الفهرس 
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Abstract
Biosurfactants are amphipathic molecules, synthesized by microorganisms. These compounds have several advantages over chemical surfactants. They are commercially important compounds with wide potential applications in the environment.
The main aim of this study was to select a local bacterial strain able to produce substantial amounts of biosurfactant; to optimize its production on a laboratory scale then upgrading the production to a larger scale level. This was accomplished by
1. Isolation of microorganisms from hydrocarbon contaminated areas & other sources for biosurfactant production.
2. Screening for a potent biosurfactant producing strain among these isolates.
3. Studying of the physiological and biochemical factors affecting the biosurfactant production of this selected isolate.
4. Identification of the potentially producing bacterial isolate as well as identification of the biosurfactant molecule produced.
5. Scaling up of the biosurfactant production to the fermentor level.
• Different oil- polluted soil samples with different levels of oil contamination were collected from different plots around oil wells of Khalda petroleum oil well, West Desert, Egypt, and from gardens of the NRC, Cairo, Egypt. These soil samples were used to isolate biosurfactant-producing bacteria using shake flasks as a batch culture.
• A total of 67 bacterial isolates, most of them were isolated from oil contaminated soil. Also we were kindly provided of a bacterial strain (NRC-1) from prof dr. abdel-mohsen saber research team that was isolated from goat skin
• All these isolates were subjected to different screening testes including two qualitative tests (DROP collapse test and blood hemolysis test) two semi quantitative tests (CTAB assay and oil spreading test) and quantitative tests (du Nouy ring method).
• The results showed that a large number of the isolated bacterial strains were very promising for the production a large amount of biosurfactant. However the strain isolated from goat skin was found to be the highest biosurfactant producer this isolate was identified using the 16s r RNA as Bacillus velezensis NRC -1.
• The biosurfactant produced by Bacillus velezensis NRC -1 isolate in mineral salts medium (MSM) was identified as surfactin, using TLC techniques.
• Media components and environmental fermentation conditions affecting growth and biosurfactant production by Bacillus velezensis NRC-1 were studied to optime the produced biosurfactant.
• Environmental conditions involved the effect of different incubation temperatures, pH values, agitation speed and inoculum size. Nutritional components included studying carbon sources concentrations, nitrogen sources concentrations, MgSO4 concentration and addition of trace elements. The results revealed that:
• The Mineral Salt medium (MSM) was the most suitable medium for BS production.
• Time course experiments revealed that biosurfactants production by Bacillus velezensis NRC-1 was growth- associated. Maximum biosurfactant production was obtained by Bacillus velezensis NRC-1 after 3 days of incubation. The surface tension reached 32 (mN/ m).
• The optimum incubation temperature was 32oC, growth reached (1.17g/l) and biosurfactant concentration reached 0.66 g/l.
• The optimum pH of the media was pH 6.8, growth reached (1.28 g/l) and biosurfactant concentration reached 0.76 g/l
• The optimum shaking speed was 200rpm, growth reached (1.3 g/l) and biosurfactant concentration reached 1 g/l
• The optimum inoculum size was 3 %, growth reached (1.7 g/l) and the biosurfactant concentration reached 1.17 g/l.
• The optimum carbon source was glucose, none of the other tested carbons was found to be superior to glucose. However alternative carbon sources could also be used effectively for high biosurfactant production, these included sucrose and molasses. The highest biosurfactant production was achieved by the use of glucose at the optimum concentration 30 g/l. growth reached (2 g/l) and biosurfactant concentration reached 1.36 g/l.
• The optimum nitrogen source was sodium nitrate; none of the other tested nitrogen’s was found to be superior to sodium nitrate. However alternative nitrogen sources could also be used effectively for high biosurfactant production, these included (NH4)2HPO4 and NH4H2PO4. The higher biosurfactant production was achieved by the use of sodium nitrate at the optimum concentration 3 g/l. Growth reached (2.56 g/l) and biosurfactant concentration reached 1.5 g/l.
• The optimum MgSO4 concentration was 0.5 g/l, growth reached (2.5 g/l) and biosurfactant concentration reached 1.57 g/l
• In addition, adding two ml of trace element solution caused enhancement of growth reached (2.68 g/l) and biosurfactant concentration reached 1.61 g/l
• Time course experiments showed that biosurfactant production by Bacillus velezensis NRC-1 in (MSM) or optimized media reached to the maximum after 48 h of incubation.
• Batch fermentation for surfactin production from Bacillus velezensis NRC-1 isolate has been applied in a 5L bioreactor. Different factors was studied including the effect of controlled and un-controlled pH, different agitation rates, different aeration volumes and bioreactor modification to avoid excess foam generation on growth and biosurfactant activity. The results revealed that:
• The surfactin production reached to the maximum production yield of 4.2 g/l in a pH-controlled medium. Also increasing the stirrer speed and aeration volume to 300 rpm and 2vvm enhanced the surfactin production about three folds.
• An improvement of the production yield is hampered by the strong foam formation, different techniques including [antifoam addition - foam collection & foam recycling] were performed.
The results of this study showed that, the modified bioreactor equipped with the foam recycler produced about 75% more surfactin compared with standard and this is an efficient method for surfactin production.
• The produced surfactin was soluble in aqueous solution at pH > 5 with optimum solubility at pH 8 – 8.5. It was also soluble in ethanol, acetone, methanol, butanol, and chloroform, but not in n-hexane, dichloromethane, ethylacetate or acetonitrile
• Surfactin showed to be highly effective biosurfactant since their crude solution could lower the surface tension of water from about 70 mN/m to 33 mN/m. Surfactin showed to be highly efficient as well, since CMC value of surfactin was found to be 15 mg/l.
• Surfactin-containing CFS of Bacillus velezensis NRC-1, showed higher emulsification indices with kerosen followed by diesel (Solar), then by vegetable oil, their indices were 72, 66 and 63%, respectively. However, it showed very low emulsification index with motor oil & linseed oil (52 and 37%, respectively).
• Surfactin solution showed very good thermal stability as there was no reduction in the surface activity to any degree upon exposure to 100°C for up to one hour.
Maximum surface activity for surfactin was obtained at pH 6.5 and further increase in pH was accompanied with a gradual reduction in surface activity up to pH 7.5 but still good surface activity was demonstrated at higher pH values. On the other hand, produced surfactin solution showed no surface activity at pH values from 2 to 4 and an abrupt increase in the surface activity was observed starting from pH 5.