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 . One of the parameters for selecting potential producer biosurfactants is the emulsification activity. Biosurfactant productivity is calculated by emulsifying activities . The marine actinomycetes isolates RG3 and RG8 had the highest emulsification indices, as shown in Table 1, of 76% ± 2.5% and 68% ± 2.2%, respectively. Korayem  and his team found that, Streptomyces isolate 5S had an emulsification index of 31.74%. After initial screening by Zambry and his team , the emulsification index test (E24) has provided positive results for all isolates of actinomycete, the results varying between 84.11% to 95.80%. Selected isolates showing positive results against the collapse of the drop and hemolytic action. In this analysis, the biosurfactant producer was screened with lipase activity. Kokare says that , lipase in the water/oil surfaces was responsible for the operation, and therefore actinomycetes have shown that lipase is capable of producing bioemulsifiers. In this work, isolates RG3 and RG8 showed zones of lysis around the colonies (Fig.2). The surface tension of the supernatant and purified 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. Arifiyanto et al.,  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  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 .
Biochemical and molecular characterization
Table 2 displays the morphological, physiological, and biochemical characteristics of the selected isolates. The sequences of 16S rRNA for the selected RG3 and RG8 isolates were compared with the Streptomyces sp. sequence using multiple sequence alignment to validate the actinomycete isolate's identification. Using agarose gel electrophoresis, an experimental study of PCR amplification was performed. 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 affiliated according to the genus Streptomyces californicus Fig.4(B).
Factors affecting the emulsification activity
There has been considerable research into the generation of biosurfactants by actinomycetes under extreme conditions for commercial use . 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-flask 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  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 specific conditions to produce high concentrations of biosurfactants . 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 findings [31, 32].
Stability of bio-surfactant activities
Effectiveness of different salt concentrations
Fig.7 indicates that the emulsification of Streptomyces althioticus RG3 was stable (70%) with 10% NaCl, and then gradually decreased to 15%–30% NaCl. Streptomycescalifornicus RG8 emulsification operation was steadily increased from 10% to 15% NaCl to 60%, and then decreased to 20%–30%. Khopade  reported that, in 4 % (w/v) NaCl, Streptomyces sp. B3 which isolated from marine resource generated the most biosurfactant (E24 = 78 %), and represented activity of emulsification (E24 = 60%) in the using of 9% (w/v) NaCl. Elkhawaga  showed, Streptomyces griseoplanus (MS1) have ability to produce biosurfactant stable at concentrations of the NaCl up to 10%. Elazzazya  stated that, in 4% (w/v) NaCl, biosurfactant synthesis was obtained.
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 Arthrobacterparaffineus 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%) . At 30°C–40°C, The surface tension of Pseudomonas sp. (MW2) culture broth was decreased, according to Dahil . Deng and his team  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. . As seen in Fig.9, the highest level of emulsification 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. . Operation for the emulsification 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 . In alkaline pH (8–9), the bio-emulsifier activity was higher than in acidic pH. (5) . 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 . According to El-Sersy, the E24 of B. subtilis was stable in a pH range of 6 to 10 , with acidic pH decreased. The pH effect for biosurfactants of various microorganisms on surface activity has been recorded .
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 . Biosurfactant biochemical composition is likely to be influenced by the substrates used in the growth medium. In the founded of refinery 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 Rufino and his colleagues . Thavasi recorded other findings, such as the biosurfactant production from L.delbrueckii containing carbohydrates in the rate of 30.0 % and lipids in the rate of 70.0 % . The biosurfactant of C. Sphaerica UCP0995 contained 15.0 % carbohydrate and 70.0 % lipids, according to Luna and his team .
Fourier Transform Infrared Spectroscopy (FTIR)
The biosurfactant developed by S.althioticus RG3 was analyzed using FTIR and showed the presence of 11 clear absorption peaks at 3343.1, 1653.7, 1401.8, 1084.9, 1007.2, 988.2, 832.6, 702.8, 663.4, 618.2, 543.8cm−1, as shown in Fig.10A. FTIR analysis of produced biosurfactant by S. californicus RG8 depicts the presence of eight clear absorption peaks at 3431.7, 2079.9, 1638, 1434.1, 1384.8, 1103.8, 983.5, 614.4 8cm−1, as shown in Fig.10B. Peaks at 3343.1 and 3431.7 cm−1were amide groups, and a diketone group were apparent in 1653.7 and 1638.01 cm−1. The peaks at 2079.9 and 1084.9cm−1 were attributed to C-O bonds. The absorption peak at 1401.8 and 1434.1 cm−1 indicates for nitrosamine presence. In 1384.8 cm−1 peaks was isopropyl, whereas that at 1103.8 cm−1 refer to ester carbonyl group indicates. In 1007.2–983.5 cm−1 peaks were polysaccharides. The peak at 832.63 cm−1was an aromatic group with absorption peaks at 702.8–543.8 cm−1. These findings agree with the results of other studies [10, 44].
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) followed by the effects against Escherichia coli ATCC 8739. Gram negative bacteria are more antagonistic to Streptomyces VITSDK1 spp. surfactants, with an inhibition zone of 10.3 mm in Klebsiellapneumoniae. Staphylococcusaureus is inhibited by Streptomyces VITSDK1 spp. surfactants by 5.3 mm . Streptomyces sp. strain AF1 has a distinct personality. It can grow at a high temperature of about 70℃ and generate an antimicrobial biosurfactant .
Figure 12 illustrated the inhibitory action of Streptomyces althioticus (RG3) biosurfactants on bacterial biofilm 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 .