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EL-Amine Bendaha et al., 1:12http://dx.doi.org/10.4172/scientificreports.544

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Volume 1 • Issue 12 • 2012

Keywords: Rhamnolipids; Hydrocarbons; Biosurfactants; Pseudomonas aeruginosa; Pseudomonas fluorescens

IntroductionRhamnolipids are one of the most interesting classes of

biosurfactants due to their advantageous features; they are the most effective biosurfactants, and are applied in several industries [1-3]. Regarding production, several carbon sources can be used by the bacteria, which include raw materials, such as waste oil or waste from the food industry [1,4]. They show higher returns compared to other biosurfactants.

The chemical structure of this biosurfactant group is composed of a hydrophilic head containing one or two molecules of rhamnose, called monorhamnolipid or dirhamnolipid, respectively, and a hydrophobic tail containing one or two fatty acids. Composition of the produced rhamnolipid molecule depends on the bacterial strain [1]. Today, recent progress in reducing production costs and the discovery of new producing strains suggests a promising future. The purpose of this study was to isolate bacteria of Pseudomonas genus and test them for rhamnolipids production, then optimize the production of the most successful strains.

Materials and MethodsSampling

The samples were taken from soils polluted by hydrocarbons at two gas stations in Mascara, and a forest in which there was discharged sewage from the second gas station. 15 soil samples were collected at the three sampling sites; about 50 g of soil was collected in sterile bags for each one. Isolation strategy

Isolation of fluorescent Isolation of fluorescent Pseudomonas begins with enrichment; environmental samples were inoculated at a rate of 1 g of soil in tubes containing 9 ml of nutrient broth. Inoculated tubes were incubated at 30°C for 24 h. 0.1 ml from enrichment tube was placed on Cetrimide agar and incubated at 30°C for 24 h [5].

Colonies producing blue-green or yellow-green pigment, revealed

*Corresponding author: Mohammed EL-Amine Bendaha, Department of Biology, Faculty of Science, Mascara University, 29000, Algeria, E-mail: biodaha@live.fr

Received July 27, 2012; Published October 22, 2012

Citation: EL-Amine Bendaha M, Mebrek S, Naimi M, Tifrit A, Belaouni HA, et al. (2012) Isolation and Comparison of Rhamnolipids Production in Pseudomonas aeruginosa P.B:2 and Pseudomonas fluorescens P.V:10. 1:544 doi:10.4172/sci-entificreports.544

Copyright: © 2012 EL-Amine Bendaha M, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

AbstractTwenty bacterial strains were isolated from soils contaminated with hydrocarbons in Mascara region (Algeria),

and tested for biosurfactants production in a nutrient broth supplemented with olive oil. The two powerful strains have been identified as Pseudomonas aeruginosa P.B:2 and Pseudomonas fluorescens P.V:10. The optimal production of rhamnolipids was obtained with the following composition: 100 ml of nutrient broth, 2% of olive oil and 2% of inoculums, with shaking at 75 rpm/min at room temperature, for an incubation period of 34 h for Pseudomonas fluorescens P.V:10 (121.120 ± 1.61 mg/L of rhamnolipids), and 38 h for Pseudomonas aeruginosa P.B:2 (101.115 ± 0.724 mg/L of rhamnolipids). After production optimization, supernatants from each bacterial culture were tested on the following hydrocarbons: C5 kerosene, C4 kerosene and diesel. Emulsion indexes obtained for Pseudomonas fluorescens P.V:10 are respectively: 59.950 ± 0.346%, 57.106 ± 0.130%, 56.863 ± 0.323%, those obtained for Pseudomonas aeruginosa P.B:2 are respectively: 57.190 ± 0.210%, 57.067 ± 0.309%, 56.323 ± 0.486%.

Isolation and Comparison of Rhamnolipids Production in Pseudomonas aeruginosa P.B:2 and Pseudomonas fluorescens P.V:10Mohammed EL-Amine Bendaha1*, Saad Mebrek2, Mostefa Naimi2, Abdelkarim Tifrit2, Hadj Ahmed Belaouni3 and Bouziane Abbouni2

1Department of Biology, Faculty of Science, Mascara University, 29000, Algeria2Department of Biology, Faculty of Science, Sidi Bel Abbes University, 22000, Algeria3E.N.S. Kouba, 16000, Algeria

under UV at 366 nm were picked and purified three times on King’s B medium at 30°C for 24 h.

Screening of potential biosurfactants producers Inoculums preparation: From petri dishes, bacterial suspensions

were prepared for each isolate in nutrient broth for 24 h; their O.D was measured at 600 nm and adjusted at 0.5 Mc.Farland to bring them under the same conditions of initial biomass, in order to select the most effective strain.

Culture medium inoculation: To maximize biosurfactant production, all isolates were grown in nutrient broth containing 2% of olive oil as the best carbon substrate [6,7].

Each 200 ml Erlenmeyer flask is inoculated with 1 ml of inoculum prepared in advance, and stirred at 75 rpm at room temperature (34 to 37°C) for 48 h, biosurfactants are recuperated in the supernatant after centrifugation at 9000 g for 15 min.

Production testing of rhamnolipids Drop collapsing: The screening for rhamnolipids production was

conducted, using the test of collapse described by Jain et al. [8]. A drop of supernatant of each isolate was placed on the surface of a glass slide covered with a thin layer of oil. The teardrop shape on the surface of the oil was observed after 1 min.

Supernatant of the culture that led to the collapse of the drop is shown as a positive result, and the drops remaining with the beads are marked as negative results, and discussed with distilled water as control.

Pseudomonas:

Citation: EL-Amine Bendaha M, Mebrek S, Naimi M, Tifrit A, Belaouni HA, et al. (2012) Isolation and Comparison of Rhamnolipids Production in Pseudomonas aeruginosa P.B:2 and Pseudomonas fluorescens P.V:10. 1:544 doi:10.4172/scientificreports.544

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Oil displacement: 15 µL of crude oil is placed on the surface of 40 ml of distilled water placed in a petri dish, and a supernatant of 10 µL of each culture was slightly put on the surface of the oil film. Diameter of the clear halo viewed under visible light, is measured after 30 s [9].

Emulsification activity: Emulsification activity was carried out using oil [10]. 4 ml of soybean oil was added to 4 ml of supernatant, i.e. a ratio of 1:1, the mixture was vortexed vigorously for 2 min. After 24 h, the emulsification index (E24) was estimated as follows: E24=HEL/HS *100

Where E24: Emulsification activity after 24 hours, HEL: Height of emulsion layer, HS: Height of total liquid column.

Biochemical profile is specific for each species, within this group of bacteria. These tests were performed according to manufacturers’ instructions, Biomerieux, France.

Probabilistic interpretation of the biochemical profiles was done by the Bacterial Identification Program [12]: software developed in the 90 years at the University of Southampton (UK), for identification of bacterial isolates, using data collected through use of probabilities.

Culture medium and growth conditions for rhamnolipids production

Each measurement is performed three times and values are expressed as mean ± standard deviation.

Determining the percentage of rhamnose: Rhamnolipids dosage is achieved through rhamnose measurement by UV spectrophotometry, using the method described by Candrasekaran and Bemiller [13]. Biosurfactant consists of a mixture of several molecules of rhamnolipids. Therefore, the determination of rhamnose concentration in a solution will follow the surfactant concentration in the different experiments. For this, we performed a calibration curve prepared from L-Rhamnose, which connects rhamnose mass in a solution with their absorbance.

Optimizing the olive oil amount: The culture medium used for rhamnolipids production by P. aeruginosa P.B:2 and P. fluorescens P.V:10 was optimized, by varying the olive oil amount from 2% to 8% (v/v) in the broth culture. Then, the culture medium was incubated at room temperature (34 to 37°C) in a shaking incubator at 75 rpm. After 48 h of incubation, bacteria were removed by centrifugation at 9000 g for 15 min. The measurement of dry weight of bacterial pellet in 1 ml of culture medium and the determination of rhamnolipids were made for each concentration of olive oil used. The dry weight was determined after centrifugation at 9000 g for 15 min, and drying at 110°C in a period of 12 h.

The optimum amount of olive oil for rhamnolipids production is used for further optimization.

Inoculums optimizationGrowth kinetics: First, a preculture of each strain was performed on

nutrient agar and incubated at 30°C. After 24 h of incubation, a colony of each culture is used to inoculate an Erlenmeyer flask containing 100 ml of nutrient broth, each flask is incubated with shaking at 75 rpm and room temperature (34 to 37°C).

Microbial growth was studied as a function of culture time by

measuring the culture medium absorbance, after each 1 h using a 600 nm UV spectrophotometer.

From graphic layout of the microbial concentration versus time, the best culture time for the inoculum preparation was determined.

Optimizing the inoculums amount: To find the appropriate amount of inoculum for rhamnolipids production, an inoculum of each strain was prepared using the best culture time obtained in advance, and by varying the inoculum amount from 2% to 8% (v/v), and then transfered into 100 ml of culture medium, containing the optimum amount of olive oil. After that, the culture was incubated at room temperature (34 to 37°C), with shaking at 75 rpm. After 48 h of incubation, measurements of bacterial pellet dry weight and rhamnolipids determination are made for each used concentration of inoculum.

The optimum inoculum amount for rhamnolipids production is used for further optimization.

Determination of the best cultivation time: To produce the maximum of biosurfactant from each selected microorganism, an optimum inoculum amount is prepared and transfered into the culture broth, containing the optimum olive oil amount. The culture is then incubated at room temperature (34 to 37°C) with shaking at 75 rpm. After that, cell dry weight and rhamnolipids determination are made for each crop, throughout the 3 h to find the best cultivation time for biosurfactant production.

Hydrocarbons emulsification: After optimization of rhamnolipids production, supernatant recovered from each culture was tested on the following hydrocarbons: Diesel, C4 kerosene and C5 kerosene.

Emulsion index (E24) was estimated for each type of hydrocarbon.

Results Isolation of fluorescent

Following the enrichment, each isolate was subcultured on Cetrimide agar, which is a medium for promoting pigmentation, and for bacterial fluorescens, it is King A medium supplemented with nalidixic acid and tétradonium bromide (Cetrimide) that inhibit the Gram positive growth, leaving the advantage to Pseudomonas sp.

Pseudomonas aeruginosa is characterized by two pigments production: pyocyanin (blue green pigment) and pyoverdin (yellow green pigment) [5].

On Cetrimide agar, two types of fluorescens were observed under UV at 366 nm. Conclusively when transplanting on King’s B medium, only one type of fluorescens is observed; this implies that some bacteria have both pyocyanin and pyoverdin pigment, while others have only pyoverdin.

20 strains with fluorescent pigments were isolated, purified and conserved; 10 of which are equipped with pyocyanin and pyoverdin (Coded P.B), and 10 others that are provided as pyoverdin (Coded P.V).

Production testing of rhamnolipidsDrop collapsing: A drop of water applied to a hydrophobic surface,

in absence of biosurfactants, form a pearl because polar molecules of water are repelled from hydrophobic surface. However, if the water drop contains biosurfactants, the latter falls and spread over the blade [14]. Results presented, show biosurfactants presence in all tested samples.

Oil displacement: The oil displacement is closely related to biosurfactant presence in the supernatant of the tested bacteria

Pseudomonas

Identification of the efficient strains: The chosen bacteria are subject to phenotypic identification, based on the following characters: Appearance of bacterial colonies on culture media, Cetrimide and King’s B, microscopic observation, respiratory type, catalase test, oxidase test and biochemical identification by API 20NE system, which is a rapid method based on the biochemical substrates degradation. This method is suitable to identify Gram-negative species level, by assessing the profile of 21 different biochemical reactions [11].

Citation: EL-Amine Bendaha M, Mebrek S, Naimi M, Tifrit A, Belaouni HA, et al. (2012) Isolation and Comparison of Rhamnolipids Production in Pseudomonas aeruginosa P.B:2 and Pseudomonas fluorescens P.V:10. 1:544 doi:10.4172/scientificreports.544

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Efficient strains identificationBoth strains P.B:2 and P.V:10 have almost the same macroscopic

and microscopic characteristics, with a slight difference in the color colonies, and a noticeable dissimilarity in pigmentation; bacterium P.B:2 holds pyoverdin and pyocianin, which is not the case of the bacterium P.V:10, which holds only pyoverdin.

Both types of strains are Gram-negative bacilli, oxidase and catalase positive, form a microbial film on the surface of the meat liver agar, and have been described as strictly aerobic bacteria.

The program suggests that strain P.B:2 is a Pseudomonas aeruginosa strain, with a identification score of 0.99999, and suggest that strain P.V:10 is a Pseudomonas fluorescens strain with an identification score of 0.98957.

Culture medium and growth conditions for rhamnolipids production

Optimizing the olive oil amount: As shown in figure 5, the increase of rhamnolipids production is obtained with 2% of olive oil used in the cultivation of Pseudomonas aeruginosa P.B:2 (89.625 ± 0.780 mg/L) and Pseudomonas fluorescens P.V:10 (105.756 ± 2.076 mg/L); it is the same for the dry weight which is 4.566 ± 0.057 g/L for Pseudomonas fluorescens P.V:10, and 4.333 ± 0.251 g/L for Pseudomonas aeruginosa P.B:2 (Figure 6).

Therefore, a nutrient broth supplemented with 2% olive oil was

[15], displacement diameter varies with biosurfactants amount in supernatant of each sample.

All tested strains showed a positive result for oil displacement with different diameters (Figures 1 and 2).

Emulsification activity: The main role of biosurfactants is to solubilize hydrophobic molecules by trapping them in a pseudohydrophobic phase formed by micelles, thereby increasing their apparent solubility [16].

The various isolated organisms solubilize the oil and form a pseudohydrophobic phase, but emulsion indexes (E24) varies from one bacterium to another, according to the rate of biosurfactants production (Figures 3 and 4).

Selection of the most efficient strainsAccording to the results obtained from figures 1-4, bacteria that

have shown great potential for rhamnolipids production are the bacteria with the maximum oil displacement diameter and the highest emulsion index (E24).

Strains P.B:2 (E24=56.320 ± 1.955% and Ø=5.733 ± 0.355 cm) and P.V:10 (E24=56.443 ± 0.885% and Ø=6.133 ± 0.321 cm) are the best biosurfactant producers, and are selected to be identified and optimized for production.

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Figure 1: Comparison of clear halos diameters due to biosurfactants of P.B strains.

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Figure 2: Comparison of clear halos diameters due to biosurfactants of P.V strains.

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Figure 3: Comparison of emulsion indexes of the different P.B strains.

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Figure 4: Comparison of emulsion indexes of the different P.V strains.

Citation: EL-Amine Bendaha M, Mebrek S, Naimi M, Tifrit A, Belaouni HA, et al. (2012) Isolation and Comparison of Rhamnolipids Production in Pseudomonas aeruginosa P.B:2 and Pseudomonas fluorescens P.V:10. 1:544 doi:10.4172/scientificreports.544

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chosen as the appropriate culture medium, for future experiments of rhamnolipids production by the two selected strains.

Inoculums optimizationGrowth kinetics: Growth kinetics of each strain was studied to find

the best culture time for an inoculum preparation, it is used to know how long it takes for each bacterium to enter in exponential phase; both strains were monitored for 26 h by measuring the O.D at 600 nm every hour at room temperature (34-37°C). From figure 7, Pseudomonas fluorescens P.V:10 reaches the exponential phase after 8 h of culture, ahead of Pseudomonas aeruginosa P.B:2 by two hours, because the lag phase and acceleration are shorter for strain P.V:10 (6 h and 2 h, respectively).

Therefore, the best culture time chosen for the inoculum preparation of Pseudomonas fluorescens P.V:10 is 8 h, the one chosen for Pseudomonas aeruginosa P.B:2 is 10 h.

Optimizing the inoculums amount: As shown in figure 8, the increase of rhamnolipids production is obtained with 2% of inoculum in Pseudomonas aeruginosa P.B:2 culture (104.826 ± 0.939 mg/L) and Pseudomonas fluorescens P.V:10 (124, 527 ± 1.071 mg/L); it is the same for the dry weight (Figure 9), which is 5.833 ± 0.115 g/L for Pseudomonas fluorescens P.V:10 and 5.500 ± 0.100 g/L for Pseudomonas aeruginosa P.B:2.

Therefore, a nutrient broth inoculated with 2% of microbial culture

was selected as a suitable culture medium for rhamnolipids production by both selected strains.

Determination of the best cultivation time: To maximize rhamnolipids production by both strains, an inoculum of Pseudomonas

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Figure 5: Effect of the olive oil amount on dry weight of Pseudomonas aeruginosa P.B: 2 and Pseudomonas fluorescens P.V: 10.

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Figure 6: Effect of the olive oil amount on L-rhamnose production by Pseudomonas aeruginosa P.B: 2 and Pseudomonas fluorescens P.V: 10.

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Figure 7: Growth kinetics of strains P. aeruginosa P.B: 2 and P. fluorescens P.V: 10 in nutrient broth at room temperature (34 to 37°C), with shaking at 75 rpm/min.

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Figure 8: Effect of inoculum amount on L-rhamnose production by Pseudomonas aeruginosa P.B: 2 and Pseudomonas fluorescens P.V: 10.

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Figure 9: Effect of inoculum amount on the dry weight of Pseudomonas aeruginosa P.B: 2 and Pseudomonas fluorescens P.V: 10.

Citation: EL-Amine Bendaha M, Mebrek S, Naimi M, Tifrit A, Belaouni HA, et al. (2012) Isolation and Comparison of Rhamnolipids Production in Pseudomonas aeruginosa P.B:2 and Pseudomonas fluorescens P.V:10. 1:544 doi:10.4172/scientificreports.544

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aeruginosa P.B:2 was prepared for 10 h and another of Pseudomonas fluorescens P.V:10 for 8 h in nutrient broth. Each bacterial culture was introduced into an environment culture containing 2% of olive oil and 2% of inoculum, and then rhamnolipids production was followed for the two candidates for 42 h (Figures 10 and 11).

As shown in figure 10, rhamnolipids production and dry weight over time has continued to increase for Pseudomonas aeruginosa P.B:2, during 34 h of culture (Rhamnolipids: 95.814 ± 0.498 mg/L, dry weight: 4.033 ± 0.057 g/L). From 34 h, a slight decrease in dry weight is due to cell lysis by its own proteases and caused a slowdown in rhamnolipids production; then from 36 h, there was resumption of growth and production.

Rhamnolipids production by Pseudomonas aeruginosa P.B:2 reach its stationary phase after 38 h of culture (101.115 ± 0.724 mg/L), even if the dry weight might continue to increase or decrease.

Therefore, the best cultivation time of Pseudomonas aeruginosa P.B:2 is 38 h.

According to results illustrated in figure 11, rhamnolipids production and dry weight over time have increased for Pseudomonas fluorescens P.V:10, during 34 h of culture (Rhamnolipids: 121.120 ± 1.61 mg/L, dry weight: 4.200 ± 0.100 g/L), and also from the 34 h, the production reached its stationary phase and its maximum, even with the increase of dry weight.

Therefore, the best cultivation time of Pseudomonas fluorescens P.V:10 is 34 h.

Hydrocarbons emulsification: Supernatant from the two

optimized cultures of Pseudomonas aeruginosa P.B:2 for 38 h and Pseudomonas fluorescens P.V:10 for 34 h tested on diesel, C4 kerosene and C5 kerosene solubilized hydrophobic molecules, and trapped them in the pseudo hydrophobic phase.

Emulsion indexes showed that rhamnolipids produced by P. fluorescens P.V:10 had better activity than those produced by P. aeruginosa P.B:2 (Figure 12). Emulsion indexes of the two strains on the different tested hydrocarbons: C5 kerosene, C4 kerosene and diesel for P. fluorescens P.V:10 are respectively: 59.950 ± 0.346%, 57.106 ± 0.130%, 56.86 ± 0.323%, and for P. aeruginosa P.B:2 they are respectively: 57.190 ± 0.210%, 57.067 ± 0.309%, 56.323 ± 0.486%.

DiscussionCetrimide agar is a selective medium for bacteria of the

Pseudomonas genus; the formula of the basic medium is a modification of king A medium, which is an environment that promotes pyocyanin and pyoverdin production. Lowbury [17] advocated the use of Cetrimide in a selective medium for Pseudomonas isolation. Due to the improvement of the inhibitor agent purity, its concentration was reduced by Lowbury and Collins [18]. The addition of nalidixic acid with a decrease of cetrimide concentration, allowed better recovery of Pseudomonas, because nalidixic acid blocks DNA replication of sensitive bacteria to this antibacterial agent [19].

Twenty bacterial strains were isolated from soil contaminated by hydrocarbons in Mascara region and tested for biosurfactants production in a nutrient broth supplemented with olive oil. Ten strains are characterized by the blue green (Pyocianin) and yellow green (pyoverdin) pigment production, the other ten are characterized by the only production of yellow green pigment. Magnesium chloride and potassium sulfate promotes pyocyanin production, and glycerol is a source of energy that accelerates the production [20]. When fluorescent Pseudomonas is grown in iron limiting conditions, they produce yellow green fluorescent siderophores called pyoverdins [21].

The nutrient broth is a culture medium rich in nitrogen compounds [22], supplemented with olive oil as the best carbon substrate [7], which allow for better biosurfactants production; insoluble substrates lead to better performance of rhamnolipids than the soluble substrates [23], more bacteria of the genus Pseudomonas are lipase positive, which facilitates fatty acids fraction assimilation contained in olive oil [7].

When testing "Drop collapsing" in biosurfactants presence, 0

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Figure 10: Concentration of L-rhamnose (mg/L) and dry weight determining for Pseudomonas aeruginosa P.B: 2 as a function of culture time.

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Figure 11: Concentration of L-rhamnose (mg/L) and dry weight determining for Pseudomonas fluorescens P.V: 10 as a function of culture time.

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Figure 12: Comparison of emulsion indexes for Pseudomonas fluorescens P.V: 10 and Pseudomonas aeruginosa P.B: 2 obtained on diesel, C4 kerosene and C5 kerosene.

Citation: EL-Amine Bendaha M, Mebrek S, Naimi M, Tifrit A, Belaouni HA, et al. (2012) Isolation and Comparison of Rhamnolipids Production in Pseudomonas aeruginosa P.B:2 and Pseudomonas fluorescens P.V:10. 1:544 doi:10.4172/scientificreports.544

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the strength or the interfacial tension between water droplet and hydrophobic surface is reduced, resulting in the spread of the water drop on hydrophobic surface [13]. Biosurfactant molecules dispersed on a petri dish containing water and a thin layer of oil adsorbed on the surface of water [24], because they have two different polarities, and density is less than water, so they float to the surface. Rhamnolipids micelles have a greater affinity for water then oil, therefore, they will return to competition with the latter to the surface occupation. If the oil volume was higher than that of biosurafactant, this latter is adsorbed at the water/oil interface [25], but because of the very small volume of oil, its hydrophobic molecules will move leaving the surface to biosurfactant molecules. Micelles are formed when hydrophobic portions unable to form hydrogen bonds in aqueous phase, unite and move towards the center leaving the hydrophilic portions outward; agitation provided by vortex was made to isolate hydrophobic molecules of oil, and trapping them inside micelles [26].

ConclusionIn this study, two types of microorganisms producing biosurfactants,

Pseudomonas fluorescens P.V:10 and Pseudomonas aeruginosa P.B:2, were isolated from soil contaminated by hydrocarbons in Mascara region (Algeria). Rhamnolipids production was made using a nutrient broth supplemented with olive oil as the best carbon source. To study microbial growth, oil displacement, drop collapsing and emulsification tests were performed. It was found that the strain P. fluorescens P.V:10 could grow in the culture medium better than the strain P. aeruginosa P.B:2, resulting in a shorter culture time for biosurfactant production (P.V:10:34h<P.B:2:38h) and a higher yield (P.V:10:121.120 ± 1.61 mg/L>P.B:2:101.115 ± 0.724 mg/L). Emulsion index measure showed that

rhamnolipids produced by P. fluorescens P.V:10 had better activity than those produced by P. aeruginosa P.B:2. By comparing results of the two strains on the different tested hydrocarbons: C5 kerosene, C4 kerosene and diesel, emulsion indexes obtained with the strain P.V:10 were higher than those obtained with the strain P.B:2. Therefore, P. fluorescens P.V:10 could be the remedy for contamination, more effectively than P. aeruginosa P.B:2.Acknowledgments

The authors gratefully acknowledge Pr. Abbouni B. and Mr. Selouani MM E-A, the owner of microbiology laboratory at the biology department, faculty of science, Sidi Bel Abbes University, Algeria for their generosity in providing us with the necessities for conducting this research.

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Both strains P.B:2 and P.V:10 are most suitable for nutrient broth supplemented with 2% of olive oil, and have best indicators of emulsion diameter and oil displacement (P.B:2:E24=56.320 ± 1.955%, Ø=5.733 ± 0.355 cm, and P.V:10:E24=56.443 ± 0.885%, Ø=6.133 ± 0.321 cm). According to the Bacterial Identification Program, strain P.B:2 is a Pseudomonas aeruginosa strain with an identification score equal to 0.99999, it also suggests that P.V:10 strain is a Pseudomonas fluorescens strain with an identification score equal to 0.98957. The greatest rhamnolipids production is obtained with 2% of olive oil concentration for Pseudomonas aeruginosa P.B:2 (89.625 ± 0.780 mg/L) and Pseudomonas fluorescens P.V: 10 (105.756 ± 2.076 mg/L); the two strains use only 2% of olive oil to achieve the best performance, this amount of olive oil is more than enough to lead to greater efficiency and saves substrate. The best culture time for inoculums preparation for Pseudomonas fluorescens P.V:10 is 8 h and the one chosen for Pseudomonas aeruginosa P.B:2 is 10 h; this is the time taken by them to reach the exponential stage where each bacterial cell gives two daughter cells, and where the growth rate is maximum and stable. Number of bacteria present in inoculum directly affects production; a cell number less than the best will probably not lead to better production, but when cells number is high, they use all the the culture medium compounds for their growth, which leads to depletion of culture medium, without achieving the desired metabolite production. After testing different inoculum concentrations, a nutrient broth inoculated with 2% of microbial culture was selected as a suitable culture medium for rhamnolipids production, by both selected bacterial strains, due to the high rate of rhamnolipids production by Pseudomonas fluorescensP.V:10, emulsion indexes obtained on the different hydrocarbons are higher than those obtained with Pseudomonas aeruginosa P.B:2. Thus, Pseudomonas fluorescens P.V:10 is a more adapted strain to grow on kerosene and diesel, and is an auxiliary of choice in bioremediation of soils contaminated by hydrocarbons.

5. Dworkin M, Falkow S, Rosenberg E, Schleifer KH, Stackebrandt E (2006) The Prokaryotes: A Handbook on the Biology of Bacteria, Third Edition, Volume 6: Proteobacteria: Gamma Subclass. Springer Science+Business Media, Singapore 654-655.

Citation: EL-Amine Bendaha M, Mebrek S, Naimi M, Tifrit A, Belaouni HA, et al. (2012) Isolation and Comparison of Rhamnolipids Production in Pseudomonas aeruginosa P.B:2 and Pseudomonas fluorescens P.V:10. 1:544 doi:10.4172/scientificreports.544

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21. Meyer JM, Abdallah MA (1978) The florescent pigment of Pseudomonas fluorescens : Biosynthesis, purification and physicochemical properties. J Gen Microbiol 107: 319-328.

22. Atlas RM (2005) Handbook of Media for Environmental Microbiology. (2nd edition), CRC Press, Taylor & Francis Group, Boca Raton, USA.

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