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Comparative approach for detection of biosurfactant-producing bacteria isolated from Ahvaz petroleum excavation areas in south of Iran

Comparative approach for detection of biosurfactant-producing bacteria isolated from Ahvaz... Annals of Microbiology, 58 (3) 555-560 (2008) Comparative approach for detection of biosurfactant-producing bacteria isolated from Ahvaz petroleum excavation areas in south of Iran 1§ 1§ 1 2 Shima AFSHAR , Tayebe Bagheri LOTFABAD , Reza ROOSTAAZAD , Abdolhossein Rouholamini NAJAFABADI , Kambiz Akbari NOGHABI * 1 2 Department of Chemical and Petroleum Engineering, Sharif University of Technology; Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences; National Institute of Genetic Engineering and Biotechnology (NIGEB), Karaj-Tehran highway, Pazhoesh Blvd. P.O.Box 14155-6343, Tehran, Iran Received 2 April 2008 / Accepted 15 June 2008 Abstract- The current study was undertaken to compare four analytical methods including drop collapse, oil spreading, surface tension (SFT) measurements, and blood agar lysis tests for detection of biosurfactant-producing bacteria. Among 32 biosurfactant-producing bacteria isolated from Ahvaz oil fields, in south of Iran, 16 isolates (50%) exhibited highest biosurfactant production. Eleven isolates (MASH.1 to MASH.11) demonstrated a reduction in surface tension from 65 mN/m to less than 41 mN/m. The results showed that about 91% of these highly biosurfactant producers had the same response levels of “++++” and “+++” in the case of both SFT and oil spreading methods. Among these, seven isolates had the haemolysis diameter less than 1 cm or between 1 and 2 cm on blood agar. As 64% of the best biosurfactant producers did not completely lyses blood, the ability of biosurfactant-producers for haemolysis may not always be trustworthy. According to our data, there is a good consistency between oil spreading technique and surface ten- sion. As a conclusion, oil spreading method is the fastest, simplest and most consistent analytical method to be suggested for accurate measurements of biosurfactant producers. Key words: biosurfactant, oil spreading, drop collapse, surface tension, blood lysis. INTRODUCTION ability to microorganisms, which is a potential problem for bac- teria. It has been assumed that surfactants would enhance the Bacteria produce a wide range of biosurfactants with diverse bioavailability of hydrophobic compounds (Ron and Rosenberg, and unlike chemical structures (Kosaric, 1992; Christofi and 2002). Because of the potential applications, many biotechno- logical studies have been focused on the mass and commercial Ivshina, 2002; Ron and Rosenberg, 2002; Das and Mukherjee, 2005). Interest in microbial surfactants has progressively been production of biosurfactants. Due to the unique properties and vast array of applications, the need for selection a simple increasing during recent years since they have higher biodegrad- and rapid as well as reliable analytical approach to detect the ability, higher specific activity at extreme temperatures, salinity, and pH levels and considered environmentally compatible with biosurfactant-producers with minimum number of false positives and/or negatives can not be ignored (Mukherjee et al., 2006). limited toxicity (Desai and Banat, 1997; Haba et al., 2000; Several analytical methods have so far been employed for Maier and Soberón-Chávez, 2000; Gautam and Tyagi, 2006). directly measurement of surface activity of biosurfactant. These Biosurfactants are interesting molecules with various biological approaches include surface and/or interfacial tension meas- functions. From a biotechnology prospective, the production of urement, drop shape analysis profile, glass slide test, method biosurfactants is important because of their numerous applica- and the oil spreading technique (Harkins, 1959; Mulligan et tions in environment, agriculture and industry (Banat, 1995; al., 1984; Banat, 1993; Van Dyke et al., 1993; Carrillo et al., Benincasa et al., 2004; Singh and Cameotra, 2004). These kind 1996; Makkar and Cameotra, 1997, 1998; Menezes Bento et of natural microbial compounds are biologically surface-active al., 2005). Each type of these aforesaid methods has their own agents and have many potential advantages over their synthetic advantages and disadvantages. Although enormous efforts have relatives. Microorganisms were considered to be detrimental to been made to develop the analytical methods for accurate meas- the petroleum industry in the past. It is now well known that urement of biosurfactant concentration and its activity, currently they can also be beneficial in terms of oil recovery. The low available reports on the comparison of these methods with each water solubility of many hydrocarbons, especially the polycyclic others to select the most reliable one is limited to a few reports aromatic hydrocarbons (PAHs), is believed to limit their avail- (Bodour and Miller-Maier, 1998; Piaza et al., 2006). The current study was undertaken to compare different analytical methods * Corresponding author. for measurement the activity of biosurfactant produced by bac- Phone: +98-21-44580352; Fax: +98-21-44580399; terial strains with unknown taxonomic affiliations isolated from E-mail: Akbari@nrcgeb.ac.ir, kambizakb@yahoo.com Both authors contributed equally to this work. oil fields in south of Iran. 556 S. Afshar et al. MATERIALS AND METHODS oil surface was inspected visually and its diameter was measured as an indicative of biosurfactant concentration; however, biosur- Media, enrichment and isolation of bacteria. The bacterial factant concentration was also measured, by using a calibration strains were isolated by the enrichment culture technique from curve based on a commercially available biosurfactant, surfactin the crude oil obtained from Ahvaz oil fields. For this purpose, 10 (Sigma, St. Louis, MO). ml crude viscous oil samples were inoculated into 90 ml of sup- plemented nutrient broth (SNB medium) of the same composi- Determination of emulsifying activities. Bacterial isolates tion as described by Francy et al. (1991). The medium incubation which were able to produce biosurfactant were evaluated for was performed at 30 °C on a rotary shaker incubator at 200 rpm emulsifying activity according to the method reported by Das et for 7 days. Then, an aliquot of culture (0.1 ml) was spread on al. (1998). For this purpose, cell-free supernatant was collected Bushnell-Haas agar(Difco, Germany) and incubated at 30 °C for by centrifugation (8000 x g, 20 min) of the culture broth. Then, 48 h. Diluted microbial suspensions in SNB were prepared and 2 ml of supernatant were mixed with 2 ml n-hexane or crude oil cultivated on Brain Heart agar (Merck, Germany) so as to obtain separately in a test tube. The mixture was vigorously stirred for 2 separate colonies. The procedure was repeated three times. min and then allowed to stand for 48 h. Relative emulsion volume Single colonies of each bacterial isolate growing well on Brain (EV, %) was measured according to the following equation: Heart agar (Merck, Germany) were designated according to their morphological and biochemical characteristics. Furthermore all Emulsion height (mm) × cross-section area (mm ) the isolates were examined by using optic microscope (Olympus EV % = Total liquid volume (mm ) Vanox AHBT3). Standard morphological and biochemical tests were used for their preliminary characterization. Among 32 bacterial strains, sixteen biosurfactant producer isolates were RESULTS AND DISCUSSION selected according to following method and maintained on nutrient agar (Merck, Germany) and Brain Heart agar slants at Table 1 shows the results of four methods that were used to 4 °C. To assess the biosurfactant production efficiency by dif- detect biosurfactant production by different isolates. Over sixteen ferent bacterial isolates, a 100 ml nutrient broth medium was isolates, seven (44%) could reduce the surface tension to 40 inoculated with a single colony of each bacterial isolates and mN/m or less; four (25%) isolates had a SFT between 40 and 42 incubated at 30 °C, 150 rpm for 24 h as seed culture. Then mN/m and five isolates (31%) exhibited a SFT of 42 mN/m or 2% (V/V) seed culture was transferred to a 500 ml Erlenmeyer higher. Oil spreading method resulted in the same response level flask containing 250 ml of E medium (Javaheri et al., 1985) with as for SFT for eleven of sixteen isolates (69%). the following components (g/l): KH PO , 2.7; K HPO , 13.9; Results in Table 2 show a good consistency between oil spreading 2 4 2 4 NaCl, 50; yeast extract, 1; NaNO , 1; (NH4) SO , 1; and 10 technique and surface tension with a Pearson rank correlation coef- 3 2 4 ml of a trace metal solution; 10 g/l glucose or crude oil were ficient, r = 0.456, and Spearman correlation coefficient, R = 0.459, added according to the experiment, to study the effect of carbon ranged between -1 (strong negative correlation) and 1 (strong posi- source on the biosurfactant production and activity. The metal tive correlation) (Table 3). Figure 1 show the relationship between solution was a modification of Wolins metal solution (Wolin et the diameter (mm) of clear zone obtained by the oil spreading al., 1963) and had the following composition (g/l): EDTA, 1; method and surface tension (mN/m) of the culture obtained by MnSO ·H O, 3; FeSO ·7H O, 0.1; CaCl ·2H O, 0.1; CoCl ·2H O, using glucose or crude oil as carbon source is consistent with the fact 4 2 4 2 2 2 2 2 0.1; ZnSO ·2H O, 0.1; CuSO ·5H O, 0.01; AlK(SO ) , 0.01; that crude oil, as a non-soluble carbon source, is better than glucose 4 2 4 2 4 2 H BO , 0.01; Na MoO ·7H O, 0.01; and MgSO , 25. (soluble carbon source) for biosurfactant production. 3 4 2 4 2 4 In the case of blood agar lysis test, as a simple and easy Biosurfactant production assay. Bacterial isolates were grown method, all the sixteen biosurfactant-producing strains were able in E medium with either glucose or crude oil as interchangeable to lyse the blood agar (Table 2). However, a consistent correla- carbon sources. The culture media were incubated at 30 °C in a tion between this test and other analytical techniques were not rotary shaker (150 rpm) for 7 days. After that, cell-free super- achieved. Complete haemolysis with a diameter > 3 cm did not natant was obtained using shear force of centrifugation (8000 x occur necessarily in the higher biosurfactant-producing isolates. g, 20 min) and collected in centrifuge tube. In addition, samples For instance; strains MASH.12 and MASH.14 could be able to lyse from culture medium containing whole cells were obtained for blood agar completely (++++), with a diameter of lysis > 3 cm, measurement of surface-active properties. but the results obtained from oil spreading technique or surface The surface tension of the samples was measured by the tension for these strains showed low activity. In contrast a weaker Ring method using a KRUSS du Nouy Tensiometer K6 at room haemolysis, with a response level of “+” or “++”, occurred for 64% temperature (McInerney et al., 1990). of the eleven strains which showed a good capability in producing Drop collapse measurement method was carried out accord- a considerable amount of biosurfactant as checked by oil spreading ing to Bodour and Miller-Maier (1998). Each bacterial isolate method (Table 2); 60% of isolates that did not exhibit a good SFT was streaked on blood agar and incubated at 37 °C for 48 h. (++) could lyse blood agar with response levels of “++++” and The plates were visually checked for the existence of clearing “+++”. Different response levels were found for haemolysis and zone around the colonies as a criterion for biosurfactant produc- oil spreading for strains MASH.12 to MASH.16. It was previously tion. With this goal, the clear zones were measured and scored reported that blood agar lysis test could be used as a simple and according to the diameter. Oil spreading measurement was done quick method for preliminary screening of biosurfactant producers by addition of 20 ml of distilled water to a Petri dish (10 cm diam- (Mulligan et al., 1984; Banat, 1993; Carrillo et al., 1996). This is a eter). Then 8 μl of crude oil was transferred to the surface of the marked contrast with our finding. Among sixteen isolates, just five water using a HPLC syringe and left to stand for a while. After strains (31%) could lyse blood agar completely (++++). Blood that, 10 μl of each bacterial isolate culture media were separately agar lysis method had a high percentage of either false-negatives added to the surface of oil; this technique was a modification of or false-positives. Therefore, blood agar lysis test is not suggested the Morikawa et al. (2000). The existence of clear zones on the as a reliable method for screening biosurfactant producers. Ann. Microbiol., 58 (3), 555-560 (2008) 557 TABLE 1 - Efficacy of the four analytical methods in predicting biosurfactant production Strain Surface tension Clear zone diameter Blood agar lysis EV % EV % (mN/m) by oil spreading (mm) diameter (cm) n-Hexane Crude Oil MASH.1 38.5 ± 0.0 3.0 ± 0.1 8.0 ± 1.0 0.0 11.45 ± 0.45 MASH.2 38.3 ± 0.3 2.5 ± 0.1 1.1 ± 0.1 0.0 0.0 MASH.3 38.8 ± 0.3 3.0 ± 0.2 5.5 ± 0.5 0.0 1.3 ± 0.2 MASH.4 38.8 ± 0.3 2.5 ± 0.2 1.5 ± 0.1 0.0 19 ± 2 MASH.5 39.5 ± 0.0 3.0 ± 0.1 2.0 ± 0.3 6.7 ± 0.2 7.6 ± 0.2 MASH.6 39.5 ± 0.0 2.5 ± 0.3 0.9 ± 0.0 0.0 10.0 ± 1 MASH.7 40.0 ± 0.0 2.5 ± 0.1 4.0 ± 0.5 0.0 5.1 ± 0.4 MASH.8 40.5 ± 0.0 1.5 ± 0.1 0.9 ± 0.1 0.0 0.7 ± 0.04 MASH.9 40.7 ± 0.6 2.0 ± 0.0 0.9± 0.0 13.3 ± 0.5 7.5 ± 1.0 MASH.10 40.8 ± 0.3 2.5 ± 0.1 0.8 ± 0.0 0.0 7.6 ± 0.2 MASH.11 40.8 ± 0.3 1.5 ± 0.0 0.9 ± 0.0 0.0 1.3 ± 0.1 MASH.12 42.0 ± 0.0 1.0 ± 0.1 6.0 ± 1.0 0.0 7.6 ± 0.4 MASH.13 42.8 ± 0.3 3.0 ± 0.1 2.5 ± 0.5 15.0 ± 0.9 1.3 ± 0.2 MASH.14 43.8 ± 0.3 1.5 ± 0.2 5.0 ± 0.8 20.0 ± 1.3 1.3 ± 0.15 MASH.15 45.8 ± 0.3 3.0 ± 0.2 1.0 ± 0.0 0.0 0.71 ± 0.08 MASH.16 46.8 ± 0.3 2.5 ± 0.1 1.0 ± 0.1 6.7 ± 0.4 7.6 ± 0.3 TABLE 2 - Comparison of response levels of the analytical methods applied for the measurement of biosurfactant activity by sixteen biosurfactant producer isolates a b c Strain Surface tension Oil spreading Blood agar lysis MASH.1 ++++ ++++ ++++ MASH.2 ++++ ++++ ++ MASH.3 ++++ ++++ ++++ MASH.4 ++++ ++++ ++ MASH.5 ++++ ++++ +++ MASH.6 ++++ ++++ + MASH.7 ++++ ++++ ++++ MASH.8 +++ +++ + MASH.9 +++ +++ + MASH.10 +++ ++++ + MASH.11 +++ +++ + MASH.12 ++ ++ ++++ MASH.13 ++ ++++ +++ MASH.14 ++ +++ ++++ MASH.15 ++ ++++ ++ MASH.16 ++ ++++ ++ ++: SFT ≥ 42 mN/m, +++: 40 mN/m ≤ SFT ≤ 42 mN/m, ++++: SFT ≤ 40 mN/m. diameter. ++: D < 1.5 mm, +++:1.5 mm ≤ D < 2.5 mm, ++++: D ≥ 2.5 mm. hemolysis, with diameter of lysis. +: incomplete hemolysis, D < 1 cm, ++: complete hemolysis, with 1 cm ≤ D < 2 cm, +++: complete hemolysis, with 2 cm ≤ D < 3 cm, ++++: complete hemolysis, with D ≥ 3 cm. TABLE 3 - Statistical correlations between analytical methods Pearson correlation coefficient (r ) Spearman correlation coefficient (R ) Surface tension Oil spreading Blood agar lysis Surface tension Oil spreading Blood agar lysis Surface tension 1 0.456 0.007 1.000 0.459 0.036 Oil spreading 0.456 1 -0.032 0.459 1.000 0.103 Blood agar lysis 0.007 -0.032 1 0.036 0.103 1.000 558 S. Afshar et al. FIG. 1 - Relationship between the diameter (mm) of the clear zone obtained by the oil spreading method and surface ten- sion (mN/m) of the culture, with two different carbon sources (glucose: open triangles; crude oil: closed circles). Utilisation of the emulsification activity assay as an analytical REFERENCES method for screening biosurfactant producers revealed that five strains (MASH.5, MASH.9, MASH.13, MASH.14 and MASH.16) over Banat I.M. (1993) The isolation of a thermophilic sixteen had some emulsification capacity with n-hexane (Table 1). biosurfactant-producing Bacillus species. Biotechnol Using crude oil instead of n-hexane, the majority (94%) of bacte- Lett., 15: 591-594. rial isolates exhibited some emulsification activity with a maximum Banat I.M. (1995). Biosurfactants production possible EV % of 19%; however, a poor correlation was found with results uses in microbial enhanced oil recovery and oil obtained from surface tension (SFT) experiments as the large pollution remediation: A Review. Bioresource Technol., euclidean distances are 153.076 and 142.476 between SFT and EV 51: 1-12. % for n-hexane and crude oil respectively, calculated by proximity- Benincasa M., Abalos A., Oliveira I., Manresa A. dissimilarity matrix. Thus the emulsification activity assay should (2004). Chemical structure, surface properties and not be considered as a consistent method for the screening of biological activities of the biosurfactant produced by biosurfactant producers as well. Pseudomonas aeruginosa LBI from soapstock. Antonie One-way analysis of variance (F-test with p < 0.05 and error van Leeuwenhoek, 85: 1-8. level A = 0.05) was performed to estimate and compare the mean Bodour A.A., Miller-Maier R.M. (1998). Application of of biosurfactant activity obtained by using different methods. a modified drop-collapse technique for surfactant Statistical tests were used calculate Pearson and Spearman corre- quantitation and screening of biosurfactant- lation coefficients; both Pearson rank correlation coefficient, r , and producing microorganisms. J. Microbiol. Methods, Spearman correlation coefficient, R , can range between -1 (strong 32: 273-280. negative correlation) and 1 (strong positive correlation). All statisti- cal analyses were performed using SPSS (version 12.0) software Carrillo P.G., Mardaraz C., Pitta-Alvarez S.J., Giulietti A.M. package. Table 3 shows the results obtained. Surface tension and (1996). Isolation and selection of biosurfactant-producing oil spreading technique were correlated with a Pearson correlation bacteria. World J. Microbiol. Biotechnol.,12: 82-84. coefficient r = 0.456 and a Spearman correlation coefficient R = Christofi N., Ivshina I.B. (2002). Microbial surfactants and 0.459. A weak negative Pearson (r = -0.032) and a weak positive their use in field studies of soil remediation. J. Appl. Spearman (R = 0.103) correlation coefficient between oil spread- Microbiol., 93: 915-929. ing technique and blood agar lysis were detected. Surface tension Das M., Das S.K., Mukherjee R.K. (1998). Surface active and blood agar lysis showed very weak Pearson (r = 0.007) and properties of the culture filtrates of a Micrococcus Spearman (R = 0.036) correlation coefficients. These analyses species grown on n-alkenes and sugars. Biores. indicate that blood agar lysis is linearly uncorrelated to the other Technol., 63: 231-235. two methods, whereas a linear correlation between surface tension and oil spreading technique exists. Das K., Mukherjee A.K. (2005). Characterization of Taking into account all results, an excellent consistency biochemical properties and biological activities of biosurfactants produced by Pseudomonas aeruginosa between oil spreading technique and SFT methods was obtained. Accordingly, we conclude that oil spreading method is the fastest, mucoid and non-mucoid strains isolated from simplest, time-saving and most reliable technique, to be suggested hydrocarbon-contaminated soil samples. Appl. Microbiol. as an analytical method for screening biosurfactant producers. Biotechnol., 69: 192-199. Ann. Microbiol., 58 (3), 555-560 (2008) 559 Desai J.D., Banat I.M. (1997). Microbial production of McInerney M.J., Javaheri M., Nagle D.N. (1990). 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Selection waste frying oils. J. Appl. Microbiol., 88: 379-387. of microbes producing biosurfactants in media without Harkins W.D., Alexander A.E. (1959). Determination of hydrocarbons. J. Ferment. Technol., 62: 311-314. surface and interfacial tension. In: Physical Methods Mukherjee S., Das P., Sen R. (2006). Towards commercial of Organic Chemistry 1. pp. 757–814, Interscience production of microbial surfactants. Trends Biotechnol., Publishers, Sydney. 24: 509-515. Javaheri M., Jenneman G. E., McInerney M.J., Knapp R.M. Piaza G.A., Zjawiony I., Banat I.M. (2006). Use of (1985). Anaerobic production of a biosurfactant by Bacillus different methods for detection of thermophilic licheniformis JF-2. Appl. Environ. Microbiol., 50: 698-700. biosurfactant-producing bacteria from hydrocarbon- Kosaric N. (1992). Biosurfactants in industry. Pure Appl. contaminated and bioremediated soils. J. Petroleum Chem., 64: 1731-1737. Science and Engineering, 50: 71-77. Maier R.M., Soberón-Chávez G. (2000). Pseudomonas Ron E.Z., Rosenberg E. (2002). Biosurfactants and oil aeruginosa rhamnolipids: biosynthesis and potential bioremediation. Environ. Biotechnol., 13: 249-252. applications. Appl. Microbiol. Biotechnol. 54: 625- Singh P., Cameotra S.S. (2004). Potential applications of microbial surfactants in biomedical sciences. Trends Makkar R.S., Cameotra S.S. (1997). Biosurfactant Biotechnol., 22: 142-146. production by a thermophilic Bacillus subtilis strain. J. Van Dyke M.I., Gulley S.L., Lee H., Trevors J.T. (1993). Ind. Microbiol. Biotechnol., 18: 37-42. Evaluation of microbial surfactants for recovery of Makkar R.S., Cameotra S.S. (1998). Biosurfactant hydrophobic pollutants from soil. J. Ind. Microbiol., production at mesophilic and thermophilic conditions 11: 163-170. by a strain of Bacillus subtilis. J. Ind. Microbiol. Wolin E., Wolin M., Wolf R. (1963). Formation of methane Biotechnol., 20: 48-52. by bacterial extracts. J. Biol. Chem., 238: 2882-2886. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Comparative approach for detection of biosurfactant-producing bacteria isolated from Ahvaz petroleum excavation areas in south of Iran

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Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
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Abstract

Annals of Microbiology, 58 (3) 555-560 (2008) Comparative approach for detection of biosurfactant-producing bacteria isolated from Ahvaz petroleum excavation areas in south of Iran 1§ 1§ 1 2 Shima AFSHAR , Tayebe Bagheri LOTFABAD , Reza ROOSTAAZAD , Abdolhossein Rouholamini NAJAFABADI , Kambiz Akbari NOGHABI * 1 2 Department of Chemical and Petroleum Engineering, Sharif University of Technology; Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences; National Institute of Genetic Engineering and Biotechnology (NIGEB), Karaj-Tehran highway, Pazhoesh Blvd. P.O.Box 14155-6343, Tehran, Iran Received 2 April 2008 / Accepted 15 June 2008 Abstract- The current study was undertaken to compare four analytical methods including drop collapse, oil spreading, surface tension (SFT) measurements, and blood agar lysis tests for detection of biosurfactant-producing bacteria. Among 32 biosurfactant-producing bacteria isolated from Ahvaz oil fields, in south of Iran, 16 isolates (50%) exhibited highest biosurfactant production. Eleven isolates (MASH.1 to MASH.11) demonstrated a reduction in surface tension from 65 mN/m to less than 41 mN/m. The results showed that about 91% of these highly biosurfactant producers had the same response levels of “++++” and “+++” in the case of both SFT and oil spreading methods. Among these, seven isolates had the haemolysis diameter less than 1 cm or between 1 and 2 cm on blood agar. As 64% of the best biosurfactant producers did not completely lyses blood, the ability of biosurfactant-producers for haemolysis may not always be trustworthy. According to our data, there is a good consistency between oil spreading technique and surface ten- sion. As a conclusion, oil spreading method is the fastest, simplest and most consistent analytical method to be suggested for accurate measurements of biosurfactant producers. Key words: biosurfactant, oil spreading, drop collapse, surface tension, blood lysis. INTRODUCTION ability to microorganisms, which is a potential problem for bac- teria. It has been assumed that surfactants would enhance the Bacteria produce a wide range of biosurfactants with diverse bioavailability of hydrophobic compounds (Ron and Rosenberg, and unlike chemical structures (Kosaric, 1992; Christofi and 2002). Because of the potential applications, many biotechno- logical studies have been focused on the mass and commercial Ivshina, 2002; Ron and Rosenberg, 2002; Das and Mukherjee, 2005). Interest in microbial surfactants has progressively been production of biosurfactants. Due to the unique properties and vast array of applications, the need for selection a simple increasing during recent years since they have higher biodegrad- and rapid as well as reliable analytical approach to detect the ability, higher specific activity at extreme temperatures, salinity, and pH levels and considered environmentally compatible with biosurfactant-producers with minimum number of false positives and/or negatives can not be ignored (Mukherjee et al., 2006). limited toxicity (Desai and Banat, 1997; Haba et al., 2000; Several analytical methods have so far been employed for Maier and Soberón-Chávez, 2000; Gautam and Tyagi, 2006). directly measurement of surface activity of biosurfactant. These Biosurfactants are interesting molecules with various biological approaches include surface and/or interfacial tension meas- functions. From a biotechnology prospective, the production of urement, drop shape analysis profile, glass slide test, method biosurfactants is important because of their numerous applica- and the oil spreading technique (Harkins, 1959; Mulligan et tions in environment, agriculture and industry (Banat, 1995; al., 1984; Banat, 1993; Van Dyke et al., 1993; Carrillo et al., Benincasa et al., 2004; Singh and Cameotra, 2004). These kind 1996; Makkar and Cameotra, 1997, 1998; Menezes Bento et of natural microbial compounds are biologically surface-active al., 2005). Each type of these aforesaid methods has their own agents and have many potential advantages over their synthetic advantages and disadvantages. Although enormous efforts have relatives. Microorganisms were considered to be detrimental to been made to develop the analytical methods for accurate meas- the petroleum industry in the past. It is now well known that urement of biosurfactant concentration and its activity, currently they can also be beneficial in terms of oil recovery. The low available reports on the comparison of these methods with each water solubility of many hydrocarbons, especially the polycyclic others to select the most reliable one is limited to a few reports aromatic hydrocarbons (PAHs), is believed to limit their avail- (Bodour and Miller-Maier, 1998; Piaza et al., 2006). The current study was undertaken to compare different analytical methods * Corresponding author. for measurement the activity of biosurfactant produced by bac- Phone: +98-21-44580352; Fax: +98-21-44580399; terial strains with unknown taxonomic affiliations isolated from E-mail: Akbari@nrcgeb.ac.ir, kambizakb@yahoo.com Both authors contributed equally to this work. oil fields in south of Iran. 556 S. Afshar et al. MATERIALS AND METHODS oil surface was inspected visually and its diameter was measured as an indicative of biosurfactant concentration; however, biosur- Media, enrichment and isolation of bacteria. The bacterial factant concentration was also measured, by using a calibration strains were isolated by the enrichment culture technique from curve based on a commercially available biosurfactant, surfactin the crude oil obtained from Ahvaz oil fields. For this purpose, 10 (Sigma, St. Louis, MO). ml crude viscous oil samples were inoculated into 90 ml of sup- plemented nutrient broth (SNB medium) of the same composi- Determination of emulsifying activities. Bacterial isolates tion as described by Francy et al. (1991). The medium incubation which were able to produce biosurfactant were evaluated for was performed at 30 °C on a rotary shaker incubator at 200 rpm emulsifying activity according to the method reported by Das et for 7 days. Then, an aliquot of culture (0.1 ml) was spread on al. (1998). For this purpose, cell-free supernatant was collected Bushnell-Haas agar(Difco, Germany) and incubated at 30 °C for by centrifugation (8000 x g, 20 min) of the culture broth. Then, 48 h. Diluted microbial suspensions in SNB were prepared and 2 ml of supernatant were mixed with 2 ml n-hexane or crude oil cultivated on Brain Heart agar (Merck, Germany) so as to obtain separately in a test tube. The mixture was vigorously stirred for 2 separate colonies. The procedure was repeated three times. min and then allowed to stand for 48 h. Relative emulsion volume Single colonies of each bacterial isolate growing well on Brain (EV, %) was measured according to the following equation: Heart agar (Merck, Germany) were designated according to their morphological and biochemical characteristics. Furthermore all Emulsion height (mm) × cross-section area (mm ) the isolates were examined by using optic microscope (Olympus EV % = Total liquid volume (mm ) Vanox AHBT3). Standard morphological and biochemical tests were used for their preliminary characterization. Among 32 bacterial strains, sixteen biosurfactant producer isolates were RESULTS AND DISCUSSION selected according to following method and maintained on nutrient agar (Merck, Germany) and Brain Heart agar slants at Table 1 shows the results of four methods that were used to 4 °C. To assess the biosurfactant production efficiency by dif- detect biosurfactant production by different isolates. Over sixteen ferent bacterial isolates, a 100 ml nutrient broth medium was isolates, seven (44%) could reduce the surface tension to 40 inoculated with a single colony of each bacterial isolates and mN/m or less; four (25%) isolates had a SFT between 40 and 42 incubated at 30 °C, 150 rpm for 24 h as seed culture. Then mN/m and five isolates (31%) exhibited a SFT of 42 mN/m or 2% (V/V) seed culture was transferred to a 500 ml Erlenmeyer higher. Oil spreading method resulted in the same response level flask containing 250 ml of E medium (Javaheri et al., 1985) with as for SFT for eleven of sixteen isolates (69%). the following components (g/l): KH PO , 2.7; K HPO , 13.9; Results in Table 2 show a good consistency between oil spreading 2 4 2 4 NaCl, 50; yeast extract, 1; NaNO , 1; (NH4) SO , 1; and 10 technique and surface tension with a Pearson rank correlation coef- 3 2 4 ml of a trace metal solution; 10 g/l glucose or crude oil were ficient, r = 0.456, and Spearman correlation coefficient, R = 0.459, added according to the experiment, to study the effect of carbon ranged between -1 (strong negative correlation) and 1 (strong posi- source on the biosurfactant production and activity. The metal tive correlation) (Table 3). Figure 1 show the relationship between solution was a modification of Wolins metal solution (Wolin et the diameter (mm) of clear zone obtained by the oil spreading al., 1963) and had the following composition (g/l): EDTA, 1; method and surface tension (mN/m) of the culture obtained by MnSO ·H O, 3; FeSO ·7H O, 0.1; CaCl ·2H O, 0.1; CoCl ·2H O, using glucose or crude oil as carbon source is consistent with the fact 4 2 4 2 2 2 2 2 0.1; ZnSO ·2H O, 0.1; CuSO ·5H O, 0.01; AlK(SO ) , 0.01; that crude oil, as a non-soluble carbon source, is better than glucose 4 2 4 2 4 2 H BO , 0.01; Na MoO ·7H O, 0.01; and MgSO , 25. (soluble carbon source) for biosurfactant production. 3 4 2 4 2 4 In the case of blood agar lysis test, as a simple and easy Biosurfactant production assay. Bacterial isolates were grown method, all the sixteen biosurfactant-producing strains were able in E medium with either glucose or crude oil as interchangeable to lyse the blood agar (Table 2). However, a consistent correla- carbon sources. The culture media were incubated at 30 °C in a tion between this test and other analytical techniques were not rotary shaker (150 rpm) for 7 days. After that, cell-free super- achieved. Complete haemolysis with a diameter > 3 cm did not natant was obtained using shear force of centrifugation (8000 x occur necessarily in the higher biosurfactant-producing isolates. g, 20 min) and collected in centrifuge tube. In addition, samples For instance; strains MASH.12 and MASH.14 could be able to lyse from culture medium containing whole cells were obtained for blood agar completely (++++), with a diameter of lysis > 3 cm, measurement of surface-active properties. but the results obtained from oil spreading technique or surface The surface tension of the samples was measured by the tension for these strains showed low activity. In contrast a weaker Ring method using a KRUSS du Nouy Tensiometer K6 at room haemolysis, with a response level of “+” or “++”, occurred for 64% temperature (McInerney et al., 1990). of the eleven strains which showed a good capability in producing Drop collapse measurement method was carried out accord- a considerable amount of biosurfactant as checked by oil spreading ing to Bodour and Miller-Maier (1998). Each bacterial isolate method (Table 2); 60% of isolates that did not exhibit a good SFT was streaked on blood agar and incubated at 37 °C for 48 h. (++) could lyse blood agar with response levels of “++++” and The plates were visually checked for the existence of clearing “+++”. Different response levels were found for haemolysis and zone around the colonies as a criterion for biosurfactant produc- oil spreading for strains MASH.12 to MASH.16. It was previously tion. With this goal, the clear zones were measured and scored reported that blood agar lysis test could be used as a simple and according to the diameter. Oil spreading measurement was done quick method for preliminary screening of biosurfactant producers by addition of 20 ml of distilled water to a Petri dish (10 cm diam- (Mulligan et al., 1984; Banat, 1993; Carrillo et al., 1996). This is a eter). Then 8 μl of crude oil was transferred to the surface of the marked contrast with our finding. Among sixteen isolates, just five water using a HPLC syringe and left to stand for a while. After strains (31%) could lyse blood agar completely (++++). Blood that, 10 μl of each bacterial isolate culture media were separately agar lysis method had a high percentage of either false-negatives added to the surface of oil; this technique was a modification of or false-positives. Therefore, blood agar lysis test is not suggested the Morikawa et al. (2000). The existence of clear zones on the as a reliable method for screening biosurfactant producers. Ann. Microbiol., 58 (3), 555-560 (2008) 557 TABLE 1 - Efficacy of the four analytical methods in predicting biosurfactant production Strain Surface tension Clear zone diameter Blood agar lysis EV % EV % (mN/m) by oil spreading (mm) diameter (cm) n-Hexane Crude Oil MASH.1 38.5 ± 0.0 3.0 ± 0.1 8.0 ± 1.0 0.0 11.45 ± 0.45 MASH.2 38.3 ± 0.3 2.5 ± 0.1 1.1 ± 0.1 0.0 0.0 MASH.3 38.8 ± 0.3 3.0 ± 0.2 5.5 ± 0.5 0.0 1.3 ± 0.2 MASH.4 38.8 ± 0.3 2.5 ± 0.2 1.5 ± 0.1 0.0 19 ± 2 MASH.5 39.5 ± 0.0 3.0 ± 0.1 2.0 ± 0.3 6.7 ± 0.2 7.6 ± 0.2 MASH.6 39.5 ± 0.0 2.5 ± 0.3 0.9 ± 0.0 0.0 10.0 ± 1 MASH.7 40.0 ± 0.0 2.5 ± 0.1 4.0 ± 0.5 0.0 5.1 ± 0.4 MASH.8 40.5 ± 0.0 1.5 ± 0.1 0.9 ± 0.1 0.0 0.7 ± 0.04 MASH.9 40.7 ± 0.6 2.0 ± 0.0 0.9± 0.0 13.3 ± 0.5 7.5 ± 1.0 MASH.10 40.8 ± 0.3 2.5 ± 0.1 0.8 ± 0.0 0.0 7.6 ± 0.2 MASH.11 40.8 ± 0.3 1.5 ± 0.0 0.9 ± 0.0 0.0 1.3 ± 0.1 MASH.12 42.0 ± 0.0 1.0 ± 0.1 6.0 ± 1.0 0.0 7.6 ± 0.4 MASH.13 42.8 ± 0.3 3.0 ± 0.1 2.5 ± 0.5 15.0 ± 0.9 1.3 ± 0.2 MASH.14 43.8 ± 0.3 1.5 ± 0.2 5.0 ± 0.8 20.0 ± 1.3 1.3 ± 0.15 MASH.15 45.8 ± 0.3 3.0 ± 0.2 1.0 ± 0.0 0.0 0.71 ± 0.08 MASH.16 46.8 ± 0.3 2.5 ± 0.1 1.0 ± 0.1 6.7 ± 0.4 7.6 ± 0.3 TABLE 2 - Comparison of response levels of the analytical methods applied for the measurement of biosurfactant activity by sixteen biosurfactant producer isolates a b c Strain Surface tension Oil spreading Blood agar lysis MASH.1 ++++ ++++ ++++ MASH.2 ++++ ++++ ++ MASH.3 ++++ ++++ ++++ MASH.4 ++++ ++++ ++ MASH.5 ++++ ++++ +++ MASH.6 ++++ ++++ + MASH.7 ++++ ++++ ++++ MASH.8 +++ +++ + MASH.9 +++ +++ + MASH.10 +++ ++++ + MASH.11 +++ +++ + MASH.12 ++ ++ ++++ MASH.13 ++ ++++ +++ MASH.14 ++ +++ ++++ MASH.15 ++ ++++ ++ MASH.16 ++ ++++ ++ ++: SFT ≥ 42 mN/m, +++: 40 mN/m ≤ SFT ≤ 42 mN/m, ++++: SFT ≤ 40 mN/m. diameter. ++: D < 1.5 mm, +++:1.5 mm ≤ D < 2.5 mm, ++++: D ≥ 2.5 mm. hemolysis, with diameter of lysis. +: incomplete hemolysis, D < 1 cm, ++: complete hemolysis, with 1 cm ≤ D < 2 cm, +++: complete hemolysis, with 2 cm ≤ D < 3 cm, ++++: complete hemolysis, with D ≥ 3 cm. TABLE 3 - Statistical correlations between analytical methods Pearson correlation coefficient (r ) Spearman correlation coefficient (R ) Surface tension Oil spreading Blood agar lysis Surface tension Oil spreading Blood agar lysis Surface tension 1 0.456 0.007 1.000 0.459 0.036 Oil spreading 0.456 1 -0.032 0.459 1.000 0.103 Blood agar lysis 0.007 -0.032 1 0.036 0.103 1.000 558 S. Afshar et al. FIG. 1 - Relationship between the diameter (mm) of the clear zone obtained by the oil spreading method and surface ten- sion (mN/m) of the culture, with two different carbon sources (glucose: open triangles; crude oil: closed circles). Utilisation of the emulsification activity assay as an analytical REFERENCES method for screening biosurfactant producers revealed that five strains (MASH.5, MASH.9, MASH.13, MASH.14 and MASH.16) over Banat I.M. (1993) The isolation of a thermophilic sixteen had some emulsification capacity with n-hexane (Table 1). biosurfactant-producing Bacillus species. 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Published: Nov 24, 2009

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