 isolate was able to grow on NA medium with a salt concentration of 20% NaCl. Isolates QSLA1, QSLA7, QSLA16, and QSLA17 were able to grow on NA medium with a salt concentration of 7% NaCl, while QSLA14 couldn't. On the other hand, only QSLA17 isolate was able to grow on NA medium without salt concentration (zero NaCl) (Table 4). It was demonstrated that halotolerant bacteria are able to growth in the absence of salt, but tolerant of varying concentrations of salt (0–5% NaCl) ‎ [ 74 ] or extremely tolerant if tolerance extends above 15% (2.5 M) NaCl ‎ [ 28 ] . On the other hand, slight halophiles (marine bacteria) grow best with 0.2 to 0.5 M NaCl (1–3% NaCl), while moderate halophiles growing optimally in medium with 0.5 to 2.5 M NaCl (3–15% NaCl), and extreme halophiles grow optimally in medium containing 2.5 to 5.2 M (saturated) NaCl (15–30% NaCl) ‎ [ 43 ] . Table 4 Salinity tolerance assay of isolates (NaCl%). Isolates NaCl concentration (%) Zero 7 12.5 20 22 QSLA1 – + + + – QSLA7 – + + – – QSLA14 – – + – – QSLA16 – + + – – QSLA17 + + + – – Therefore, the ability of QSLA17 isolate to grow in the absence and the presence of NaCl confirmed that it is a halotolerant bacterium, while the other four isolates are halophiles as they weren't able to grow in the absence of NaCl and grew optimally in medium with 7–22% NaCl. Among them, two isolates, QSLA7 and QSLA16, grew only on NA medium with 7 and 12.5% NaCl, while the colonies of QSLA14 isolate appeared only on NA medium with 12.5% NaCl, so they were considered as moderately halophilic bacteria, while QSLA1 isolate classified as extremely halophilic bacteria as it grown on NA medium with 20%. Although QSLA16 strains were isolated from a saline water pond with 24.2% salt concentration, they couldn't grow on NA medium prepared using distilled water and 20–22% NaCl. In this respect, it was demonstrated that the NaCl requirement of these organisms has been known for many years, and NaCl could not be replaced by other salts. Also, potassium was essential for optimum growth and required for normal pigmentation. Magnesium was another essential ion for optimal growth; more of the rods appeared "normal" on medium containing the original concentration of magnesium. At lower concentrations, the organisms grew as spheres and seemed to adapt to the low concentrations of Mg 2+ . The spherical form was retained when these cells were returned to medium containing the original concentration of magnesium ‎ [ 24 ] . Using shake cultures, it was founded ‎that 10 ppm of Fe 2+ increased the yield of cells. Small amounts of manganese (0.05 ppm) provided some stimulation of growth and a marked increase in pigmentation [ 65 ] . So, isolate QSLA16 couldn’t grow on NA medium prepared using distilled water and high salt concentration of NaCl (20%) because of the absence of other essential elements such as potassium, magnesium, iron, and manganese, which are all required for optimum growth. Bacteria inhabiting hypersaline environments, like solar salterns, generally are well adapted to growth in the presence of high salt concentrations ‎ [ 71 ] . Isolates obtained from saline regions of Karnataka were able to grow in salt concentrations ranging from 5–25% ‎ [ 31 ] . Forty-six halobacterial isolates were isolated from Sambhar Lake soil and water samples. All of them were tolerant to 10% NaCl concentration, while forty-four cultures were tolerant to 15% NaCl concentration. Out of the ten cultures selected for further studies, three were tolerant to 25% salt ‎ [ 51 ] . Twenty-eight moderately halophilic bacteria were obtained from three salterns in Tamil Nadu, Kerala, and Goa, India. All isolates, except one isolate, grew rapidly on halophilic medium (HM) agar plates containing 5% NaCl, 21 isolates demonstrated heavy growth at 10–15% salt content, followed by moderate and low growth when the salt concentration was increased. From the results of maximum tolerable salt concentration (MTSC), it was observed that among 28 isolates, one grew maximally up to 5% (slight halophiles), four grew up to 10%, seventeen grew up to 15% (moderately halophilic) six grew up to 20% NaCl containing medium and also without salt (halotolerant) ‎ [ 55 ] . Molecular Identification of the Isolates Besides morphological and biochemical characterization of the isolates, the 16S rDNA gene of the isolates was sequenced and analyzed. The resultant sequences were compared with those identified and deposited in the NCBI BLAST database. The sequence analysis of the 16S rRNA gene of the isolates demonstrated that QSLA1 showed the highest similarity to strain Halomonas sp. RS-17 with 91.26%. Isolate QSLA7showed similarity of 94% to Salinivibrio sp. pr6. Isolate QSLA14 was identified as Lentibacillus sp. strain BCHS25 with 87.66% similarity. Isolate QSLA16 was shown to be a strain of Uncultured bacterium clone QAMU23 and Sphingomonas sp. LE-239 with 80.15 and 79.9% similarity, respectively, whereas QSLA17 showed the highest similarity to strain Bacillus sp. strain 6 with 74.05% similarity (Table 5). Table (5) The blasted isolates, nearest neighbors in the Genebank database, and their % relatedness. Isolates Organisms Closest strains in Genebank Identity (%) QSLA1 Halomonas Halomonas sp. RS-17 91.26 QSLA7 Salinivibrio Salinivibrio sp. pr6 94.00 QSLA14 Lentibacillus Lentibacillus sp. strain BCHS25 87.66 QSLA16 Uncultured bacterium ( Sphingomonas ) Uncultured bacterium clone QAMU23. ( Sphingomonas sp. LE-239) 80.15 (79.9) QSLA17 Bacillus Bacillus sp. strain 6 74.05 Out of twenty-eight moderately halophilic bacteria were isolated from three salterns in Tamil Nadu, Kerala and Goa, India, Nine isolates demonstrated 97–99% similarity to genus Bacillus and one isolate showed the highest similarity to Oceanobacillus with 98% similarity, while twelve isolates showing 96–99% similarity to genus Staphylococcus , two isolates belonged to Pseudomonas with 98% similarity, one isolate demonstrated 98% similarity to genus Enterobacter and three isolates were most closely related to Enterobacter , Ochrabactrum and Stenotrophomonas with 98, 98 and 96% similarity respectively ‎ [ 55 ] . 74 halophilic bacteria were isolated from Algerian saline lakes ecosystems (Sebkha and Chott) located in arid and semi-arid ecoclimate zones. Out of them, 16 isolates were most closely related to Halomonas , 18 isolates belonged to Bacillus , 7 strains were similar to Oceanobacillus , 5 isolates were identified as Virgibacillus , 10 isolates were related to Halobacillus , 2 isolates belonged to Thalassobacillus , and one isolate showed the highest similarity to Lentibacillus with 99–100% similarity ‎ [ 42 ] . Sequence deposition in GenBank (NCBI) of isolated halophilic bacteria Among 5 isolates, two resultant sequences of isolated bacteria were deposited in NCBI Genebank under accession numbers shown in Table 6. However, the other three isolates that demonstrated low/no similarity to those of the Genebank were rejected during deposition in the NCBI database. Previous studies also described similar bacterial communities in solar salterns and other salted environments. 231 moderately halophilic Bacteria and 49 extremely halophilic microorganisms were obtained from different areas of Howz Soltan playa, a hypersaline lake in the central desert zone of Iran. The isolates were identified as members of the following genera: Salicola , Halovibrio , Halomonas , Bacillus , Oceanobacillus , Thalasobacillus , Virgibacillus , Gracilibacillus , Halobacillus , Piscibacillus , and Salinicoccus ‎ [ 57 ] . Species of the genera Halobacillus , Halomonas , Thalassobacillus , Brevibacterium , and Bacillus were isolated from the Karak saline mine soil in Pakistan ‎ [ 58 ] . Table (6) Accession numbers of submitted isolate sequences deposited in GenBank (NCBI). Isolates Organisms Strains in Genebank Accession numbers QSLA1 Halomonas Halomonas sp. strain QSLA1 OP442496 QSLA14 Lentibacillus Lentibacillus sp. strain QSLA14 OP443587 Phylogenetic affiliation of bacterial isolates The resultant sequences were aligned using the Clustal W program with those identified in the GenBank, and the output was used to construct the phylogenetic tree. The result of the sequencing analysis and the closest phylogenetic bacterial isolates are presented in Table 5. Phylogenetically diverse halophilic bacteria, including Gram-negative and Gram-positive bacteria, were isolated from four ponds of different salt concentrations belonging to Qarun Lake. The sequences of the isolates fell within three major groups; two strains of the isolates fell under Firmicutes, while Gammaproteobacteria included one isolate, and one isolate fell into Alphaproteobacteria. The Firmicutes group includes three families, Bacillaceae, Planococcaceae, and Staphylococcaceae, two of which are isolated strains affiliated with the family Bacillaceae, with two different genera, including Bacillus and Lentibacillus . Gammaproteobacteria comprise several medically and scientifically important groups of bacteria, such as the families Halomonadaceae, Vibrionaceae, and Pseudomonadaceae. Alphaproteobacteria includes several families, such as Sphingomonadaceae. The general phylogenetic associations of the isolated strains are shown separately in Fig. 2. Grouping our isolates with these identified halophilic bacteria in the Genebank confirms again the halophilic characters of the isolates. Detection of hydrolytic enzymes As a result of their extreme adaptation, halophilic bacteria have attracted the attention of the scientific community due to their ability to produce active enzymes, exo polysaccharides, and pigments with potential biotechnology applications ‎ [ 7 ] . Isolates were examined for lipase, esterase, casease, gelatinase (protease), and chitinase production. Enzymatic activity index was measured ‎ [ 37 ] . For this purpose, the colony diameter and the clear zone diameter around colonies were measured. Assays were performed in triplicate (n = 3), and average and Standard error values are shown in the graphs. Lipolytic enzyme assay on phenol red agar medium supplemented with olive oil The lipolytic activities of all isolates were qualitatively determined on plates containing phenol red agar medium; among the isolates, QSLA1 and QSLA7 demonstrated different lipolytic activity on phenol red agar plates (Table 7 and Fig. 3). Table (7) screening isolates for lipase activity using olive oil (Means + Std. Error). Lipase isolates Colony diameter(cm) Clear zone diameter(cm) Lipolytic Enzymatic Rate QSLA1 0.8 2.0 3.50 ± 0.06 a QSLA7 0.9 2.5 3.64 ± 0.06 a Values are Means ± Standard Errors of three replicates. The isolate QSLA7, which had 94% similarity to Salinivibrio sp. pr6 demonstrated the highest lipolytic enzymatic rate (3.64), followed by QSLA1. There were no significant differences between them, as shown in Table 7 and Fig. 4. The change in color of phenol red was used as an indicator for lipase activity, where lipase-producing bacteria will turn the dye into a yellow color‎ [ 38 ] . An extracellular lipase was characterized from moderately halophilic Salinivibrio sp. strain SA-2. The enzyme remained active in the presence of 17% NaCl ‎ [ 5 ] . Marinobacter lipolyticus , a novel moderate halophile, was isolated from a hypersaline habitat with lipolytic activity that grows optimally at 7.5% NaCl. Many moderately halophilic bacteria belonging to the genera Marinobacter , Virgibacillus , Halobacillus , Geomicrobium , Chromohalobacter , Oceanobacillus , Bacillus , Halomonas , and Staphylococcus were identified with the ability to produce industrially important enzymes and a salt requirement of 3–20% (w/v) NaCl‎ [ 40 ] . 54 Halobacterial strains were isolated from waters of the El Golea Sebkha, which is located in the middle of Algerian Sahara and contains salt concentrations up to 300 g/L. 35 isolates demonstrated lipolytic activity on sterilized medium (NaCl 25% w/v) supplemented with 2.5% olive oil (w/v)‎ [ 10 ] . Marinobacter sp. SWJ2, which isolated from Seawater in Tanjung Piai National Park, Johor Bahru, Malaysia, demonstrated lipase production ability ‎ [ 47 ] . It was illustrated that olive oil is suitable due to the high concentration (greater than 70%) of oleic acid and has been used to assay the activity of various microbial lipases‎ [ 70 ] . Lipolytic enzyme assay on Tween 80 agar medium The results of lipolytic activity of the isolates on Tween 80 agar medium demonstrated that, all of the five bacterial isolates were able to release lipolytic enzymes, demonstrated by precipitation of the calcium salt of the fatty acids released by hydrolysis of tweens around the lipase producing bacteria as shown in Fig. (5). The highest lipolytic enzymatic rate was shown by isolate QSLA16 (2.99), which identified as uncultured bacterium clone QAMU23 with 80.15% similarity, followed by QSLA1 (2.62), QSLA17 (2.60), QSLA7 (2.43), and QSLA14 (1.72) as shown in table (8). There were significant differences between QSLA16 and the other isolates, while significant differences were not recorded between QSLA1, QSLA7, and QSLA17 isolates; however, significant differences were shown between them and QSLA14, as observed in Table 8 and Fig. 6. Table (8) Screening of the isolates for lipase activity (using Tween 80) (Means + Std. Error). Lipase isolates Colony diameter (cm) Clear zone diameter(cm) lipolytic Enzymatic rate QSLA1 1.4 2.4 2.62 ± 0.06 b QSLA7 1.8 2.7 2.43 ± 0.06 b QSLA14 1.0 1.8 1.72 ± 0.06 a QSLA16 1.4 2.9 2.99 ± 0.06 c QSLA17 2.4 4.1 2.60 ± 0.06 b Values are Means ± Standard Errors of three replicates c = higher value a = lower value Lipase-producing microorganisms were detected, isolated from different counties of the Amazon region, using Tween 80 as a substrate‎ [ 77 ] . Detection of esterase Three isolates, QSLA14, QSLA16, and QSLA17, demonstrated esterase activity (Table 9 and Fig. 7) as precipitation of the calcium salt of the fatty acids released by hydrolysis of tweens around the lipase-producing bacteria‎ [ 35 ] . The isolate QSLA17 possessed the highest value of esterolytic enzymatic rate (3.21), followed by QSLA16 (2.61) and QSLA14 (2.1). There were significant differences between all of the isolates, as shown in Table 9 and Fig. 8. Table (9) Screening of the isolates for esterase activity using Tween 20 as substrate (Means + Std. Error). Esterase isolates Colony diameter (cm) Clear (precipitation) zone diameter (cm) Esterase Enzymatic rate QSLA14 1.1 1.3 2.10 ± 0.06 a QSLA16 1.9 3.2 2.61 ± 0.06 b QSLA17 1.0 2.3 3.21 ± 0.06 c Values are Means ± Standard Errors of three replicates c = higher value a = lower value The use of Tweens as lipase substrates in screening procedures has been criticized because tweens may be hydrolyzed by esterase (tween 20). Nonetheless, Tweens are attractive because they are very readily incorporated into growth medium, promoting optimal contact between cells and/or enzymes and the substrate ‎ [ 70 ] . Twenty-four halo alkali bacterial isolates with esterolytic activity were isolated from Lake Magadi, Kenya. The isolates were screened for their ability to produce estrase enzyme by spotting them onto the basal medium supplemented with Tween 20 as a substrate‎ [ 34 ] . Detection of casease Isolates QSLA1, QSLA7, and QSLA14 demonstrated caseinase activity in modified skim milk agar medium (Fig. 9) in the form of clear zones around the isolates on plates with skim milk as a substrate, as observed by Cappuccino and Sherman (2002) and Kiplimo et al. (2019). Among caseae producer isolates, QSLA7 gives the highest value of proteolytic enzymatic rate (3.11), followed by QSLA1 (3) and QSLA14 (2.61). Significant differences between isolates QSLA7 and QSLA1 weren't found, but were found between them and the QSLA14 isolate, as observed in Table 10 and Fig. 10. Table (10) Screening of the isolates for caseinase activity (Means + Std. Error). Isolates Colony diameter (cm) Clear zone diameter (cm) Proteolytic Enzymatic Rate QSLA1 2.0 4.1 3.00 ± 0.06 b QSLA7 1.8 4.0 3.11 ± 0.06 b QSLA14 1.7 2.9 2.63 ± 0.06 a Values are Means ± Standard Errors of three replicates b = higher value a = lower value 37 halophilic archaeal strains were obtained from the marine solar saltern of Mulund, Mumbai. Screening for protease production using the skimmed milk agar plates method demonstrated that 5 have produced halophilic proteases‎ [ 50 ] . Detection of gelatinase (protease) Screening of isolates for gelatinase activity was carried out using nutrient gelatin medium. Isolates QSLA1, QSLA7, QSLA14, and QSLA17 demonstrated gelatinase activity when bacterial growth liquefied gelatin medium after incubation at 30°C for 7 days and being placed in a refrigerator at 4°C for 60 min (until the non-inoculated gelatin solidified) as illustrated in previous studies‎ [78] , also it was observed that gelatin hydrolysis, as evidenced by liquefaction of the nutrient gelatin after being placed in cold water/refrigerator at 4°C‎ [ 13 ] . As shown, all isolates able to producing casease analyzed gelatin, as a nitrogen source in medium, except isolate QSLA17, which was identified as Bacillus sp. (74.05%), that hydrolyzed gelatin only. We thought that maybe because casein and gelatin are 2 contrasting proteins with respect to their amino acid composition. Casein is a complete protein because it contains all indispensable amino acids (IAA), whereas gelatin is an incomplete protein because it lacks the essential amino acid tryptophan and contains low amounts of, e.g., methionine and histidine, as it was demonstrated‎ [ 27 ] . Chitinase activity For chitinase detection, LB colloidal chitin agar medium was prepared. Bacterial isolates QSLA1, QSLA7, QSLA14, and QSLA16 (Table 11) demonstrated chitinase activity as a zone of clearance around the colony due to the breakdown of chitin to N-acetylglucosamine (Fig. 11) as it was clarified ‎ [ 37 , 2 ] Table (11): Screening of the isolates for chitinase activity (Means + Std. Error). Isolates Colony diameter (cm) Clear zone diameter (cm) Chitinolytic Enzymatic Rate QSLA1 1.8 3.4 2.74 ± 0.06 c QSLA7 1.1 1.6 2.38 ± 0.06 b QSLA14 2.3 3.5 2.44 ± 0.06 b QSLA16 3.6 4.0 2.09 ± 0.06 a Values are Means ± Standard Errors of three replicates c = higher value a = lower value Table (11), and Fig. (12) show that QSLA1 scored the best chitinolytic enzymatic rate (2.74) and was significantly different from other chitinase-producing isolates. QSLA14 came in second with an enzymatic rate of 2.44, followed by QSLA7 and QSLA16 with chitinolytic enzymatic rates of 2.38 and 2.09, respectively. Moderately halophilic bacteria were isolated from shallow salt lakes with the ability to produce chitinase. Those moderately halophilic bacteria were Virgibacillus marismortui , B. subtilis , Bacillus pumilus , Bacillus licheniformis , Terribacillus halophilus , Planococcus rifietoensis , Staphylococcus equorum , and Staphylococcus sp‎ [ 23 ] . In a study evaluating the antimicrobial activity of QSLA1 isolate against highly aggressive plant pathogens, the results showed that QSLA1 isolate possessed antifungal activity against Fusarium oxysporium and Alternaria solani , as well as antibacterial activity against Ralstonia solanacearum ‎ [ 46 ] . This ability to resist plant pathogens may be due to its ability to produce one or more of these enzymes, each of which plays an important role in the process of killing and decomposing plant pathogens. Detection and quantitative screening for biosurfactant production A number of screening methods have been developed to detect biosurfactant production amongst microorganisms‎. Assays such as the Du-Nouy ring tensiometer assay, axisymmetric drop shape assay, and pendant drop shape assay measure the surface and interfacial tensions of biosurfactants directly; whereas the oil spreading and emulsification capacity give an indirect measurement of surfactant production [ 75 ] . In this study, screening for biosurfactant production was carried out by oil displacement test, emulsification index, and measurement of surface tension using a Stalagometer Instrument for five randomly selected isolates. The four halophilic isolates QSLA1, QSLA7, QSLA14, and QSLA16 were grown in Landy Medium under 2 different salt concentrations (3.5 and 7%), while the halotolerant isolate QSLA17 was grown under different salt concentrations (zero, 3.5, and 7%). Assays were performed in triplicate (n = 3), and average and Standard error values are shown in the graphs. Results are shown in Table 20. Table (12): Screening of isolates for biosurfactant production by the selected isolates (Means + Std. Error). Treatments NaCl concentration of the Landy broth medium Oil spill zone (cm) Emulsification on index E24% Surface tension γ (dyne/cm 2 ) QSLA1 3.5% 3.77 ± 0.06 cd 76.08 ± 0.66 h 50.49 ± 1.04 bcd QSLA1 7% 4.97 ± 0.06 f 77.01 ± 2.11 h 49.14 ± 0.58 b QSLA7 3.5% 3.80 ± 0.06 cd 64.35 ± 0.93 f 51.77 ± 0.52 def QSLA7 7% 3.93 ± 0.06 cd 66.32 ± 0.89 f 50.89 ± 0.80 bcd QSLA14 3.5% 3.83 ± 0.06 cd 64.02 ± 1.28 f 50.83 ± 0.57 bcd QSLA14 7% 4.03 ± 0.06 cd 65.08 ± 0.66 f 49.86 ± 0.16 bc QSLA16 3.5% 4.17 ± 0.06 de 55.62 ± 0.60 e 50.58 ± 0.17 bcd QSLA16 7% 4.53 ± 0.06 e 70.69 ± 0.60 g 49.73 ± 0.25 bc QSLA17 Zero 4.20 ± 0.06 de 52.10 ± 0.48 d 50.50 ± 0.64 bcd QSLA17 3.5% 3.64 ± 0.06 c 43.67 ± 0.80 b 52.50 ± 0.75 ef QSLA17 7% 3.10 ± 0.06 b 32.70 ± 2.75 a 53.43 ± 1.16 f Landy Broth Medium (LBM) * 7% 0.50 a – 67.76 g Tween 20 (1%) in LBM (7%) ** 7% 7.17 ± 0.06 g 81.65 ± 0.45 i 37.04 ± 1.43 a Values are Means ± Standard Errors of three replicates i, g = higher value a = lower value * = Negative control. ** = Positive control. Oil displacement test A zone of displacement in the oil was observed by QSLA1, QSLA7, QSLA14, QSLA16, and QSLA17 (Figs. 13 & 14), and the diameter of oil displacement (cm) was measured (Table 12). The results demonstrated that QSLA1 had higher activity (4.97 cm) with respect to oil displacement at 7% NaCl concentration. A significant difference was observed between QSLA1 and QSLA16 grown on Landy Medium with 7% salt concentration of medium and other treatments as shown in Fig. (15). Plates treated only with Landy Broth Medium (7% NaCl) were used as negative control, while plates treated with tween 20 (1%) added to Landy Broth Medium (7% NaCl) were used as positive control. The oil displacement test, also known as the oil spreading assay, relies on the displacement of oil by the biosurfactant‎ [ 44 ] . Displacement of the oil, as indicated by a zone of clearing, is considered to be a positive indication of the presence of surfactant compounds. The diameter of the displacement corresponds to the level of biosurfactant activity‎ [ 75 ] . The oil spreading method is simple, fast, requires a small amount of sample, and does not require specialized equipment. It is also sufficiently sensitive to detect biosurfactant activity at low concentrations, so several studies have used the oil spreading technique as a reliable Means to screen for biosurfactant compound production amongst various microorganisms‎ [ 52 ] . Emulsification index (E24) The E24 test, also known as the emulsification capacity assay, relies on the ability of a surfactant molecule to form a stable emulsion (Cooper and Goldenberg, 1987). This assay aids in quantitative screening of biosurfactant producers‎ [ 64 ] . The results in Table 20 demonstrated that the emulsification index of QSLA1 isolate was the best amongst other isolates (77.01%), in 7% NaCl concentration. A significant difference was observed between QSLA1 and QSLA16 grown on 7% salt concentration of medium and other treatments, as shown in Figs. 49, 50, and 51. This assay is carried out to determine the emulsification property of surfactant compounds. It involves the formation of micelles where hydrophobic liquids become dispersed within hydrophilic liquids (e.g., water), thereby forming an emulsion of two substances, which are normally immiscible‎ [ 8 ] . Measurement of surface tension using the stalagmometric assay Using the stalagometric assay, all tested isolates demonstrated decreased surface tension compared to the negative control sample (un-inoculated Landy Medium 7% salt) (Table 20). The best result in decreasing surface tension (49.14 dyne/cm 2 ) was observed in QSLA1 supernatant with 7% NaCl concentration. A significant difference was observed between QSLA1 grown on 7% salt concentration and QSLA17 grown on 7% salt concentration, as shown in Fig. 52. Concerning the ability of studies isolates to produce halo biosurfactants; it was noticeable that the results of the oil displacement test are similar to those of the emulsification index assay and surface tension measurement. Where isolate QSLA17 recorded a high level of emulsification zone percentage and displacement zone diameter in the absence of salt, then decreased with increasing salt concentrations, while a low level of surface tension was recorded in the absence of salt, then increased with increasing salt concentrations. On the other hand, the percentage of emulsification zone and displacement zone diameter for QSLA1, QSLA7, QSLA14, and QSLA16 isolates was increased with increasing salt concentrations, while surface tension decreased. This is due to QSLA17 being a halotolerant bacterium, and QSLA1, QSLA7, QSLA14, and QSLA16 are halophilic isolates. The results observed that the two isolates QSLA1 and QSLA16, which were identified as Halomonas sp. RS-17 and Uncultured bacterium clone QAMU23, respectively, demonstrated the best results of biosurfactant production with the used methods, especially in high salt concentrations, as they were halophilic bacteria, followed by isolate QSLA7, which was identified as Salinivibrio sp. While isolate QSLA17 demonstrated the lowest results, it demonstrated high biosurfactant production in the absence of salt, as it was a halotolerant bacterium. In a previous study, the ability of QSLA16 and QSLA17 isolates to produce halo biosurfactants was examined using PCR technique to ensure the presence of the genes encoding for non-ribosomal lipopeptides (NRPs). It was revealed that QSLA16 and QSLA17 contain surfactin and fengycin genes while mycosubtilin (iturin) gene was detected only in QSLA17 isolate. Moreover, HPLC results showed that isolate QSLA16 produced surfactin and fengycins, with concentrations of 98 mg/l and 45 mg/l, respectively, while isolate QSLA17 produced surfactin, fengycins and iturin, with concentrations of 112 mg/l, 38 mg/l and 67 mg/l, respectively. Therefore, QSLA16 showed antibacterial activity against salmonella typhi, Acinetobacter baumanni and Staphylococcus aureus but it had no effect on E. coli , Klebsiella pneumonia and Proteus mirabilis ‎ [ 1 ] . In another study conducted, GC-MS analysis showd that metabolites of isolates QSLA1 and QSLA7 contain bioactive compounds such as Decanoic Acid, Ethyl Este and its derivatives ‎ [ 46 ] which are classified as fatty acids and fatty acid derivatives, biosurfactants ‎ [ 63 , 17 ] . Members of the Rhodobacteraceae, Rhodospirillaceae, Halomonadaceae, Oceanospirillaceae, Pseudomonadaceae, and Shewanellaceae families have also been reported to be oil-degraders and biosurfactants producers‎ [ 6 ] . Halomonas sp. BS4 (a halophilic bacterium) was isolated from Thamaraikulam solar salt works in India. After routine biosurfactant screening by various methods, it was found to be a biosurfactant-producing bacterium. Biosurfactants containing compound 1, 2-Ethanediamine N, N, N0, N0-tetra, 8-Methyl-6-nonenamide, (Z)-9-octadecenamide, and fatty acid derivatives retrieved from Halomonas sp. BS4‎ [ 20 ] . Sari et al. (2020) Halomonas meridiana BK-AB4 was obtained from the Bledug Kuwu mud volcano in Central Java, Indonesia, and studied it for microbial-enhanced oil recovery (MEOR) applications using crude oil. It was found to be a biosurfactant producer. The main biosurfactant compounds are 9(E), 11(Z)-Octadecadienoic acid, 13-hydroxy-, methyl ester (C19H34O3); 9(E), 11(E)-Octadecadienoic acid, 13-oxo-, methyl ester (C19H32O2); 6,9,12-Octadecatrienoic acid, methyl ester (C19H32O2); and 3-Oxononadecanoic acid (C19H36O3). Those four compounds consist of 77.7% of the biosurfactant produced ‎ [ 63 ] . A biosurfactant-producing bacterium, designated 3B-2, was isolated from marine sediment and identified it as Vibrio sp. by 16S rRNA gene sequencing. The produced biosurfactant was characterized in terms of protein concentration, surface tension, and oil-displacement efficiency‎ [ 29 ] . Halophilic Bacillus sp. BS3 was identified from solar salt works, identified by 16S rRNA sequencing, was used for screening their biosurfactant production. The biosurfactants were characterized by TLC, FTIR, and GC-MS analysis and identified as lipopeptide type. GC-MS analysis revealed that the biosurfactant had various compounds, including 13- 13-Docosenamide, (Z); Mannosamine, 9- and N, N, N', N'-tetramethyl‎ [ 19 ] . Genera Thalassobacillus , Halobacillus , Lentibacillus , and Bacillus belonged to family Bacillaceae, which are known as NRPs producers, such as surfactin ‎ [ 39 ] that increase the bioavailability of hydrocarbons and improve their degradation‎ [ 32 ] . It was illustrated that the biosurfactant, such as surfactin, increases the bioavailability of hydrocarbons/oils (such as Tween 80) in culture‎ [ 32 ] . Liberated fatty acids bind with the calcium incorporated into the medium. The calcium laurate complex is visible as insoluble crystals around the inoculation site ‎ [ 35 ] . And this explains the ability of the isolates to analyze olive oil and Tweens, and how to detect the occurrence of this analysis. Conclusion This study highlights the remarkable diversity and biotechnological relevance of halo enzymes and halo biosurfactants derived from halophilic microorganisms. The investigated halo biosurfactants and halo enzymes demonstrated stability and catalytic efficiency under extreme saline conditions, underscoring their potential for sustainable industrial applications such as bio catalysis, pharmaceuticals, food processing, and environmental applications. These findings provide valuable insights into the adaptive strategies of halophiles and emphasize the untapped potential of their halo enzymes and halo biosurfactants in biotechnology. Looking ahead, future studies should aim to integrate genomic and proteomic analyses to discover novel halo enzymes with unique structural and functional properties, while advances in protein engineering and directed evolution may further enhance their stability, catalytic performance, and substrate specificity. Moreover, efforts to scale up biosurfactants and halo enzyme production under industrially relevant conditions, coupled with investigations into synergistic enzyme systems for complex bio catalytic pathways, will be crucial to bridging laboratory findings with real-world applications. Beyond industrial use, exploring the medical and environmental potential of halo biosurfactants and halo enzymes, particularly in antimicrobial development and bioremediation of saline or contaminated habitats, represents a promising frontier. Altogether, this work not only deepens our understanding of halophilic adaptations but also lays a foundation for exploiting halo enzymes as sustainable and versatile tools in modern biotechnology. Declarations Funding details If the paper is accepted the publishing will be funded according to the Open Access Agreement for Egypt between Springer Nature &Science, Technology& Innovation Funding Authority (STDF) in cooperation with Egyptian Knowledge Bank (EKB). Author Contribution Methodology, results &discussions & statistical analysis, A. A.A.M., Abstract & introduction, A.E.E. Phylogenetic analysis & figures preparing, B.M.E.E. All authors have read &agreed to the published version of the manuscript. 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Genetic diversity &salt tolerance of bacterial communities from two Tunisian soils. Ann. Microbiol. 59 , 25–32 (2009). Van Der Lelie, D. et al. Use of Single-Point Genome Signature Tags as a Universal Tagging Method for Microbial Genome Surveys. Appl. &Environmental Microbiol. 72 (3), 2092–2101 (2011). Ventosa, A. & &Arahal, D. R. Physico-Chemical Characteristics of Hypersaline Environments &Their Biodiversity. Encyclopedia of Life Support Systems (EOLSS). Extremophiles , Vol. II. (2011). Ventosa, A. & &Nieto, J. J. Biotechnological applications &potentialities of halophilic microorganisms. World J. Microbiol. &Biotechnology , 11 . (1995). Walter, V., Syldatk, C. & &Hausmann, R. Screening concepts for the isolation of biosurfactant-producing microorganisms. Biosurfactants , pp. 1–13. (2010). Whittenbury, R. Hydrogen Peroxide Formation &Catalase Activity in the Lactic Acid Bacteria. J. Gen. Microbiol. 35 , 13–26 (1964). Willerding, A. L., Antonio, O. L., Moreira, F. W., Germano, M. G. & &Chagas, J. A. F. Lipase Activity among Bacteria Isolated from Amazonian Soils. SAGE-Hindawi Access to Research Enzyme , Research Volume 2011, Article ID 720194, 5 pages (2011). 10.4061/2011/720194 Ingroff, A. E., Goldson, P. R., Mcginnis, M. R. & &Kerkering, T. M. Evaluation of proteolytic activity to differentiate some dematiaceous fungi. Am. Soc. Microbiol. J. Clin. Microbiol. 26 (2), 301–307 (1988). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7456751","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":505815819,"identity":"330328b7-93a8-442b-868e-53ea16f45a60","order_by":0,"name":"Ahmed Ahmed Abdelmonaem Mousa","email":"data:image/png;base64,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","orcid":"","institution":"Menoufia University","correspondingAuthor":true,"prefix":"","firstName":"Ahmed","middleName":"Ahmed Abdelmonaem","lastName":"Mousa","suffix":""},{"id":505815820,"identity":"9a7a6afe-30c9-44cd-b138-00416b26ff0d","order_by":1,"name":"Basma Mahmoud Ebraheim Elkot","email":"","orcid":"","institution":"Menoufia 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calibrated scale.\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7456751/v1/d9f3ef202ce7f80a7ceb0f54.jpeg"},{"id":89974600,"identity":"fd87d459-71de-43d2-af54-4d68c8797fe4","added_by":"auto","created_at":"2025-08-27 05:54:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":7247054,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree constructed using neighbor-joining analysis based on 16S rDNA sequences of the isolates and more closely related sequences deposited in the Genebank.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7456751/v1/3942f2d7de9b1a26bd251a9a.png"},{"id":89974595,"identity":"3de15a6d-51ad-4c8f-a74d-c4aae52f3bc1","added_by":"auto","created_at":"2025-08-27 05:54:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":261640,"visible":true,"origin":"","legend":"\u003cp\u003ePhenol red, olive oil agar plates showing yellow zones around positive isolates (concentration 12.5% NaCl).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7456751/v1/9c84579e0f47198846faa7fc.png"},{"id":89974621,"identity":"25863966-06e9-4447-9827-b9b1230ddf2f","added_by":"auto","created_at":"2025-08-27 05:54:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":11722,"visible":true,"origin":"","legend":"\u003cp\u003eRates of lipase determination using olive oil.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7456751/v1/ad872f312398b86fe5bc8f96.png"},{"id":89976347,"identity":"d45f9270-5549-400a-a679-0d3d4ff99f11","added_by":"auto","created_at":"2025-08-27 06:02:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":154000,"visible":true,"origin":"","legend":"\u003cp\u003eTween 80 agar plates for lipase detection showing insoluble crystal zones around the inoculation site due to the calcium complex precipitation (concentration 12.5% NaCl).\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7456751/v1/ed4281ffbd52f3c97cca6e03.png"},{"id":89974619,"identity":"209c26e4-8a7a-40c5-8f73-0863d3d4669a","added_by":"auto","created_at":"2025-08-27 05:54:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":13140,"visible":true,"origin":"","legend":"\u003cp\u003eRates of lipase determination on Tween 80 agar medium.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-7456751/v1/467573be83a58416bf89e580.png"},{"id":89976354,"identity":"7cee838c-e2bd-49d7-9a79-e667b6c96e51","added_by":"auto","created_at":"2025-08-27 06:02:46","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":140265,"visible":true,"origin":"","legend":"\u003cp\u003eTween 20 agar plates showing zones of insoluble crystals around the inoculation site due to the calcium complex precipitation (concentration 12.5% NaCl).\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-7456751/v1/7ebe54625b6891da2cff9035.png"},{"id":89974597,"identity":"7ccf2256-bdd7-4b32-bb14-0cf8e1e6c95c","added_by":"auto","created_at":"2025-08-27 05:54:44","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":11337,"visible":true,"origin":"","legend":"\u003cp\u003eRates of esterase determination on Tween 20 agar medium.\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-7456751/v1/1646a26befefc2528596ee17.png"},{"id":89976344,"identity":"8a6d9ff9-6a1b-490c-afae-7699f42b8e4f","added_by":"auto","created_at":"2025-08-27 06:02:44","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":125202,"visible":true,"origin":"","legend":"\u003cp\u003eSkim milk agar plates (for casease detection) showing clear zones around positive treatments (concentration 12.5% NaCl).\u003c/p\u003e","description":"","filename":"image11.png","url":"https://assets-eu.researchsquare.com/files/rs-7456751/v1/e186130c48487b362669eca5.png"},{"id":89976342,"identity":"3284c8f5-48ca-40ee-9a26-3cc69607da14","added_by":"auto","created_at":"2025-08-27 06:02:44","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":11592,"visible":true,"origin":"","legend":"\u003cp\u003eRates of caseinase determination on skim milk agar medium.\u003c/p\u003e","description":"","filename":"image12.png","url":"https://assets-eu.researchsquare.com/files/rs-7456751/v1/0076a5c65ea8ea60a59f7e28.png"},{"id":89974628,"identity":"c64356f8-990d-4340-a667-6fcd676f6a82","added_by":"auto","created_at":"2025-08-27 05:54:44","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":134788,"visible":true,"origin":"","legend":"\u003cp\u003eChitin agar plates showing clear zones around positive treatments (concentration 12.5% NaCl).\u003c/p\u003e","description":"","filename":"image13.png","url":"https://assets-eu.researchsquare.com/files/rs-7456751/v1/82a5a7bf9a44ad5c1ed614f6.png"},{"id":89974635,"identity":"e8d92a92-3cbf-4fb6-964d-def3e596bcd0","added_by":"auto","created_at":"2025-08-27 05:54:45","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":11444,"visible":true,"origin":"","legend":"\u003cp\u003eRates of chitinase production using colloidal chitin agar plates.\u003c/p\u003e","description":"","filename":"image14.png","url":"https://assets-eu.researchsquare.com/files/rs-7456751/v1/a9463fa30138f5eadba66968.png"},{"id":89977675,"identity":"37ac66f6-d80b-4367-9cf3-af43b1553c2b","added_by":"auto","created_at":"2025-08-27 06:10:45","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":126181,"visible":true,"origin":"","legend":"\u003cp\u003eOil displacement test showing a zone of displacement in the oil for isolate QSLA17 grown on Landy Medium in two salt concentrations (3.5 and 7% NaCl).\u003c/p\u003e","description":"","filename":"image15.png","url":"https://assets-eu.researchsquare.com/files/rs-7456751/v1/4ac8bde32f51eba3184ac5fe.png"},{"id":89977676,"identity":"635289ac-b9b7-4044-9f97-937c2c0a93c1","added_by":"auto","created_at":"2025-08-27 06:10:46","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":167056,"visible":true,"origin":"","legend":"\u003cp\u003eOil displacement test showing zone of displacement in the oil for isolates QSLA1, QSLA7, QSLA14, and QSLA16 grown on Landy Medium in two salt concentrations (3.5 and 7% NaCl).\u003c/p\u003e","description":"","filename":"image16.png","url":"https://assets-eu.researchsquare.com/files/rs-7456751/v1/e2f154c3a5388d3c168d1372.png"},{"id":89974665,"identity":"3c55fbbd-10a6-4d9c-8a76-8ba53e2197cd","added_by":"auto","created_at":"2025-08-27 05:54:46","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":16862,"visible":true,"origin":"","legend":"\u003cp\u003eBiosurfactant production as indicated by oil displacement test.\u003c/p\u003e","description":"","filename":"image17.png","url":"https://assets-eu.researchsquare.com/files/rs-7456751/v1/b544bcbab563b3779edc3f78.png"},{"id":89976346,"identity":"fc67c7f3-b201-438d-bd4a-aa1fc1ca3eba","added_by":"auto","created_at":"2025-08-27 06:02:45","extension":"png","order_by":16,"title":"Figure 16","display":"","copyAsset":false,"role":"figure","size":184377,"visible":true,"origin":"","legend":"\u003cp\u003eEmulsification test showing a zone of emulsion for isolates QSLA16 and QSLA17 grown on Landy Medium in different salt concentrations.\u003c/p\u003e","description":"","filename":"image18.png","url":"https://assets-eu.researchsquare.com/files/rs-7456751/v1/4c45a496425f3fec112a79c2.png"},{"id":89974671,"identity":"88bcd15e-3a39-4535-b4a2-d69fce71dee6","added_by":"auto","created_at":"2025-08-27 05:54:47","extension":"png","order_by":17,"title":"Figure 17","display":"","copyAsset":false,"role":"figure","size":341696,"visible":true,"origin":"","legend":"\u003cp\u003eEmulsification test showing a zone of emulsion for isolates QSLA1, QSLA7, and QSLA14 grown on Landy Medium in two salt concentrations (3.5 and 7% NaCl).\u003c/p\u003e","description":"","filename":"image19.png","url":"https://assets-eu.researchsquare.com/files/rs-7456751/v1/0643325ade00b1fbd2ed11ef.png"},{"id":89976345,"identity":"4822c074-bf7d-41ce-9798-0ffd5d300e99","added_by":"auto","created_at":"2025-08-27 06:02:44","extension":"png","order_by":18,"title":"Figure 18","display":"","copyAsset":false,"role":"figure","size":17779,"visible":true,"origin":"","legend":"\u003cp\u003eEmulsification index for the tested bacterial isolates under different salt concentration ratios.\u003c/p\u003e","description":"","filename":"image20.png","url":"https://assets-eu.researchsquare.com/files/rs-7456751/v1/47ee0ec0ad620d644cdf69a3.png"},{"id":89974632,"identity":"11e485ed-3f22-4710-8419-03997915841a","added_by":"auto","created_at":"2025-08-27 05:54:44","extension":"png","order_by":19,"title":"Figure 19","display":"","copyAsset":false,"role":"figure","size":17588,"visible":true,"origin":"","legend":"\u003cp\u003eMeasurement of surface tension test using a stalagometer for selected isolates under different salt concentrations.\u003c/p\u003e","description":"","filename":"image21.png","url":"https://assets-eu.researchsquare.com/files/rs-7456751/v1/9c77ad550b8b31620f6bd077.png"},{"id":97900895,"identity":"e470e3fa-0aee-4e74-8e71-0f41a7f4ed49","added_by":"auto","created_at":"2025-12-10 15:46:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10365479,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7456751/v1/e5c1edce-9285-43ca-940a-0eb6babe2c98.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exploring halo enzymes and halo biosurfactants production from halophilic and halotolerant bacteria isolated from Lake Qarun solar salterns","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHypersaline ecosystems are widely distributed habitats, including a variety of terrestrial lakes and deep-sea basins with salt concentrations exceeding three times seawater up to saturation. Hypersaline habitats can be divided into two main types, thalassohaline and athalassohaline waters\u0026lrm; \u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e. Thalassohaline waters or brines are of marine origin and have ionic composition similar to that of seawater. Athalassohaline waters or brines, such as the Dead Sea and soda lakes, are often found inland and therefore not directly connected to marine waters. Hypersaline environments are spreading all over the world, in arid, coastal, and deep-sea locations, underground salt mines, and artificial salterns \u003csup\u003e\u0026lrm;[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. Solar salterns are widely distributed and can be found at sea level in arid and semiarid regions, both as naturally occurring or human-made salterns \u0026lrm;\u003csup\u003e[\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eHalophiles are salt-loving microorganisms present in saline habitats as normal inhabitants \u0026lrm;\u003csup\u003e[\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]\u003c/sup\u003e and can be found in all three domains of life: Archaea, Bacteria, and Eukarya \u0026lrm;\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. They are an excellent source of a great variety of extracellular hydrolytic enzymes such as protease, lipase, esterase, gelatinase, and chitinase. Such enzymes are not only salt-tolerant but also can withstand and carry out reactions efficiently under extreme conditions \u0026lrm;\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. The isolation of moderate and extreme halophiles able to produce hydrolases will provide the possibility to have optimal activities at different salt concentrations that could be useful in some industrial processes \u0026lrm;\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. The halophilic members in the genera \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eHalomonas\u003c/em\u003e, \u003cem\u003eSalinovibrio\u003c/em\u003e, \u003cem\u003eChromohalobacter\u003c/em\u003e, and \u003cem\u003eSalinicoccus\u003c/em\u003e have been identified with potential in the production of a variety of exo-enzymes under hypersaline habitats \u0026lrm;\u003csup\u003e[\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]\u003c/sup\u003e. Also, they considered a highly promising source for discovering novel biosurfactant molecules that are used currently for pharmacological, food, and cosmetics applications \u003csup\u003e\u0026lrm;[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eQarun Lake is located in the north of Fayoum Governorate and is currently saline, turbid, and without surface outflow \u0026lrm;\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Solar salterns of Qarun Lake typically consist of several ponds interconnected to form the so-called multipond system. These ponds are used by a local company for salt and minerals production. These different ponds have different salt concentrations, therefore have different microbial communities.\u003c/p\u003e\u003cp\u003eThis study was undertaken with the aim of isolating and identifying halophilic/halotolerant bacterial communities that can reside in the solar saltern ponds of Qarun Lake and test their ability to produce salt-tolerant hydrolytic enzymes and biosurfactants.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eBacterial isolates\u003c/h2\u003e\n \u003cp\u003eDuring September 2019, water samples were collected from solar saltern ponds. The collected water samples were filtered to remove impurities, analyzed for salt concentration by refractometer and pH by pH meter, before use. Ten liters of water were collected from each pond in sterile plastic jars, placed in ice packs, and transferred to the Biotechnology Laboratory, Botany Department, Faculty of Agriculture, Menoufia University.\u003c/p\u003e\n \u003cp\u003eA sterilized Nutrient agar medium (NA) was used for bacterial isolation by streaking 100 \u0026micro;l of the water sample on NA plates. The plates were incubated at 30\u0026deg;C for 7\u0026ndash;30 days \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. A subsequent streaking and sub-culturing were performed on NA medium prepared by pond water for purification, and the purified isolates were kept in 30% glycerol in the freezer till further use.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eMorphological and physiological characterization of the isolates\u003c/h3\u003e\n\u003cp\u003eColonies developed on the plates were morphologically examined for their shape, pigmentation, elevation, and optical properties. In addition, cell shape, Gram staining, and endospore formation procedure \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e, motility by hanging drop method \u003csup\u003e\u0026lrm;[\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e, and catalase activity method \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e76\u003c/span\u003e]\u003c/sup\u003e, were also evaluated.\u003c/p\u003e\n\u003ch3\u003eSalinity tolerance of the isolates\u003c/h3\u003e\n\u003cp\u003eBacterial isolates were screened for salt tolerance using NA medium supplemented with various levels of NaCl (0, 7, 12.5, 20, and 22%). The plates were incubated for 7 days at 30\u0026deg;C, and the growth was recorded\u003csup\u003e\u0026lrm;[\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e]\u0026lrm;\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eMolecular Identification of the isolates by 16S rDNA\u003c/h3\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eDNA Extraction and Polymerase Chain Reaction (PCR)\u003c/h2\u003e\n \u003cp\u003eGenomic DNA was extracted from bacterial cells grown aerobically in nutrient broth\u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. The extracted DNA was purified and visualized under UV by staining with ethidium bromide \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e61\u003c/span\u003e]\u003c/sup\u003e and kept at -20\u0026deg;C until use \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e. Amplification of the 16S rRNA gene sequence, using bacterial primers 27F and 1492R (\u003cstrong\u003eTable\u0026nbsp;1\u003c/strong\u003e), was performed using a model PTC-100 thermal cycler (MJ Research Inc., USA) \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e]\u003c/sup\u003e. Presence of amplified products was confirmed by applying 7 \u0026micro;l of PCR product on 1% agarose gel in 1X TAE buffer containing ethidium bromide stain, then visualized by Gel documentation system (Bio-Rad Laboratories) \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e61\u003c/span\u003e]\u003c/sup\u003e. The PCR products obtained were purified using the QIAquick PCR purification Kit protocol (Qiagen) and sent for sequencing.\u003c/p\u003e\n \u003cp\u003eTable\u0026nbsp;(1) primers used for 16S rRNA sequencing analysis (PCR) \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e72\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Taba\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePrimer name\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eOrientation\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePriming site\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSequence (5\u0026acute;- 3\u0026acute;)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eForward\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u0026ndash;27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAGAGTTTGATCCTGGCTCAG\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1492R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eReverse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1492\u0026ndash;1513\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGGTTACCTTGTTACGACTT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e16S rRNA sequencing and evolutionary relationships of taxa\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe sequence was carried out by Colors Laboratories (El-Etihad Square, Maadi, Cairo, Egypt) according to the laboratory instructions. The sequences were edited by eliminating all gaps (Complete deletion option) using the CHROMAS PRO software version 1.5.\u003c/p\u003e\n \u003cp\u003eThe 16S rRNA gene sequences were then compared with those published in GenBank databases using Basic Local Alignment Search Tool (BLAST) in the National Centre for Biotechnology Information (NCBI) website (htt//\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.ncbi.nih.gov\u003c/span\u003e\u003c/span\u003e). Alignment was done using CLUSTAL W 1.6 software \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. The evolutionary history was inferred using the Neighbor-Joining method \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e]\u003c/sup\u003e. Evolutionary analyses were conducted in MEGA X \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. The sequences were aligned using the embedded muscle algorithm, and the output was used to build a phylogenetic tree by calculating distance matrices for Neighbor-Joining (NJ) analysis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eDetection of hydrolytic enzymes\u003c/h2\u003e\n \u003cp\u003eIsolates were examined for lipase, esterase, casease, gelatinase (protease), and chitinase production. Bacterial inoculum used for the enzymatic screening was prepared by growing the isolates in 10 ml NB medium with 12.5% total salt under shaking at 120 rpm for about 72 h. Tests were done by the disc diffusion method \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. Plates were incubated for seven days at 30\u0026deg;C. All assays were carried out in triplicate. An enzymatic rate determination was used, with the following formula \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e:\u003c/p\u003e\n \u003cp\u003e\u003cimg 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UJl9/vnnffeM7LcfZIq6A9vut5kjtKyineFDvXJbt24tr/bSRdYZHl5BOc6YwX6Oa9asibvk02pJobLA0aIAVSYireDayuVQK6rNnpFt9rPUWBAzFNlKbE/L/TZzNO0xmdJF1hkeXkE5zphBC4X9LumWYlq5raSomLR+TvssqiKjK2t6enpOJZKTY+9GdbP1g1bUX3/91bhnJF166N62bVtsndShPTH3798fu+1Ev/02U5Qm9sFkEgWQN7nFwF1knSETHgbHccYQTXTgNZdjsoPFyjAZgQkATArQPos6l8oxSUGTB3Qu3eNRExmgbnKERZMaCK/9IPkthz9UDHECgz2Ps/KpXOrHxtAa7LEfR97MzMxEW5h8wTlNfsjJEofVYdPrDAdfqOs4zrxCy8b3jHQGwbv4HMeZV3zPSGdQvIJyHGde8D0jnSPFu/gcx3GcEaQo/ge3+KbrqFBhpAAAAABJRU5ErkJggg==\" width=\"424\" height=\"39\"\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eLipolytic enzyme assay on phenol red agar medium supplemented with olive oil\u003c/h3\u003e\n\u003cp\u003eBacterial isolates were plated on phenol red agar plates containing 1% olive oil \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/sup\u003e. Phenol red has an endpoint at pH 7.3\u0026ndash;7.4, where a slight decrease in pH will turn its color from pink to yellow. Lipase-producing bacteria will turn the dye into yellow \u003csup\u003e\u0026lrm;[\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eLipolytic enzyme assay using Tween 80 agar medium\u003c/h3\u003e\n\u003cp\u003eBacterial isolates were plated on Tween 80 agar plates and incubated as described above \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Tween 80 (fatty acid esters of polyoxyethylene sorbitan) is mostly hydrolyzed by the lipase as it contains esters of oleic acid and rarely by esterases \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eDetection of esterase\u003c/h2\u003e\n \u003cp\u003eFor the detection of esterase-producing halophilic bacteria, Tween 20 (fatty acid esters of polyoxyethylene sorbitan) has been used as a substrate in Tween 20 agar medium \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Tween20 is easily hydrolyzed by esterase as it contains esters of lower-chain fatty acids \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eScreening using tweens (Tween 20 and Tween 80) agar plates demonstrated precipitation around the esterase/lipase-producing bacteria. This is based on the precipitation of the calcium salt of the fatty acids released by the hydrolysis of the Tweens.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eDetection of casease\u003c/h2\u003e\n \u003cp\u003eTo detect casease enzyme, the isolates were plated on modified skim milk agar medium using skim milk agar disc diffusion test \u003csup\u003e\u0026lrm;[\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. For this purpose, a solution of defatted skim milk powder was autoclaved separately for five minutes at 115\u0026deg;C, then well mixed with NA medium and poured onto petri dishes \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Positive results for casease production were indicated by clear zones around the isolates on the plates, whereas for the negative results, the isolates lacked zones of clearance \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eDetection of gelatinase (protease)\u003c/h2\u003e\n \u003cp\u003eGelatin liquefaction detects the breakdown of gelatin to polypeptides and amino acids by enzyme gelatinase \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Gelatin is a solid at room temperature, but above 25\u0026deg;C it liquefies. When gelatinase hydrolyses this protein into amino acids, it remains liquefied even at the low temperatures of an ice bath \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eThe method described by Ingroff et al. (1988) was used for testing gelatinase (protease) activity was tested by preparing nutrient gelatin medium which was sterilized at 100\u0026deg;C for 20 min, then cooled to 40\u0026deg;C and inoculated with 1ml of each isolate, then incubated at 30\u0026deg;C for 7 days. Proteolytic activity was detected after being placed in a refrigerator at 4\u0026deg;C for 60 min (until the non-inoculated gelatin solidified) \u0026lrm;\u003csup\u003e\u003cstrong\u003e[78]\u003c/strong\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eChitinolytic activity\u003c/h2\u003e\n \u003cp\u003eThe detection was carried out using Luria Bertani (LB) colloidal chitin agar medium. Colloidal chitin suspension was prepared \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e]\u003c/sup\u003e, mixed with LB medium, and autoclaved at 15 lbs. and 121\u0026deg;C for 20 minutes \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e and inoculated with isolates using the disc diffusion method \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. Plates were incubated at 30\u0026deg;C for 7 days. Clear zone diameter was measured in (cm) at the average of three-dimensional measures and used as an indicator of chitinase activity \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eBiosurfactant production\u003c/h2\u003e\n \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\n \u003ch2\u003eGrowth of isolated bacteria on spent engine oil\u003c/h2\u003e\n \u003cp\u003eFifty milliliter of modified Landy broth medium in 250 ml Erlenmeyer flask was amended with 1% of spent engine oil \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e, supplemented with salt water in different concentrations, was inoculated with 1/2 ml (1%) of bacterial inoculum and incubated at 30\u0026deg;C for 7 days to determine the ability of bacterial isolates to grow on spent engine oil which enhance biosurfactant production. Bacterial cultures were centrifuged at 3000 rpm for 20 min. to use the supernatant for determining the ability of selected strains for biosurfactant production \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e66\u003c/span\u003e]\u003c/sup\u003e. The halophilic isolates QSLA1, QSLA7, QSLA14, and QSLA16 were examined for biosurfactant production on Landy medium with 3.5 and 7% salt concentrations, while isolate QSLA17 was examined for biosurfactant production on Landy medium with 0, 3.5, and 7% salt concentrations, as it was a halotolerant bacterium.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eDetection and quantitative screening for biosurfactant production\u003c/h2\u003e\n \u003cp\u003eThe quantitative screening for biosurfactant production from the isolates was carried out by oil displacement test, emulsification index, and measurement of surface tension \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e68\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eOil displacement activity\u003c/h2\u003e\n \u003cp\u003eA modification was carried out on the oil spreading technique \u003csup\u003e\u003cstrong\u003e\u0026lrm;\u003c/strong\u003e[\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e. Ten \u0026micro;l of spent engine oil was previously put onto the surface of 20 ml of distilled water in a petri dish (70 mm in diameter) to form a thin membrane. The culture of selected isolates was centrifuged at 3000 rpm, then ten \u0026micro;l of bacterial culture supernatant was added on top of the spent engine oil layer. A zone of displacement in the oil was observed, and the diameter of oil displacement (cm) was measured as the activity of biosurfactants. The method was effective for measuring biosurfactant activity, especially when the activity and the quantity of biosurfactant were not high enough.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eEmulsification index (E24%)\u003c/h2\u003e\n \u003cp\u003eUsing the method described by Selim et al. (2019), two mL of cell-free supernatant were added to 2 mL of spent engine oil in a screw capped tube and mixed vigorously by vortex at high speed for 2 min. The tubes were kept standing at room temperature for 24 hrs. \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e66\u003c/span\u003e]\u003c/sup\u003e. The emulsification index (E24%) was calculated as the ratio of emulsion zone height to the total height of the three phases using the equation described below \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e67\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"264\" height=\"37\"\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003eSurface tension measurement using a stalagometer\u003c/h2\u003e\n \u003cp\u003eThe surface tension of supernatant broth containing biosurfactant was measured at 25\u0026deg;C using a stalagometer (Fig.\u0026nbsp;1) (Stalagometer Rohr B Abgew) by the drop method \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e69\u003c/span\u003e]\u003c/sup\u003e. In this assay, a Traube stalagometer was used to measure the surface tension of a liquid \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. A Traube stalagometer is an instrument that allows for the consistent formation of uniform volumes of liquid droplets and subjects them to gravitational force within a modified capillary tube. The number of drops that fall per unit of volume is compared to a control liquid (i.e., water) of known surface tension. This information is then used to calculate the surface tension of the sample according to the following equation:\u003c/p\u003e\n \u003cp\u003e\u0026gamma;0\u0026thinsp;=\u0026thinsp;\u0026gamma; (n/n0)\u003c/p\u003e\n \u003cp\u003eWhere \u0026gamma;0 and \u0026gamma; are the surface tensions of the reference solvent (for water, \u0026gamma;0\u0026thinsp;=\u0026thinsp;72 dyne/cm2) and biosurfactant-containing broth solution, and n0 and n are the drop numbers of the reference solvent and biosurfactant-containing solution, respectively. The reference solution was taken as uninoculated Modified Landy broth medium \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e69\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eThe data were further subjected to statistical analysis using analysis of variance (ANOVA), and the differences between Means were compared by a high-range statistical domain using Tukey\u0026rsquo;s test and Tukey\u0026apos;s post hoc analysis in order to distinguish homogeneous\u0026ndash;heterogeneous groups among different variables. The multiple comparisons of Means were considered at a probability level p\u0026thinsp;=\u0026thinsp;0.05. The results are presented as the average means\u0026thinsp;\u0026plusmn;\u0026thinsp;Standard Errors (SE) of triplicate.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\n \u003ch2\u003eIsolation and purification of halophilic bacteria from solar salterns\u0026apos; water\u003c/h2\u003e\n \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e\n \u003ch2\u003eBacterial isolates\u003c/h2\u003e\n \u003cp\u003eThis study included five halo-bacterial isolates; QSLA7, that was isolated from the first pond with 4% salinity, QSLA1 and QSLA17 which were obtained from the second pond with 8.2% salt concentration, while QSLA14 was isolated from the third pond with 17.2% salt concentration and QSLA16 that obtained from the fourth pond with 24.2% salinity on nutrient agar (NA) medium that prepared by dissolving their component in saline water taken from the pond water samples instead of distilled water. Results were shown in Table\u0026nbsp;4.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\n \u003ch2\u003eCultural, morphological, and biochemical characterization of the isolates\u003c/h2\u003e\n \u003cp\u003eColonial, morphological and biochemical characterizations, which aids in partial identification of microorganism \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e69\u003c/span\u003e]\u003c/sup\u003e, were checked and it was observed that QSLA1, QSLA7, and QSLA16 isolates were gram negative and non-spore forming bacteria, while QSLA17 was gram positive and QSLA14 was gram variable, also both of them were spore forming. All isolates were catalase producers and motile. Moreover, QSLA1, QSLA14, QSLA16, and QSLA17 were rod-shaped, while QSLA7 was curved rod-shaped, as shown in Table\u0026nbsp;2.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cimg 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\" width=\"584\" height=\"145\"\u003e\u003c/div\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\n \u003ch2\u003eColony morphology\u003c/h2\u003e\n \u003cp\u003eAll colonies of the isolates were translucent, flat, shiny, and round. The colonies\u0026apos; edge of QSLA7, QSLA14, QSLA16, and QSLA17 isolates were ragged, while the colony edge of QSLA1 was lobate. Concerning colony size, QSLA1 and QSLA14 were pinpoint-sized, while the others were small. Moreover, QSLA1, QSLA7, and QSLA17 have a creamy color, while QSLA14 and QSLA16 were pink and yellow colored, respectively, as described in Table\u0026nbsp;3.\u003c/p\u003e\n \u003cp\u003e\u003cimg 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\" width=\"584\" height=\"202\"\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\n \u003ch2\u003eSalinity tolerance assay\u003c/h2\u003e\n \u003cp\u003eFor the halo-tolerance assay, the isolates demonstrated varied tolerance to different NaCl concentrations, 0, 7, 12.5, 20, and 22% NaCl. All isolates appeared in NA medium with salt concentrations of 12.5% NaCl and did not appear using 22% NaCl in the same culture medium, while only the QSLA1 isolate was able to grow on NA medium with a salt concentration of 20% NaCl. Isolates QSLA1, QSLA7, QSLA16, and QSLA17 were able to grow on NA medium with a salt concentration of 7% NaCl, while QSLA14 couldn\u0026apos;t. On the other hand, only QSLA17 isolate was able to grow on NA medium without salt concentration (zero NaCl) (Table\u0026nbsp;4).\u003c/p\u003e\n \u003cp\u003eIt was demonstrated that halotolerant bacteria are able to growth in the absence of salt, but tolerant of varying concentrations of salt (0\u0026ndash;5% NaCl) \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e74\u003c/span\u003e]\u003c/sup\u003eor extremely tolerant if tolerance extends above 15% (2.5 M) NaCl \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. On the other hand, slight halophiles (marine bacteria) grow best with 0.2 to 0.5 M NaCl (1\u0026ndash;3% NaCl), while moderate halophiles growing optimally in medium with 0.5 to 2.5 M NaCl (3\u0026ndash;15% NaCl), and extreme halophiles grow optimally in medium containing 2.5 to 5.2 M (saturated) NaCl (15\u0026ndash;30% NaCl) \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eSalinity tolerance assay of isolates (NaCl%).\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eIsolates\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003eNaCl concentration (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eZero\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e12.5\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eTherefore, the ability of QSLA17 isolate to grow in the absence and the presence of NaCl confirmed that it is a halotolerant bacterium, while the other four isolates are halophiles as they weren\u0026apos;t able to grow in the absence of NaCl and grew optimally in medium with 7\u0026ndash;22% NaCl. Among them, two isolates, QSLA7 and QSLA16, grew only on NA medium with 7 and 12.5% NaCl, while the colonies of QSLA14 isolate appeared only on NA medium with 12.5% NaCl, so they were considered as moderately halophilic bacteria, while QSLA1 isolate classified as extremely halophilic bacteria as it grown on NA medium with 20%.\u003c/p\u003e\n \u003cp\u003eAlthough QSLA16 strains were isolated from a saline water pond with 24.2% salt concentration, they couldn\u0026apos;t grow on NA medium prepared using distilled water and 20\u0026ndash;22% NaCl. In this respect, it was demonstrated that the NaCl requirement of these organisms has been known for many years, and NaCl could not be replaced by other salts. Also, potassium was essential for optimum growth and required for normal pigmentation. Magnesium was another essential ion for optimal growth; more of the rods appeared \u0026quot;normal\u0026quot; on medium containing the original concentration of magnesium. At lower concentrations, the organisms grew as spheres and seemed to adapt to the low concentrations of Mg\u003csup\u003e2+\u003c/sup\u003e. The spherical form was retained when these cells were returned to medium containing the original concentration of magnesium \u003csup\u003e\u003cstrong\u003e\u0026lrm;\u003c/strong\u003e[\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Using shake cultures, it was founded \u0026lrm;that 10 ppm of Fe\u003csup\u003e2+\u003c/sup\u003e increased the yield of cells. Small amounts of manganese (0.05 ppm) provided some stimulation of growth and a marked increase in pigmentation \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e65\u003c/span\u003e]\u003c/sup\u003e. So, isolate QSLA16 couldn\u0026rsquo;t grow on NA medium prepared using distilled water and high salt concentration of NaCl (20%) because of the absence of other essential elements such as potassium, magnesium, iron, and manganese, which are all required for optimum growth.\u003c/p\u003e\n \u003cp\u003eBacteria inhabiting hypersaline environments, like solar salterns, generally are well adapted to growth in the presence of high salt concentrations \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e71\u003c/span\u003e]\u003c/sup\u003e. Isolates obtained from saline regions of Karnataka were able to grow in salt concentrations ranging from 5\u0026ndash;25% \u003csup\u003e\u003cstrong\u003e\u0026lrm;\u003c/strong\u003e[\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. Forty-six halobacterial isolates were isolated from Sambhar Lake soil and water samples. All of them were tolerant to 10% NaCl concentration, while forty-four cultures were tolerant to 15% NaCl concentration. Out of the ten cultures selected for further studies, three were tolerant to 25% salt \u003csup\u003e\u003cstrong\u003e\u0026lrm;\u003c/strong\u003e[\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eTwenty-eight moderately halophilic bacteria were obtained from three salterns in Tamil Nadu, Kerala, and Goa, India. All isolates, except one isolate, grew rapidly on halophilic medium (HM) agar plates containing 5% NaCl, 21 isolates demonstrated heavy growth at 10\u0026ndash;15% salt content, followed by moderate and low growth when the salt concentration was increased. From the results of maximum tolerable salt concentration (MTSC), it was observed that among 28 isolates, one grew maximally up to 5% (slight halophiles), four grew up to 10%, seventeen grew up to 15% (moderately halophilic) six grew up to 20% NaCl containing medium and also without salt (halotolerant) \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\n \u003ch2\u003eMolecular Identification of the Isolates\u003c/h2\u003e\n \u003cp\u003eBesides morphological and biochemical characterization of the isolates, the 16S rDNA gene of the isolates was sequenced and analyzed. The resultant sequences were compared with those identified and deposited in the NCBI BLAST database. The sequence analysis of the 16S rRNA gene of the isolates demonstrated that QSLA1 showed the highest similarity to strain Halomonas sp. RS-17 with 91.26%. Isolate QSLA7showed similarity of 94% to Salinivibrio sp. pr6. Isolate QSLA14 was identified as Lentibacillus sp. strain BCHS25 with 87.66% similarity. Isolate QSLA16 was shown to be a strain of Uncultured bacterium clone QAMU23 and Sphingomonas sp. LE-239 with 80.15 and 79.9% similarity, respectively, whereas QSLA17 showed the highest similarity to strain Bacillus sp. strain 6 with 74.05% similarity (Table\u0026nbsp;5).\u003c/p\u003e\n \u003cp\u003eTable (5) The blasted isolates, nearest neighbors in the Genebank database, and their % relatedness.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tabc\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIsolates\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eOrganisms\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eClosest strains in Genebank\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIdentity (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eHalomonas\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eHalomonas\u003c/em\u003e sp. RS-17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e91.26\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eSalinivibrio\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eSalinivibrio\u003c/em\u003e sp. pr6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e94.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eLentibacillus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eLentibacillus\u003c/em\u003e sp. strain BCHS25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e87.66\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eUncultured bacterium\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e(\u003cem\u003eSphingomonas\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eUncultured bacterium\u003c/em\u003e clone QAMU23. (\u003cem\u003eSphingomonas\u003c/em\u003e sp. LE-239)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e80.15 (79.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus\u003c/em\u003e sp. strain 6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e74.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eOut of twenty-eight moderately halophilic bacteria were isolated from three salterns in Tamil Nadu, Kerala and Goa, India, Nine isolates demonstrated 97\u0026ndash;99% similarity to genus \u003cem\u003eBacillus\u003c/em\u003e and one isolate showed the highest similarity to \u003cem\u003eOceanobacillus\u003c/em\u003e with 98% similarity, while twelve isolates showing 96\u0026ndash;99% similarity to genus \u003cem\u003eStaphylococcus\u003c/em\u003e, two isolates belonged to \u003cem\u003ePseudomonas\u003c/em\u003e with 98% similarity, one isolate demonstrated 98% similarity to genus \u003cem\u003eEnterobacter\u003c/em\u003e and three isolates were most closely related to \u003cem\u003eEnterobacter\u003c/em\u003e, \u003cem\u003eOchrabactrum\u003c/em\u003e and \u003cem\u003eStenotrophomonas\u003c/em\u003e with 98, 98 and 96% similarity respectively \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003e74 halophilic bacteria were isolated from Algerian saline lakes ecosystems (Sebkha and Chott) located in arid and semi-arid ecoclimate zones. Out of them, 16 isolates were most closely related to \u003cem\u003eHalomonas\u003c/em\u003e, 18 isolates belonged to \u003cem\u003eBacillus\u003c/em\u003e, 7 strains were similar to \u003cem\u003eOceanobacillus\u003c/em\u003e, 5 isolates were identified as \u003cem\u003eVirgibacillus\u003c/em\u003e, 10 isolates were related to \u003cem\u003eHalobacillus\u003c/em\u003e, 2 isolates belonged to \u003cem\u003eThalassobacillus\u003c/em\u003e, and one isolate showed the highest similarity to \u003cem\u003eLentibacillus\u003c/em\u003e with 99\u0026ndash;100% similarity \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\n \u003ch2\u003eSequence deposition in GenBank (NCBI) of isolated halophilic bacteria\u003c/h2\u003e\n \u003cp\u003eAmong 5 isolates, two resultant sequences of isolated bacteria were deposited in NCBI Genebank under accession numbers shown in Table\u0026nbsp;6. However, the other three isolates that demonstrated low/no similarity to those of the Genebank were rejected during deposition in the NCBI database.\u003c/p\u003e\n \u003cp\u003ePrevious studies also described similar bacterial communities in solar salterns and other salted environments. 231 moderately halophilic Bacteria and 49 extremely halophilic microorganisms were obtained from different areas of Howz Soltan playa, a hypersaline lake in the central desert zone of Iran. The isolates were identified as members of the following genera: \u003cem\u003eSalicola\u003c/em\u003e, \u003cem\u003eHalovibrio\u003c/em\u003e, \u003cem\u003eHalomonas\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eOceanobacillus\u003c/em\u003e, \u003cem\u003eThalasobacillus\u003c/em\u003e, \u003cem\u003eVirgibacillus\u003c/em\u003e, \u003cem\u003eGracilibacillus\u003c/em\u003e, \u003cem\u003eHalobacillus\u003c/em\u003e, \u003cem\u003ePiscibacillus\u003c/em\u003e, and \u003cem\u003eSalinicoccus\u003c/em\u003e \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/sup\u003e. Species of the genera \u003cem\u003eHalobacillus\u003c/em\u003e, \u003cem\u003eHalomonas\u003c/em\u003e, \u003cem\u003eThalassobacillus\u003c/em\u003e, \u003cem\u003eBrevibacterium\u003c/em\u003e, and \u003cem\u003eBacillus\u003c/em\u003e were isolated from the Karak saline mine soil in Pakistan \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eTable (6) Accession numbers of submitted isolate sequences deposited in GenBank (NCBI).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tabd\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIsolates\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eOrganisms\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStrains in Genebank\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAccession numbers\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eHalomonas\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eHalomonas\u003c/em\u003e sp. strain QSLA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOP442496\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eLentibacillus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eLentibacillus\u003c/em\u003e sp. strain QSLA14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOP443587\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003ePhylogenetic affiliation of bacterial isolates\u003c/h3\u003e\n\u003cp\u003eThe resultant sequences were aligned using the Clustal W program with those identified in the GenBank, and the output was used to construct the phylogenetic tree. The result of the sequencing analysis and the closest phylogenetic bacterial isolates are presented in Table\u0026nbsp;5. Phylogenetically diverse halophilic bacteria, including Gram-negative and Gram-positive bacteria, were isolated from four ponds of different salt concentrations belonging to Qarun Lake. The sequences of the isolates fell within three major groups; two strains of the isolates fell under Firmicutes, while Gammaproteobacteria included one isolate, and one isolate fell into Alphaproteobacteria.\u003c/p\u003e\n\u003cp\u003eThe Firmicutes group includes three families, Bacillaceae, Planococcaceae, and Staphylococcaceae, two of which are isolated strains affiliated with the family Bacillaceae, with two different genera, including \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003eLentibacillus\u003c/em\u003e. Gammaproteobacteria comprise several medically and scientifically important groups of bacteria, such as the families Halomonadaceae, Vibrionaceae, and Pseudomonadaceae. Alphaproteobacteria includes several families, such as Sphingomonadaceae. The general phylogenetic associations of the isolated strains are shown separately in Fig. 2. Grouping our isolates with these identified halophilic bacteria in the Genebank confirms again the halophilic characters of the isolates.\u003c/p\u003e\n\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\n \u003ch2\u003eDetection of hydrolytic enzymes\u003c/h2\u003e\n \u003cp\u003eAs a result of their extreme adaptation, halophilic bacteria have attracted the attention of the scientific community due to their ability to produce active enzymes, exo polysaccharides, and pigments with potential biotechnology applications \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Isolates were examined for lipase, esterase, casease, gelatinase (protease), and chitinase production. Enzymatic activity index was measured \u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. For this purpose, the colony diameter and the clear zone diameter around colonies were measured. Assays were performed in triplicate (n\u0026thinsp;=\u0026thinsp;3), and average and Standard error values are shown in the graphs.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec32\" class=\"Section2\"\u003e\n \u003ch2\u003eLipolytic enzyme assay on phenol red agar medium supplemented with olive oil\u003c/h2\u003e\n \u003cp\u003eThe lipolytic activities of all isolates were qualitatively determined on plates containing phenol red agar medium; among the isolates, QSLA1 and QSLA7 demonstrated different lipolytic activity on phenol red agar plates (Table\u0026nbsp;7 and Fig.\u0026nbsp;3).\u003c/p\u003e\n \u003cp\u003eTable (7) screening isolates for lipase activity using olive oil (Means\u0026thinsp;+\u0026thinsp;Std. Error).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tabe\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLipase isolates\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eColony diameter(cm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eClear zone diameter(cm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLipolytic Enzymatic Rate\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.50\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.64\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eValues are Means \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e Standard Errors of three replicates.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eThe isolate QSLA7, which had 94% similarity to \u003cem\u003eSalinivibrio\u003c/em\u003e sp. pr6 demonstrated the highest lipolytic enzymatic rate (3.64), followed by QSLA1. There were no significant differences between them, as shown in Table 7 and Fig. 4. The change in color of phenol red was used as an indicator for lipase activity, where lipase-producing bacteria will turn the dye into a yellow color\u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eAn extracellular lipase was characterized from moderately halophilic \u003cem\u003eSalinivibrio\u003c/em\u003e sp. strain SA-2. The enzyme remained active in the presence of 17% NaCl\u003csup\u003e\u003cstrong\u003e\u0026lrm;\u003c/strong\u003e[\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eMarinobacter lipolyticus\u003c/em\u003e, a novel moderate halophile, was isolated from a hypersaline habitat with lipolytic activity that grows optimally at 7.5% NaCl. Many moderately halophilic bacteria belonging to the genera \u003cem\u003eMarinobacter\u003c/em\u003e, \u003cem\u003eVirgibacillus\u003c/em\u003e, \u003cem\u003eHalobacillus\u003c/em\u003e, \u003cem\u003eGeomicrobium\u003c/em\u003e, \u003cem\u003eChromohalobacter\u003c/em\u003e, \u003cem\u003eOceanobacillus\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eHalomonas\u003c/em\u003e, and \u003cem\u003eStaphylococcus\u003c/em\u003e were identified with the ability to produce industrially important enzymes and a salt requirement of 3\u0026ndash;20% (w/v) NaCl\u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003e54 Halobacterial strains were isolated from waters of the El Golea Sebkha, which is located in the middle of Algerian Sahara and contains salt concentrations up to 300 g/L. 35 isolates demonstrated lipolytic activity on sterilized medium (NaCl 25% w/v) supplemented with 2.5% olive oil (w/v)\u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eMarinobacter\u003c/em\u003e sp. SWJ2, which isolated from Seawater in Tanjung Piai National Park, Johor Bahru, Malaysia, demonstrated lipase production ability\u003csup\u003e\u003cstrong\u003e\u0026lrm;\u003c/strong\u003e[\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eIt was illustrated that olive oil is suitable due to the high concentration (greater than 70%) of oleic acid and has been used to assay the activity of various microbial lipases\u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e70\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e\n \u003ch2\u003eLipolytic enzyme assay on Tween 80 agar medium\u003c/h2\u003e\n \u003cp\u003eThe results of lipolytic activity of the isolates on Tween 80 agar medium demonstrated that, all of the five bacterial isolates were able to release lipolytic enzymes, demonstrated by precipitation of the calcium salt of the fatty acids released by hydrolysis of tweens around the lipase producing bacteria as shown in Fig. (5). The highest lipolytic enzymatic rate was shown by isolate QSLA16 (2.99), which identified as \u003cem\u003euncultured bacterium\u003c/em\u003e clone QAMU23 with 80.15% similarity, followed by QSLA1 (2.62), QSLA17 (2.60), QSLA7 (2.43), and QSLA14 (1.72) as shown in table (8). There were significant differences between QSLA16 and the other isolates, while significant differences were not recorded between QSLA1, QSLA7, and QSLA17 isolates; however, significant differences were shown between them and QSLA14, as observed in Table 8 and Fig. 6.\u003c/p\u003e\n \u003cp\u003eTable (8) Screening of the isolates for lipase activity (using Tween 80) (Means\u0026thinsp;+\u0026thinsp;Std. Error).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tabf\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLipase isolates\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eColony diameter (cm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eClear zone diameter(cm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003elipolytic Enzymatic rate\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.62\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.43\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.72\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.99\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.06\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.60\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eValues are Means \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e Standard Errors of three replicates\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003ec\u0026thinsp;=\u0026thinsp;higher value a\u0026thinsp;=\u0026thinsp;lower value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eLipase-producing microorganisms were detected, isolated from different counties of the Amazon region, using Tween 80 as a substrate\u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e77\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e\n \u003ch2\u003eDetection of esterase\u003c/h2\u003e\n \u003cp\u003eThree isolates, QSLA14, QSLA16, and QSLA17, demonstrated esterase activity (Table\u0026nbsp;9 and Fig.\u0026nbsp;7) as precipitation of the calcium salt of the fatty acids released by hydrolysis of tweens around the lipase-producing bacteria\u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. The isolate QSLA17 possessed the highest value of esterolytic enzymatic rate (3.21), followed by QSLA16 (2.61) and QSLA14 (2.1). There were significant differences between all of the isolates, as shown in Table\u0026nbsp;9 and Fig.\u0026nbsp;8.\u003c/p\u003e\n \u003cp\u003eTable (9) Screening of the isolates for esterase activity using Tween 20 as substrate (Means\u0026thinsp;+\u0026thinsp;Std. Error).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tabg\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eEsterase isolates\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eColony diameter (cm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eClear (precipitation) zone diameter (cm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eEsterase Enzymatic rate\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.10\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.61\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.21\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.06\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eValues are Means \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e Standard Errors of three replicates\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003ec\u0026thinsp;=\u0026thinsp;higher value a\u0026thinsp;=\u0026thinsp;lower value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eThe use of Tweens as lipase substrates in screening procedures has been criticized because tweens may be hydrolyzed by esterase (tween 20). Nonetheless, Tweens are attractive because they are very readily incorporated into growth medium, promoting optimal contact between cells and/or enzymes and the substrate\u003csup\u003e\u003cstrong\u003e\u0026lrm;\u003c/strong\u003e[\u003cspan class=\"CitationRef\"\u003e70\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eTwenty-four halo alkali bacterial isolates with esterolytic activity were isolated from Lake Magadi, Kenya. The isolates were screened for their ability to produce estrase enzyme by spotting them onto the basal medium supplemented with Tween 20 as a substrate\u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003ch3\u003eDetection of casease\u003c/h3\u003e\n\u003cp\u003eIsolates QSLA1, QSLA7, and QSLA14 demonstrated caseinase activity in modified skim milk agar medium (Fig.\u0026nbsp;9) in the form of clear zones around the isolates on plates with skim milk as a substrate, as observed by Cappuccino and Sherman (2002) and Kiplimo et al. (2019). Among caseae producer isolates, QSLA7 gives the highest value of proteolytic enzymatic rate (3.11), followed by QSLA1 (3) and QSLA14 (2.61). Significant differences between isolates QSLA7 and QSLA1 weren\u0026apos;t found, but were found between them and the QSLA14 isolate, as observed in Table\u0026nbsp;10 and Fig.\u0026nbsp;10.\u003c/p\u003e\n\u003cp\u003eTable (10) Screening of the isolates for caseinase activity (Means\u0026thinsp;+\u0026thinsp;Std. Error).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tabh\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIsolates\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eColony diameter (cm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eClear zone diameter (cm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eProteolytic Enzymatic Rate\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.00\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.11\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.63\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eValues are Means \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e Standard Errors of three replicates\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eb\u0026thinsp;=\u0026thinsp;higher value a\u0026thinsp;=\u0026thinsp;lower value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e37 halophilic archaeal strains were obtained from the marine solar saltern of Mulund, Mumbai. Screening for protease production using the skimmed milk agar plates method demonstrated that 5 have produced halophilic proteases\u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eDetection of gelatinase (protease)\u003c/h3\u003e\n\u003cp\u003eScreening of isolates for gelatinase activity was carried out using nutrient gelatin medium. Isolates QSLA1, QSLA7, QSLA14, and QSLA17 demonstrated gelatinase activity when bacterial growth liquefied gelatin medium after incubation at 30\u0026deg;C for 7 days and being placed in a refrigerator at 4\u0026deg;C for 60 min (until the non-inoculated gelatin solidified) as illustrated in previous studies\u0026lrm;\u003csup\u003e\u003cstrong\u003e[78]\u003c/strong\u003e\u003c/sup\u003e, also it was observed that gelatin hydrolysis, as evidenced by liquefaction of the nutrient gelatin after being placed in cold water/refrigerator at 4\u0026deg;C\u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAs shown, all isolates able to producing casease analyzed gelatin, as a nitrogen source in medium, except isolate QSLA17, which was identified as \u003cem\u003eBacillus\u003c/em\u003e sp. (74.05%), that hydrolyzed gelatin only. We thought that maybe because casein and gelatin are 2 contrasting proteins with respect to their amino acid composition. Casein is a complete protein because it contains all indispensable amino acids (IAA), whereas gelatin is an incomplete protein because it lacks the essential amino acid tryptophan and contains low amounts of, e.g., methionine and histidine, as it was demonstrated\u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003cdiv id=\"Sec37\" class=\"Section2\"\u003e\n \u003ch2\u003eChitinase activity\u003c/h2\u003e\n \u003cp\u003eFor chitinase detection, LB colloidal chitin agar medium was prepared. Bacterial isolates QSLA1, QSLA7, QSLA14, and QSLA16 (Table\u0026nbsp;11) demonstrated chitinase activity as a zone of clearance around the colony due to the breakdown of chitin to N-acetylglucosamine (Fig.\u0026nbsp;11) as it was clarified\u003csup\u003e\u003cstrong\u003e\u0026lrm;\u003c/strong\u003e[\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003eTable (11): Screening of the isolates for chitinase activity (Means\u0026thinsp;+\u0026thinsp;Std. Error).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tabi\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIsolates\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eColony diameter (cm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eClear zone diameter (cm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eChitinolytic Enzymatic Rate\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.74\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.06\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.38\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.44\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.09\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eValues are Means \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e Standard Errors of three replicates\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003ec\u0026thinsp;=\u0026thinsp;higher value a\u0026thinsp;=\u0026thinsp;lower value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eTable\u0026nbsp;(11), and Fig.\u0026nbsp;(12) show that QSLA1 scored the best chitinolytic enzymatic rate (2.74) and was significantly different from other chitinase-producing isolates. QSLA14 came in second with an enzymatic rate of 2.44, followed by QSLA7 and QSLA16 with chitinolytic enzymatic rates of 2.38 and 2.09, respectively.\u003c/p\u003e\n \u003cp\u003eModerately halophilic bacteria were isolated from shallow salt lakes with the ability to produce chitinase. Those moderately halophilic bacteria were \u003cem\u003eVirgibacillus marismortui\u003c/em\u003e, \u003cem\u003eB. subtilis\u003c/em\u003e, \u003cem\u003eBacillus pumilus\u003c/em\u003e, \u003cem\u003eBacillus licheniformis\u003c/em\u003e, \u003cem\u003eTerribacillus halophilus\u003c/em\u003e, \u003cem\u003ePlanococcus rifietoensis\u003c/em\u003e, \u003cem\u003eStaphylococcus equorum\u003c/em\u003e, and \u003cem\u003eStaphylococcus\u003c/em\u003e sp\u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eIn a study evaluating the antimicrobial activity of QSLA1 isolate against highly aggressive plant pathogens, the results showed that QSLA1 isolate possessed antifungal activity against \u003cem\u003eFusarium oxysporium\u003c/em\u003e and \u003cem\u003eAlternaria solani\u003c/em\u003e, as well as antibacterial activity against \u003cem\u003eRalstonia solanacearum\u003c/em\u003e\u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e. This ability to resist plant pathogens may be due to its ability to produce one or more of these enzymes, each of which plays an important role in the process of killing and decomposing plant pathogens.\u003c/p\u003e\n \u003cdiv id=\"Sec38\" class=\"Section3\"\u003e\n \u003ch2\u003eDetection and quantitative screening for biosurfactant production\u003c/h2\u003e\n \u003cp\u003eA number of screening methods have been developed to detect biosurfactant production amongst microorganisms\u0026lrm;. Assays such as the Du-Nouy ring tensiometer assay, axisymmetric drop shape assay, and pendant drop shape assay measure the surface and interfacial tensions of biosurfactants directly; whereas the oil spreading and emulsification capacity give an indirect measurement of surfactant production \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e75\u003c/span\u003e]\u003c/sup\u003e. In this study, screening for biosurfactant production was carried out by oil displacement test, emulsification index, and measurement of surface tension using a Stalagometer Instrument for five randomly selected isolates. The four halophilic isolates QSLA1, QSLA7, QSLA14, and QSLA16 were grown in Landy Medium under 2 different salt concentrations (3.5 and 7%), while the halotolerant isolate QSLA17 was grown under different salt concentrations (zero, 3.5, and 7%). Assays were performed in triplicate (n\u0026thinsp;=\u0026thinsp;3), and average and Standard error values are shown in the graphs. Results are shown in Table\u0026nbsp;20.\u003c/p\u003e\n \u003cp\u003eTable (12): Screening of isolates for biosurfactant production by the selected isolates (Means\u0026thinsp;+\u0026thinsp;Std. Error).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tabj\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTreatments\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNaCl concentration of the Landy broth medium\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eOil spill\u003c/p\u003e\n \u003cp\u003ezone (cm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eEmulsification\u003c/p\u003e\n \u003cp\u003eon index E24%\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSurface tension \u0026gamma; (dyne/cm\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.77\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.06\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e76.08\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.66\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50.49\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;1.04\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.97\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.06\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e77.01\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;2.11\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e49.14\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.58\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.80\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.06\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e64.35\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.93\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e51.77\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.52\u003csup\u003edef\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.93\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.06\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e66.32\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.89\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50.89\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.80\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.83\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.06\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e64.02\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;1.28\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50.83\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.57\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.03\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.06\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e65.08\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.66\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e49.86\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.16\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.17\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.06\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55.62\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.60\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50.58\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.17\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.53\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.06\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70.69\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.60\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e49.73\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.25\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eZero\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.20\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.06\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e52.10\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.48\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50.50\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.64\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.64\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.06\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e43.67\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.80\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e52.50\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.75\u003csup\u003eef\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSLA17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.10\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.70\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;2.75\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e53.43\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;1.16\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLandy Broth Medium (LBM) \u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.50\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e67.76\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTween 20 (1%) in LBM (7%) \u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.17\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.06\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e81.65\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.45\u003csup\u003ei\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37.04\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;1.43\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003eValues are Means \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e Standard Errors of three replicates\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003ei, g\u0026thinsp;=\u0026thinsp;higher value a\u0026thinsp;=\u0026thinsp;lower value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e* = Negative control. ** = Positive control.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec39\" class=\"Section2\"\u003e\n \u003ch2\u003eOil displacement test\u003c/h2\u003e\n \u003cp\u003eA zone of displacement in the oil was observed by QSLA1, QSLA7, QSLA14, QSLA16, and QSLA17 (Figs. 13 \u0026amp; 14), and the diameter of oil displacement (cm) was measured (Table 12). The results demonstrated that QSLA1 had higher activity (4.97 cm) with respect to oil displacement at 7% NaCl concentration. A significant difference was observed between QSLA1 and QSLA16 grown on Landy Medium with 7% salt concentration of medium and other treatments as shown in Fig. (15). Plates treated only with Landy Broth Medium (7% NaCl) were used as negative control, while plates treated with tween 20 (1%) added to Landy Broth Medium (7% NaCl) were used as positive control.\u003c/p\u003e\n \u003cp\u003eThe oil displacement test, also known as the oil spreading assay, relies on the displacement of oil by the biosurfactant\u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. Displacement of the oil, as indicated by a zone of clearing, is considered to be a positive indication of the presence of surfactant compounds. The diameter of the displacement corresponds to the level of biosurfactant activity\u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e75\u003c/span\u003e]\u003c/sup\u003e. The oil spreading method is simple, fast, requires a small amount of sample, and does not require specialized equipment. It is also sufficiently sensitive to detect biosurfactant activity at low concentrations, so several studies have used the oil spreading technique as a reliable Means to screen for biosurfactant compound production amongst various microorganisms\u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n \u003cdiv id=\"Sec40\" class=\"Section3\"\u003e\n \u003ch2\u003eEmulsification index (E24)\u003c/h2\u003e\n \u003cp\u003eThe E24 test, also known as the emulsification capacity assay, relies on the ability of a surfactant molecule to form a stable emulsion (Cooper and Goldenberg, 1987). This assay aids in quantitative screening of biosurfactant producers\u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e64\u003c/span\u003e]\u003c/sup\u003e. The results in Table 20 demonstrated that the emulsification index of QSLA1 isolate was the best amongst other isolates (77.01%), in 7% NaCl concentration. A significant difference was observed between QSLA1 and QSLA16 grown on 7% salt concentration of medium and other treatments, as shown in Figs. 49, 50, and 51.\u003c/p\u003e\n \u003cp\u003eThis assay is carried out to determine the emulsification property of surfactant compounds. It involves the formation of micelles where hydrophobic liquids become dispersed within hydrophilic liquids (e.g., water), thereby forming an emulsion of two substances, which are normally immiscible\u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003ch3\u003eMeasurement of surface tension using the stalagmometric assay\u003c/h3\u003e\n\u003cp\u003eUsing the stalagometric assay, all tested isolates demonstrated decreased surface tension compared to the negative control sample (un-inoculated Landy Medium 7% salt) (Table\u0026nbsp;20). The best result in decreasing surface tension (49.14 dyne/cm\u003csup\u003e2\u003c/sup\u003e) was observed in QSLA1 supernatant with 7% NaCl concentration. A significant difference was observed between QSLA1 grown on 7% salt concentration and QSLA17 grown on 7% salt concentration, as shown in Fig. 52.\u003c/p\u003e\n\u003cp\u003eConcerning the ability of studies isolates to produce halo biosurfactants; it was noticeable that the results of the oil displacement test are similar to those of the emulsification index assay and surface tension measurement. Where isolate QSLA17 recorded a high level of emulsification zone percentage and displacement zone diameter in the absence of salt, then decreased with increasing salt concentrations, while a low level of surface tension was recorded in the absence of salt, then increased with increasing salt concentrations. On the other hand, the percentage of emulsification zone and displacement zone diameter for QSLA1, QSLA7, QSLA14, and QSLA16 isolates was increased with increasing salt concentrations, while surface tension decreased. This is due to QSLA17 being a halotolerant bacterium, and QSLA1, QSLA7, QSLA14, and QSLA16 are halophilic isolates.\u003c/p\u003e\n\u003cp\u003eThe results observed that the two isolates QSLA1 and QSLA16, which were identified as \u003cem\u003eHalomonas\u003c/em\u003e sp. RS-17 and \u003cem\u003eUncultured bacterium\u003c/em\u003e clone QAMU23, respectively, demonstrated the best results of biosurfactant production with the used methods, especially in high salt concentrations, as they were halophilic bacteria, followed by isolate QSLA7, which was identified as \u003cem\u003eSalinivibrio\u003c/em\u003e sp. While isolate QSLA17 demonstrated the lowest results, it demonstrated high biosurfactant production in the absence of salt, as it was a halotolerant bacterium.\u003c/p\u003e\n\u003cp\u003eIn a previous study, the ability of QSLA16 and QSLA17 isolates to produce halo biosurfactants was examined using PCR technique to ensure the presence of the genes encoding for non-ribosomal lipopeptides (NRPs). It was revealed that QSLA16 and QSLA17 contain surfactin and fengycin genes while mycosubtilin (iturin) gene was detected only in QSLA17 isolate. Moreover, HPLC results showed that isolate QSLA16 produced surfactin and fengycins, with concentrations of 98 mg/l and 45 mg/l, respectively, while isolate QSLA17 produced surfactin, fengycins and iturin, with concentrations of 112 mg/l, 38 mg/l and 67 mg/l, respectively. Therefore, QSLA16 showed antibacterial activity against \u003cem\u003esalmonella typhi, Acinetobacter baumanni\u003c/em\u003e and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e but it had no effect on \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eKlebsiella pneumonia\u003c/em\u003e and \u003cem\u003eProteus mirabilis\u003c/em\u003e\u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn another study conducted, GC-MS analysis showd that metabolites of isolates QSLA1 and QSLA7 contain bioactive compounds such as Decanoic Acid, Ethyl Este and its derivatives \u003csup\u003e\u003cstrong\u003e\u0026lrm;\u003c/strong\u003e[\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e which are classified as fatty acids and fatty acid derivatives, biosurfactants\u003csup\u003e\u003cstrong\u003e\u0026lrm;\u003c/strong\u003e[\u003cspan class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Members of the Rhodobacteraceae, Rhodospirillaceae, Halomonadaceae, Oceanospirillaceae, Pseudomonadaceae, and Shewanellaceae families have also been reported to be oil-degraders and biosurfactants producers\u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eHalomonas\u003c/em\u003e sp. BS4 (a halophilic bacterium) was isolated from Thamaraikulam solar salt works in India. After routine biosurfactant screening by various methods, it was found to be a biosurfactant-producing bacterium. Biosurfactants containing compound 1, 2-Ethanediamine N, N, N0, N0-tetra, 8-Methyl-6-nonenamide, (Z)-9-octadecenamide, and fatty acid derivatives retrieved from \u003cem\u003eHalomonas\u003c/em\u003e sp. BS4\u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eSari et al. (2020) \u003cem\u003eHalomonas meridiana\u003c/em\u003e BK-AB4 was obtained from the Bledug Kuwu mud volcano in Central Java, Indonesia, and studied it for microbial-enhanced oil recovery (MEOR) applications using crude oil. It was found to be a biosurfactant producer. The main biosurfactant compounds are 9(E), 11(Z)-Octadecadienoic acid, 13-hydroxy-, methyl ester (C19H34O3); 9(E), 11(E)-Octadecadienoic acid, 13-oxo-, methyl ester (C19H32O2); 6,9,12-Octadecatrienoic acid, methyl ester (C19H32O2); and 3-Oxononadecanoic acid (C19H36O3). Those four compounds consist of 77.7% of the biosurfactant produced \u003csup\u003e\u003cstrong\u003e\u0026lrm;\u003c/strong\u003e[\u003cspan class=\"CitationRef\"\u003e63\u003c/span\u003e]\u003c/sup\u003e. A biosurfactant-producing bacterium, designated 3B-2, was isolated from marine sediment and identified it as \u003cem\u003eVibrio\u003c/em\u003e sp. by 16S rRNA gene sequencing. The produced biosurfactant was characterized in terms of protein concentration, surface tension, and oil-displacement efficiency\u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Halophilic \u003cem\u003eBacillus\u003c/em\u003e sp. BS3 was identified from solar salt works, identified by 16S rRNA sequencing, was used for screening their biosurfactant production. The biosurfactants were characterized by TLC, FTIR, and GC-MS analysis and identified as lipopeptide type. GC-MS analysis revealed that the biosurfactant had various compounds, including 13- 13-Docosenamide, (Z); Mannosamine, 9- and N, N, N\u0026apos;, N\u0026apos;-tetramethyl\u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Genera \u003cem\u003eThalassobacillus\u003c/em\u003e, \u003cem\u003eHalobacillus\u003c/em\u003e, \u003cem\u003eLentibacillus\u003c/em\u003e, and \u003cem\u003eBacillus\u003c/em\u003e belonged to family Bacillaceae, which are known as NRPs producers, such as surfactin\u003csup\u003e\u003cstrong\u003e\u0026lrm;\u003c/strong\u003e[\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e that increase the bioavailability of hydrocarbons and improve their degradation\u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIt was illustrated that the biosurfactant, such as surfactin, increases the bioavailability of hydrocarbons/oils (such as Tween 80) in culture\u0026lrm;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Liberated fatty acids bind with the calcium incorporated into the medium. The calcium laurate complex is visible as insoluble crystals around the inoculation site\u003csup\u003e\u003cstrong\u003e\u0026lrm;\u003c/strong\u003e[\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. And this explains the ability of the isolates to analyze olive oil and Tweens, and how to detect the occurrence of this analysis.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study highlights the remarkable diversity and biotechnological relevance of halo enzymes and halo biosurfactants derived from halophilic microorganisms. The investigated halo biosurfactants and halo enzymes demonstrated stability and catalytic efficiency under extreme saline conditions, underscoring their potential for sustainable industrial applications such as bio catalysis, pharmaceuticals, food processing, and environmental applications. These findings provide valuable insights into the adaptive strategies of halophiles and emphasize the untapped potential of their halo enzymes and halo biosurfactants in biotechnology. Looking ahead, future studies should aim to integrate genomic and proteomic analyses to discover novel halo enzymes with unique structural and functional properties, while advances in protein engineering and directed evolution may further enhance their stability, catalytic performance, and substrate specificity. Moreover, efforts to scale up biosurfactants and halo enzyme production under industrially relevant conditions, coupled with investigations into synergistic enzyme systems for complex bio catalytic pathways, will be crucial to bridging laboratory findings with real-world applications. Beyond industrial use, exploring the medical and environmental potential of halo biosurfactants and halo enzymes, particularly in antimicrobial development and bioremediation of saline or contaminated habitats, represents a promising frontier. Altogether, this work not only deepens our understanding of halophilic adaptations but also lays a foundation for exploiting halo enzymes as sustainable and versatile tools in modern biotechnology.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2 dir=\"LTR\"\u003eFunding details\u003cstrong\u003e\u003c/strong\u003e\u0026nbsp;\u003c/h2\u003e\n\u003cp dir=\"LTR\"\u003eIf the paper is accepted the publishing will be funded according to the Open Access Agreement for Egypt between Springer Nature \u0026amp;Science, Technology\u0026amp; Innovation Funding Authority (STDF) in cooperation with Egyptian Knowledge Bank (EKB).\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMethodology, results \u0026amp;discussions \u0026amp; statistical analysis, A. A.A.M., Abstract \u0026amp; introduction, A.E.E. Phylogenetic analysis \u0026amp; figures preparing, B.M.E.E. All authors have read \u0026amp;agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003esequence data that support the findings of this study have been deposited in NCBI Datasets with the primary accession codes:OP442496: (https://www.ncbi.nlm.nih.gov/search/all/?term= OP442496).OP443587: (https://www.ncbi.nlm.nih.gov/search/all/?term= OP443587).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbd-Elmonaem, A. A., Mahmoud, W. H., Elsaied, H. \u0026amp; Elbeltagy, A. E. Efficiency of non-ribosomal lipopeptides (nrps) produced by salt tolerant bacteria against some pathogenic bacteria. \u003cem\u003eMenoufia J. Agric. Biotechnol.\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e (Issue 1), 1\u0026ndash;18 (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAkeed, Y., Atrash, F. \u0026amp; Naffaa, W. 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Microbiol.\u003c/em\u003e \u003cb\u003e26\u003c/b\u003e (2), 301\u0026ndash;307 (1988).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7456751/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7456751/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHalophilic and halotolerant bacteria are microorganisms that thrive in hypersaline environments. They have much potential in several fields in life, such as Industry and agriculture. In this study, five isolates were obtained from four saline ponds of Qarun Lake using Nutrient Agar (NA) medium prepared with water from the ponds. Among them, QSLA17 was considered a halotolerant bacterium, while QSLA1, QSLA7, QSLA14, and QSLA16 were considered halophilic bacteria. The 16S rRNA analysis demonstrated that the isolates were affiliated with three groups: Firmicutes, Gammaproteobacteria, and Alphaproteobacteria. The ability of isolates to produce hydrolytic enzymes and biosurfactants was tested. After initial screening, all isolates were found to have positive lipase activity using Tween 80 as a substrate, while QSLA1 and QSLA7 isolates were able to produce lipase using olive oil. QSLA14, QSLA16, and QSLA17 isolates demonstrated the ability for esterase production, while QSLA1, QSLA7, and QSLA14 isolates succeeded in producing casease. Gelatinase (protease) was excreted by isolates QSLA1, QSLA7, QSLA14, and QSLA17, while chitinase was produced by isolates QSLA1, QSLA7, QSLA14, and QSLA16. Moreover, all isolates demonstrated their ability to produce biosurfactants while growing in Modified Landy Medium with different salt concentrations, and in this context, the QSLA1 isolate demonstrated the best results compared to the other isolates.\u003c/p\u003e","manuscriptTitle":"Exploring halo enzymes and halo biosurfactants production from halophilic and halotolerant bacteria isolated from Lake Qarun solar salterns","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-27 05:54:38","doi":"10.21203/rs.3.rs-7456751/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7d8559db-c2b7-4c29-ba06-984231360ed0","owner":[],"postedDate":"August 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":53726177,"name":"Biological sciences/Biochemistry"},{"id":53726178,"name":"Biological sciences/Biotechnology"},{"id":53726179,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2025-12-10T11:39:23+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-27 05:54:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7456751","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7456751","identity":"rs-7456751","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}