Biosurfactant Production by Marine Actinomycetes Isolates Streptomyces Althioticus RG3 and Streptomyces Californicus RG8 as a Promising Source of Antimicrobial and Antifouling Effects


 Background: Because of the ability of their bioactive metabolites production, many researchers were attracted to investigate and isolating marine actinomycetes from unique location with a unique environment. Information on antimicrobial activity and antifouling agents by Streptomyces sp. from the Ras Garib area, Gulf of Suez, Egypt is limited. One of the metabolites produced by the actinomycetes was biosurfactant. This paper describes the possibility of marine actinomycetes isolates for the production of biosurfactants, In addition to the possibility of using it as antimicrobial and antifouling agents.Results: Marine actinobacterial isolates RG3 and RG8 had emulsification indexes of 76 and 68%, respectively. The two marine actinobacterial isolates were identified using 16srDNA as Streptomyces althioticus RG3 and Streptomyces californicus RG8, and submitted in the database of genetic information with accession number MW661230 and MW661234, respectively. Biosurfactants were stable at 10% NaCl, in case of Streptomyces althioticus RG3 and stable at 10%–15% NaCl in the case of Streptomyces californicus RG8. A temperature of 35℃ was suitable for the stability of biosurfactants produced by both strains. Both strains produced the most biosurfactant when exposed to alkaline conditions. We characterized the biosurfactants produced by both strains including features such as the chemical composition of the biosurfactants and FTIR analysis. The antimicrobial activity of biosurfactants extract evaluated using a well diffusion method against Vibrio alginolyticus MK170250, Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 4027, and Staphylococcus aureus ATTC 25923. Streptomyces althioticus RG3 biosurfactants have been shown to have better antimicrobial activity than Streptomyces californicus RG8, indicating that they may be used in pharmaceutical industries and in the manufacture of antifouling products.Conclusions: Streptomyces althioticus RG3 and Streptomyces californicus RG8, isolated from Ras Garib, Gulf of Suez, Egypt, were able to develop very stable biosurfactants under stress conditions, which could be useful in a variety of industries such as pharmaceuticals and antifouling manufacturing.


Background
Surfactants help to form stable gels and foams by reducing surface tension at phase interfaces, emulsifying oil in water and water in oil mixtures, and emulsifying oil in water [1]. Synthetic precursors from the petrochemical and/or oleochemical industries can be used to make these amphiphilic compounds; however, surfactants can be produced by microbial organisms in a variety of forms.
Biosurfactants and bioemulsiers are surface-compounds that are active derived from sources biological such as yeasts, bacteria, and yeasts [2]. Microbial surface-active compounds' molecular structure is used to classify them. Lipoproteins, lipopolysaccharides, heteropolysaccharides, and proteins are examples of large molecular mass compounds known as bioemulsiers. Compounds with a lower molecular weight known as biosurfactants include glycolipids such as rhamnolipids, sophorolipids, mannosylerythritol lipids, and trehalose lipids, as well as lipopeptides like surfactant and fengycin. [3]. High and low molecular weight surface active compounds may be utilised in a broad range of utilizations comprising treatments for individuals and at home; chemicals used in agriculture; improved recuperation of microbials from oil; textiles and biomedicines. Bio-factants and bio-emulsi ers are an alternative to synthetically produced surfactants because of their low toxic, biodegradable content and an increased consumer awareness of sustainability and environmental protection [4]. Actinobacteria are members of the order Actinomycetales. Actinomycetes with characteristic laments are gram-positive bacteria. Actinomycetes encompass a wide variety of habitats. They are present in different soil types, marine and ground water, and ecosystems of mangrove [5]. They can be alkali-and thermophilic [6], halophilic [7], or acidophilic [8]. Due to their ability to produce metabolite combinations that differ in structure and function, they played a signi cant position in several industries. The quest for metabolites of actinomycetes is made possible by exploration of certain areas, unique ecosystems and combinations of isolation methods [9]. Surfactants, active metabolites that reduce surface tension, are thought to be formed by the majority of microorganisms, including actinomycetes. The purpose of the research was to isolate actinomycetes from marine environments and explore their capability for producing commercially signi cant biosurfactants.

Sampling and isolation of marine actinomycetes
In sterile plastic bags, samples of marine sediment were obtained from Ras Garib area of the Gulf of Suez in Egypt (Fig.1). The coordinates and map work were obtained at the Marine Physics Laboratory, (NIOF), Red Sea, using the Surfer program version 15.2.305. This sediment sample was found to be high in oils and was therefore used to monitor for actinomycetes containing biosurfactants. A dry heating method was used to isolate actinomycetes. For eight days, sediment samples were dried in a 70°C oven, then mashed and ltered through a 1-5 mm sieve. The samples were shaken for two minutes and then left for one minute before being suspended in nine ml of sterilized seawater [10]. From these solutions, serial dilutions were made, up to 10 6 , then each dilution was added to the starch nitrate medium in 0.1ml increments. with the following components (gl −1 ): 20 starch, 0.5 K 2 HPO 4 , 1 KNO 3 , 0.5 MgSO 4 7H 2 O, 0.01 FeSO 4 , 15 agar [11] at pH 7.0. It was forti ed with griseofulvin and 0.05 ppm chloramphenicol. After that, the culture 7 days at 28°C± 2°C was incubated. Preliminary selection of actinomycete colonies was made based on a morphology of colony, and they were streaked on the previous medium. For further identi cation tests, in the refrigerator at 2°C isolates were held.

Screening of biosurfactant producers
The selected isolates have been inoculated into a 500 mL ask of Erlenmeyer with 100 mL Kim biosurfactant medium. It consisting of (gl −1 ): NaNO 3 , 1.0; KH 2 PO 4 , 0.1; MgSO 4 7H 2 O, 0.1; CaCl 2 , 0.1; yeast extract, 0.2% and 3% olive oil as the carbon source at pH 6.0 [12]. For 5 days, the cultures of broth were incubated at 32°C in a shaker incubator at 120 rpm min. Positive results were considered actinomycete cultures that emulsi ed oil as evidence of biosurfactant producing activity. Further tests were carried out to validate biosurfactant output by the screened strains. The existence of biosurfactant in cell-free supernatant was investigated using drop collapsing tests, hemolytic tests, blue agar plates, emulsifying capacity, and surface tension measurements.

Drop collapsing test
After 1 minute of adding ve mL of supernatant of culture to two mL of mineral oil surface, on the surface of oil, the drop shape was examined. Cultures that produced positive (+) biosurfactant produced at drops, but cultures that produced round drops produced negative (-) results [13].

Hemolytic test
Actinomycetes isolates are grown in Blood Agar. 1.5 mL sterile human blood was pipetted into an Erlenmeyer ask containing 100 mL mineral agar medium. Until it became frozen and solid, the sterile blood medium was poured. The potential biosurfactant isolates were inoculated on blood medium, which was incubated at 32℃ for 7 days. The hemolysis zone and changed of color produced by the behavior actinomycetes were tested [14].

Blood agar plate
To a glycerol yeast extract agar medium with methylene blue (0.2 mg/ml), carbon sources (2 % w/v) and cetyl trimethyl ammonium bromide (0.5 mg/ml) were added. A dark blue halo around the culture was deemed favorable for biosurfactant processing. [15].

Production of Lipase
Using tributyrin agar plates, the lipase formed by pure marine actinomycetes cultures was determined. After being inoculated, the plates were incubated at 30°C for 7 days. After the incubation period, A clear zone around the colonies was tested on the plates [16].

Emulsifying capacity
Emulsi cation index (E24) supernatant after adding volume of para n oil (v:v) was determined, then vortexed at highest speeds two min, to the sample cultures and allowed to stand for 24 hours. The E24 percentage with the following equation was determined [17].

Actinomycetes Isolate's identi cation
Morphological, physiological, biochemical, and molecular approaches were used to identify the most potent marine actinobacterial isolates [18]. DNA was isolated and puri ed from the selected actinomycetes. 16S sequence analysis was used to characterize the genotype., Finally, Bioedit was used to perform several alignments of the sequences of the members that are the most closely related [11].
Medium optimization for highest production of biosurfactant By using series experiments, the medium was optimized by changing one variable at a time while maintaining the other variables constant at a set of conditions. To increase biosurfactant productivity, two factors were chosen: the source of carbon and the source of nitrogen. The sources of carbon which used is dextrose, sucrose, mannitol, xylose, maltose, glycerol, and glucose (20g/l). Separate ltration using bacteriological lters was used to sterilize the carbon sources, and then applied under aseptic conditions to the production medium. In order to determine the best nitrogen sources in biosurfactant production at concentrations of 10g/l with the optimal carbon source, yeast extract, urea, peptone, tryptone, ammonium sulfate, and beef extract were used [19].

Biosurfactant extraction
A solvent extraction method [21] was used to extract the biosurfactants provided by Streptomyces althioticus RG3 and Streptomyces californicus RG8 from the ltrate. The biosurfactant was extracted and puri ed using 4L of culture broth centrifuged for the rst time at 7000 rpm and 4°C for 25 minutes. The crude material was extracted by combining it with a similar equal quantity of chloroform: methanol at a 2:1 (v/v) ratio in a separator ask, shaking it vigorously, and letting it sit for 30 minutes. The bottom aqueous layer of the separation funnel was withdrawn, white bio-emulsi er layer immediately above it was collected in petri dishes and evaporated in a 40°C-45°C oven [10].

The biosurfactant's chemical composition
Lowry's method was used to assess concentration of protein from isolated biosurfactant [22], using bovine serum albumin as standard. By using the method of phenol sulfuric acid, carbohydrate content was determined [23]. D-glucose was the traditional tool for measured total lipid content [24].

Fourier Transform Infrared Spectroscopy (FTIR)
In (SRTA-City), the functional classes of isolated biosurfactants from Streptomyces althioticus RG3 and Streptomyces californicus RG8 were identi ed using (FTIR) using a spectrum 400 instrument (PerkinElmer, Waltham Massachusetts MA, USA). Under ambient conditions, 400-4000 cm 1 were the spectral resolution and the precision of the wave number.

Antimicrobial Assay
The antimicrobial activity of the biosurfactants production against Vibrioalginolyticus MK170250, Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 4027, and Staphylococcus aureus ATTC 25923 was determined using agar well diffusion method. In a nutshell, 100µl of aqueous biosurfactant solutions is poured into 6-mm-diameter wells in the pour plated nutrient agar. The plates were incubated at 37 °C for 24 h. The presence of a clear zone around the wells indicated antimicrobial activity [25].
These bacterial pathogens were kindly provided by the staff members of the National Institute of Oceanography and Fisheries (NIOF), Red Sea branch, Hurghada city, Egypt.

Antifouling activity
In a conical ask (50 ml) containing sterilized cover glass, seawater (1 ml) was mixed with nutrient broth medium (20 ml) (Oxoid Ltd, England) and incubated overnight at 28°C. About 500 ul of aqueous solutions of the biosurfactant was added into the ask (as an antifouling agent). The ask was lled with 500 µl of biosurfactant aqueous solutions (as an antifouling agent). The cover glass was washed in water, dried at room temperature, and examined under the microscope after ten minutes of dying with crystal violet solution (0.4 %) [11].

Data analysis
For data analysis, the R software (version 3.0.1) for Windows was used, and the results were interpreted as the mean ± standard deviation (SD) of the quintuple's samples.

Results And Discussion
Isolation and production of biosurfactants Nine actinomycetes isolates were isolated from soil that has been tainted with fuel oil in the Ras Garib region of Egypt's Gulf of Suez and screened for biosurfactant production. Extracellular biosurfactant production of 3% olive oil from Kim's medium was initially screened for all these isolates as the only source of carbon. Out of nine isolates, only two isolates, RG3 and RG8, showed positive results. During the screening process, olive oil was used as the sole carbon source, as the biosurfactant production was increased using actinomycetes [26]. One of the parameters for selecting potential producer biosurfactants is the emulsi cation activity. Biosurfactant productivity is calculated by emulsifying activities [15]. The marine actinomycetes isolates RG3 and RG8 had the highest emulsi cation indices, as shown in Table 1 [27], lipase in the water/oil surfaces was responsible for the operation, and therefore actinomycetes have shown that lipase is capable of producing bioemulsi ers. In this work, isolates RG3 and RG8 showed zones of lysis around the colonies (Fig.2). The surface tension of the supernatant and puri ed biosurfactant was measured using a surface tensiometer.
Both marine actinomycetes isolates (RG3 and RG8) reduced surface tension to values of 77.24 ± 2.4 mN/m and 69.27 ± 2.4 respectively, recording positive results for biosurfactant activity. Fig.3 shows the measurements of the surface tension effects and those caused by a decrease in the surface tension value. Ari yanto et al., [14] reported that three actinomycetes isolates belong to Streptomyces sp. named AF6, AB8, and AA1, which were 56.22, 57.27 and 57.33 mN/m surface tension values, respectively. If a bacterial isolate surface tension decreased to 40 mN/m or less, Cooper and Goldenberg [28] suggest that it could be a promising producer of biosurfactants. The marine organism Virgibacillussalaries displayed a decrease in tension on the surface (30 mN/m), an E24 = 80% of twenty, and three morphologically distinct colonies, collapse of drop , distribution of oil , and hemolysis of blood all showed promising effects [29].
Biochemical and molecular characterization  Table 3 shows the GenBank accession numbers of the closest neighbors of isolates RG3 and RG8. Isolate RG3 was assigned as Streptomyces althioticus RG3 with the accession number (MW661230) Fig.4(A). Isolate RG8 was given the name Streptomyces californicus RG8 with accession number (MW661234) as it is a liated according to the genus Streptomyces californicus Fig.4(B).

Factors affecting the emulsi cation activity
There has been considerable research into the generation of biosurfactants by actinomycetes under extreme conditions for commercial use [30]. We investigated the effect of extreme conditions, including carbon sources, nitrogen sources, higher salt, temperature, and pH, on the production of biosurfactant from the marine isolates of Streptomyces althioticus RG3 and Streptomyces californicus RG8, to assess its stability.

Effect of various sources of carbon
The amount of biosurfactant generated was measured and found to be relying on the composition of medium. Changes in the carbon source in material was caused by biosurfactant secretion in shaken-ask experiments. Sucrose, dextrose, and glucose were found to be the most effective sources of carbon for production biosurfactant. Glucose (2% w/v) in the two strains Streptomyces althioticus RG3 and Streptomyces californicus RG8 were the carbon sources producing the most biosurfactants (Fig.5). Nutrient substrates were screened, and we found that these isolates supported growth in all substrates, although xylose was limited in yield. Khopade and his team [19] said that, production of biosurfactant though marine Streptomyces sp. isolate B3 was decreased surface tension to 29 mN/m and the emulsifying activity showed 80%. Streptomyces sp. generated the most biosurfactants when sucrose was used as the carbon source, according to previous research [16,19].

Effectiveness of nitrogen sources
Production of biosurfactant is affected by the nitrogen source, as shown in Fig. 6. Macro-nutrients need speci c conditions to produce high concentrations of biosurfactants [19]. In Streptomyces althioticus RG3 we discovered that the better use of nitrogen is required for growth and production of biosurfactant was yeast extract, which had an E24 of 69.25%., while ammonium sulfate was best for the isolation of Streptomyces californicus RG8, with an E24 of 59.21%. Other studies in which organic nitrogen sources were favored over inorganic sources produced similar ndings [31,32].

Various temperatures effectiveness
One of the most critical parameters in the bioprocess was temperature. At 35℃, the highest emulsifying activity of Streptomyces althioticus RG3 and Streptomyces californicus RG8 was observed, at emulsifying indices of 78% and 65%, respectively. Even so, there was still stable biosurfactant output from 45°C to 50°C, implying that both isolates were thermotolerant (Fig.8), and indicating that the biosurfactant is moderately thermostable. The composition of the biosurfactant in Arthrobacterpara neus and Pseudomonas sp. changed as the temperature changed [13,33]. A similar study indicated that the biosurfactant activity was highest when the marine Streptomyces species B3 was cultivated at 30℃ (E24 = 80%) [19]. At 30°C-40°C, The surface tension of Pseudomonas sp. (MW2) culture broth was decreased, according to Dahil [34]. Deng and his team [35] showed that rising level of biosurfactant was generated by Achromobacter sp. HZ01 at 40℃, 60℃, and 80℃.

Effect of pH
Due to the large amount of use of biosurfactants in the production of detergents, it was necessary to choose an alkaline biosurfactant from isolated bacteria. [20]. As seen in Fig.9, the highest level of emulsi cation operation for Streptomyces althioticus RG3 and Streptomyces californicus RG8 it was the alkaline (pH 10) and reached 69% and 65%, respectively. This may be attributed to greater surfactant micelle stability with fatty acids in the precent of NaOH and secondary metabolites precipitation with rising values of pH. [19]. Operation for the emulsi cation was decreased due to a decrease in the value of pH moving from the basal to the acidic zone (9 to 5 pH), because of partial biosurfactant precipitation [36]. In alkaline pH (8-9), the bio-emulsi er activity was higher than in acidic pH. (5) [10]. Nadem et al, stated that Streptomyces sp. SS 20, isolated , soil contaminated with hydrocarbons has a high activity of bioemulsifying and stability over in a wide temperature spectrum (30℃-100℃) and pH level of 3-7 [37]. According to El-Sersy, the E24 of B. subtilis was stable in a pH range of 6 to 10 [38], with acidic pH decreased. The pH effect for biosurfactants of various microorganisms on surface activity has been recorded [39].

Biosurfactant chemical characterization
Chemical composition of the biosurfactants S. althioticus RG3 produces a biosurfactants had a chemical structure of 30% protein, 20% carbohydrate, and 50% lipids. S.californicus RG8 provided a biosurfactant with a chemical composition of 18% proteins, 45% carbohydrates, and 37% lipids. Lipids, glycolipids, lipopeptides, and polysaccharide protein complexes are examples of biosurfactants isolated from microorganisms [40]. Biosurfactant biochemical composition is likely to be in uenced by the substrates used in the growth medium. In the founded of re nery soybean oil, The biosurfactant production from C. lipolytica UCP0988 contained proteins in the rate of 50.0 %, carbohydrates in the rate of 8.0 %, and lipids in the rate of 20.0 %, according to Ru no and his colleagues [41]. Thavasi recorded other ndings, such as the biosurfactant production from L.delbrueckii containing carbohydrates in the rate of 30.0 % and lipids in the rate of 70.0 % [42].  [10,44].

Antimicrobial activity
Because of their anti-adhesive agents and enzyme inhibitors, biosurfactants have operated as fungicidal, bactericidal, insecticidal, and antiviral ingredients [14,19,45]. In this study, we observed that an increase in the biosurfactants concentration led to improvement of its antimicrobial effects ( Table 4). The highest antimicrobial effects of biosurfactants were observed against Vibrioalginolyticus MK170250 from Streptomyces althioticus RG3 (Fig.11) [14].
Antifouling activity Figure 12 illustrated the inhibitory action of Streptomyces althioticus (RG3) biosurfactants on bacterial bio lm formation. The biosurfactants production by Streptomyces althioticus RG3 reduced the density of bacterial cells and acted as an anti-biofouling agent, while Streptomycescalifornicus RG8 not achieved that. Napyradiomycin which production from marine Streptomyces aculeolatus isolate PTM-029 exhibited the higher antibacterial activity, the higher microfouling inhibitory activity, and the most potent antimicrofouling activity [47].    "+" sign demonstrates microbial growth inhibition, whereas "−" The failure to prevent the growth of the microorganisms under investigation. (+++) strongest effect, (++) moderate effect. Figure 1 Map of the Ras Gharib area and the location of sampling site. Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors. Emulsion formation using para n oil and inoculated marine actinomycetes supernatant for isolates (RG3 and RG8). Effect of pH on biosurfactant production by Streptomyces althioticus RG3 and Streptomyces californicus RG8.

Figure 11
Antagonistic effects of biosurfactants production by Streptomyces althioticus RG3 against Vibrio alginolyticus MK170250 Figure 12 Photographs illustrating the antifouling effect of the biosurfactants from Streptomyces althioticus (RG3) on the bio lm formation, the emergence of a small number of bacterial colonies (A) and the effect without the biosurfactant compounds, the emergence of large numbers of bacterial colonies (B).