Ann Microbiol (2011) 61:605613 DOI 10.

1007/s13213-010-0179-0

ORIGINAL ARTICLE

Response surface methodology (Box-Behnken) to improve a liquid media formulation to produce biosurfactant and phenanthrene removal by Pseudomonas putida
Angeles Martnez-Toledo & Refugio Rodrguez-Vzquez

Received: 24 August 2010 / Accepted: 29 November 2010 / Published online: 21 December 2010 # Springer-Verlag and the University of Milan 2010

Abstract A culture medium formulation was established using a Box-Behnken experimental design aimed at enhancing biosurfactant production and phenanthrene removal by Pseudomonas putida CB-100. The independent variables selected (+, 0, , levels) were: glucose (9.1, 13.6, and 18.2 g/l), NH4Cl (0.5, 0.75, and 1.0 g/l), and yeast extract (0.005, 0.0075, and 0.01 g/l), with 200 mg/l phenanthrene at 37C, shaken at 150 rpm, at pH 7, for 5 days. Analyses of results by one-way analysis of variance showed that biosurfactant production was improved by the glucose ammonium chloride interaction at high values (p<0.004). Phenanthrene removal was enhanced by the ammonium chloride-yeast extract interaction at low and high values, respectively (p<0.02). The highest phenanthrene removal (82.4%) was observed in formulation 11, which had a C:N ratio of 5:1 and a C:P ratio of 10:1. In addition, 47.5 mN/m surface tension, 20% emulsion capacity, and 23.5 mg/l biosurfactant production were obtained. With this medium, we followed the kinetic growth of P. putida for 118 h, the culture conditions were the same as those used in the experimental design. At 46 h, we obtained 57.8% phenanthrene removal and 27 mg/l biosurfactant production. The critical micelle concentration of the biosurfactant was 430 mg/l. The biosurfactant produced by P. putida was characterized as a rhamnolipid type.
A. Martnez-Toledo Facultad de Ingeniera, Universidad Autnoma de San Luis Potos, Av. Dr. Manuel Nava # 8, Zona Universitaria, C.P. 78290 San Luis Potos, SLP, Mexico R. Rodrguez-Vzquez (*) Departamento de Biotecnologa y Bioingeniera, Centro de Investigacin y de Estudios Avanzados del IPN, Av. Instituto Politcnico Nacional #2508, Col. San Pedro Zacatenco C. P. 07360 DF, Mexico e-mail: rrodrig@cinvestav.mx

Keywords Cell culture . Experimental design . HPLC . Kinetic . Rhamnolipid

Introduction In 2008, several cities in Mexico were reported to have areas that were severely polluted with different toxic organic compounds, such as pesticides, dioxins, polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), etc. (SEMARNAT 2008). PAHs, products of industrial processes that result from incomplete combustion of biomass and fossil fuels, are hydrophobic, recalcitrant, and toxic to the environment. The United States Environmental Protection Agency lists PAHs as priority pollutants that must be removed from contaminated soils due to their mutagenic and carcinogenic characteristics (ATSDR 1995). Bioremediation methodologies with microorganisms are widely applied for the removal of toxic organic compounds from the environment (Hardman 1991; Hwang and Cutright 2002). PAHs form epoxides through the addition of a molecule of oxygen (Cerniglia 1984) in the metabolism of fungi and bacteria in which oxygenases have an important role. These epoxides are highly reactive and carcinogenic because they can be substituted for the 5 phosphate in the DNA chain. Phenanthrene is a model compound for PAH degradation (Samanta et al. 2002) Pseudomonades are isolated from polluted environments and are used in many bioremediation studies because of their ability to degrade or transform several toxic compounds (Ambramowicz 1990; Perales and Harwood 2002). Pseudomonas putida, in particular, has a great metabolic capacity because, it used as an energy source many compounds, including recalcitrant organic compounds (Walker and Keasling 2002; Wackett 2003) but it is also able

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to produce molecules (intra- and extracellular) that function as biosurfactants (Bongolo 1999; Borjana et al. 2002; AmzcuaVega et al. 2004) which are of interest in bioremediation of environments contaminated with different hydrophobic compounds, such as PCBs (Golyshin et al. 1999) and PAHs (Zhang et al. 1997; Barkay et al. 1999; Schippers et al. 2000). Rhamnolipids, produced by Pseudomonas aeruginosa, have a molecular structure that contains one or two molecules of hydroxydecanoic acid (Cameotra and Makkar 1998) and are one type of biosurfactant employed in the bioremediation of environments contaminated with diverse toxicants (Volkering et al. 1998) The objective of the present work was to improve the culture medium formulation for the removal of phenanthrene and production of rhamnolipids by P. putida, for future application in bioremediation systems. For this purpose, a response surface methodology (RSM) was selected; this tool is used when only a few significant factors are involved in optimization. The Box-Behnken design (a type of RSM) is an independent rotatable or nearly rotatable quadratic design; it requires fewer runs (15) in a three-factor (variables) experimental design. In addition, it creates empirical model equations that correlate the relationship between the variables and the response (Myers 1971) Further, we aimed to observe the kinetic growth of P. putida to determine glucose consumption through phenanthrene removal at the time that biosurfactant is produced, as well as to identify the biosurfactant using high performance liquid chromatography (HPLC). These findings could provide information about the optimal time to use P. putida to produce a higher yield of biosurfactant, thereby improving phenanthrene removal.

analyzed with gas chromatographyflame ionization detector), and emulsion capacity (percentage of the emulsion layer of the cell-free media mixed with diesel fuel). The abiotic controls for the experimental design and kinetic studies were performed in sterile culture media with 200 mg/l phenanthrene and without any inoculum. We used the destructive bottle sampling method for the kinetic study. Box-Behnken experimental design The Box-Behnken experimental design (Montgomery 1991) was performed with three factors at three levels (+1, 0, 1): glucose (18.2, 13.6, and 9.1 g/l); yeast extract (0.01, 0.0075, and 0.005 g/l); and ammonium chloride (1.0, 0.75, and 0.5 g/l); the coded and natural variables are presented in Table 1. Other components (g/l) of the liquid media were: Fe2SO47H2O (0.0005), FeCl36H2O (0.0049), KH2PO4 (0.2), K2HPO4 (2.0), MgSO47H2O (1.0) NaNO3 (5.0) KCl (1.1), NaCl (1.1), CaCl22H2O (0.05), ZnSO47H2O (0.006), MnSO4H2O (0.006), CoCl26H2O (0.0006), CuSO45H2O (0.0006), and 200 mg/l phenanthrene. Phenanthrene was dissolved in acetone, 200 mg/l was added to a sterilized flask and the acetone was allowed to evaporate before adding the sterilized medium. Culture conditions were: pH 7, shaken at 150 rpm, 10% (v/v) inoculum, 37C, for 5 days. Thirteen different liquid media (according to the experimental design) without phenanthrene were formulated to produce the initial inoculum under the same culture conditions.

Table 1 Media formulation obtained from Box-Behnken design (coded and natural variables) Formulatin Coded variables X1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 X2 1 1 1 1 0 0 0 0 1 1 1 1 0 0 0 X3 0 0 0 0 1 1 1 1 1 1 1 1 0 0 0 Natural variables (g/l) Glucose 9.1 9.1 18.2 18.2 9.1 9.1 18.2 18.2 13.6 13.6 13.6 13.6 13.6 13.6 13.6 Yeast extract 0.005 0.01 0.005 0.01 0.0075 0.0075 0.0075 0.0075 0.005 0.005 0.01 0.01 0.0075 0.0075 0.0075 NH4Cl 0.75 0.75 0.75 0.75 0.5 1 0.5 1 0.5 1 0.5 1 0.75 0.75 0.75

Materials and methods The following experimental procedures were performed based on a previous report (Martnez-Toledo et al. 2006). Strain maintenance (pregrown on TSAtryptic soy casein in Petri dishes), biomass quantification (10 ml of cell-free culture media were using to weigh the biomass),surface tension measurements (a DuNoy tensiometer was used to analyze 20 ml of cell-free culture media), extraction and quantification of phenanthrene (liquidliquid extraction with dichloromethane was used to extract phenanthrene from 30 ml of cell-free culture media, and then the extract was analyzed by HPLC), biosurfactant extraction (acetone was used to precipitate the biosurfactant from 50 ml of cellfree media, the precipitate was collected by Millipore filtration), destructive analysis of the biosurfactant (the biosurfactant was hydrolyzed, extracted with hexane, and then fatty acid methyl esters in the non-polar fraction and trimethylsilylated-methyl sugars in the polar fraction were

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The design includes three replicates (formulations 1315), which were the central point and were used to estimate the pure error sum of squares in the statistical analyses. A BoxBehnken statistical screening design was used to optimize and evaluate the main effects, interaction effects, and quadratic effects of the independent variables. A second-order polynomial (quadratic polynomial) model was fitted to the response data obtained from the design. The polynomial equation proposed for the response (Y1 to Y4) was:
Yi b0 b1 X1 b2 X2 b3 X3 b11 X1 2 b22 X2 2 b33 X3 2 b12 X1 X2 b13 X1 X3 b23 X2 X3 b123 X1 X2 X3 "

Where Yi (i=1 to 4) are the predicted values for change in surface tension (ST), biomass (BIOM), biosurfactant production (BP), and phenanthrene removal (PR), respectively. 0 is an intercept; X1(glucose), X2(yeast extract), and X3(ammonium chloride) are the independent variables; 1, 2, and 3 are the linear coefficients; 11, 22, and 33 are the quadratic coefficients; and 12, 13, 23, and 123 are the cross-product coefficients. Kinetics The culture medium selected from the experimental design for the kinetic study was the one in which P. putida could remove the highest percentage of phenanthrene. The liquid media composition was formulated with (g/l): glucose (13.6); yeast extract (0.01); NH4Cl (0.5); Fe2SO47H2O (0.0005); KH2PO4 (0.2); K2HPO4 (2.0); MgSO47H2O (1.0); NaNO3 (5.0); FeCl36H2O (0.0049); KCl (1.1); NaCl (1.1); CaCl22H2O (0.05); ZnSO47H2O (0.006); MnSO47H2O (0.006); CoCl26H2O (0.0006); CuSO45H2O (0.0006), and 200 mg/l phenanthrene. The culture conditions were: pH 7, shaken at 150 rpm, at 37C, for 5 days. The inoculum was added at 10% (v/v). Three replicates were used for each experimental unit. The initial inoculum was obtained with the liquid media without phenanthrene under the same culture conditions. Kinetics were monitored at 2, 6, 10, 14, 22, 34, 46, 70, 94, and 118 h after inoculation. The variables analyzed were: biomass, glucose consumption, biosurfactant production, and surface tension. Biosurfactants were analyzed by HPLC, which permits analysis of the entire molecule; the critical micelle concentration (CMC) was determined in the cell-free media with the maximum surface tension change, which was obtained at 46 h of the kinetic study. Non-destructive analysis of the biosurfactant The biosurfactant extracted from the cell-free liquid cultures was dried under a nitrogen stream to eliminate the solvent. The biosurfactant was then mixed with 400 l ethyl acetate, 125 l p-bromoacetophenone, and 75 mg

triethylamine in reaction vials. The mixture was heated at 70C for 1 h, then cooled to ambient temperature (25 2.0C), and analyzed with HPLC; the samples that could not be analyzed on the same day were stored at 20C, as reported by Schenk et al. (1995). HPLC analysis was performed in a Varian chromatograph, with a VIDAC reversed phase column 201TP54 (5 m, 250 mm4.6 mm), an inert pump (Varian, Model 9012), automatic sample injector (Varian, Model 9300), and diode-array detector (Varian, Polychrom Model 9065). The mobile phase comprised water (solvent A) and acetonitrile (solvent B); the gradient elution profile was solvents A:B at 30:70 from 0 to 7 min, at 0:100 from 8 to 16 min, and at 30:70 from 17 to 18 min. The injection volume was 50 l, with a flow rate of 0.8 ml/min at 230 and 320 nm wavelengths. Critical micelle concentration determination The CMC was determined as reported by Sustersick-Caas (2004). Briefly, dilutions of the cell-free culture were made. Each dilution was then scanned in a UV-vis Perkin-Elmer chromatograph (Model Lamda 3), the absorbance scale was set from 0 to 1 to find the wavelength at which the dilutions exhibited maximum absorbance. Once the optimal wavelength was selected, dilutions versus their transmittances were plotted on a graph. In this graph, lines from the lowest point to the highest point and vice versa were drawn, and the CMC was determined from their intersection, as reported by Sheppard and Mulligan (1987) Glucose analysis Glucose was analyzed in the cell-free culture from the kinetic study. Measurements were performed with an HPLC using a calibrated standard glucose solution. The analytical chromatographic system comprised an LDC analytical pump, an infrared detector (Model 350), and an integrator (Varian, Model 4400). Chromatography was performed with a Benson Carbohydrate Column Ca2+ at 45C with a flow rate of 4 ml/min. The injection volume was 50 l. The mobile phase was water: acetonitrile (80:20; v/v) with an isocratic gradient. Statistical analysis was performed using the SAS (Statistical Analysis Systems) version 6.01 and Design Expert software system. All data were analyzed with oneway analysis of variance (ANOVA), and a p value of less than 0.05 was considered statistically significant.

Results and discussion Box-Behnken results The results of the Box-Behnken experimental design are presented in Table 2. SAS and Design expert software was

608 Table 2 Biosurfactant concentration, phenanthrene removal, and biomass production by P. putida obtained in the BoxBehnken experimental design Treatment 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Standard deviation STi (mN/m) 74.0 75.3 76.0 74.5 69.0 71.6 75.0 73.8 75.8 74.6 66.5 66.0 66.6 66.6 66.6 STf (mN/m) 59.8 64.9 72.3 73.0 52.3 63.0 58.1 59.5 61.0 68.1 45.7 56.3 57.4 56.9 54.9 1.32

Ann Microbiol (2011) 61:605613 NCG (mg/l) 0.0a 0.1 0.0a 0.0a 0.1 0.1 0.0a 0.0a 0.2 0.0a 0.5 0.1 0.2 0.3 0.3 0.058 BP (mg/l) 19.0 7.3 14.7 15.6 26.7 13.1 8.3 25.0 25.8 25.8 23.5 22.0 7.6 7.2 13.8 3.79 PR (%) 54.2 42.8 40.0 21.5 15.9 67.4 40.3 32.5 42.9 44.7 82.4 20.4 50.5 57.5 60.4 4.58

STi initial surface tension; STf final surface tension; NCG net cellular growth; BP biosurfactant production; PR phenanthrene removal
Non-cellular growth was observed; only the initial inoculum remained
a

used to fit the polynomial quadratic model for the response variables through multiple regression analysis. The ANOVA analysis (Table 3) showed that glucose, yeast extract, and ammonium chloride had significant effects (p<0.0143) only in biosurfactant production, the quadratic component of ammonium chloride had the highest significant effect (p<0.0019) on biosurfactant production, supporting the hypothesis that the variables examined induced biosurfactant production in P. putida. The regression analysis produced the following equation with R=0.94 and p<0.01 for biosurfactant production:
YBP 9:53 0:31X1 2:11X2 0:20X3 3:15X1 X2 7:57X1 X3 0:37X2 X3 0:69X1 2 5:31X2 2 9:43X3 2

The polynomial equation represents the quantitative effect of process variable (X1, X2, and X3) and their
Table 3 ANOVA results of the Box-Behnken experimental design to improve biosurfactant production (BP) Source Model X1 X2 X3 X1X2 X1X3 X2X3 X1X1 X2X2 X3X3 Pure error Corrected total df 9 1 1 1 1 1 1 1 1 1 2 14

interactions on the response YBP. The values of the coefficients X1, X2, and X3 are related to the effect of these variables on the response YBP. Coefficients with more than 1 factor term and those with higher order terms represent interaction terms and quadratic relationship, respectively. A positive value represents an effect that favors the optimization, while a negative value indicates an antagonistic effect. The values of X1, X2, and X3 were substituted in the equation to obtain the theoretical values of YBP. The predicted values and the observed values were found to be in good agreement. Phenanthrene removal was higher at the low glucose concentration (9.1 g/l). Biomass production was low, but the statistical analysis indicated a significant correlation between phenanthrene removal and biomass production (R=0.68, p<0.005). Phenanthrene removal was not correlated with biosurfactant production. This last result was similar to that obtained by Martnez-Toledo et al.

Sum of squares 722.228 0.78 35.70 0.32 39.69 229.52 0.56 1.77 104.04 328.57 27.93 771.09

Mean square 80.247 0.78 35.70 0.32 39.69 228.52 0.56 1.77 104.04 328.57 13.69

F value 8.64 0.084 3.83 0.034 4.26 24.64 0.06 0.19 11.17 35.25

Pr > F 0.01a 0.78 0.11 0.86 0.09 0.004a 0.82 0.68 0.02a 0.001a

a Significant at 5% level, R2 =0.9396, adj-R2 =0.8309, C.V=17.92, BP Mean=17.03

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(2006) who reported that biosurfactant production increased at the same time as phenanthrene removal diminished. Willumsen and Karlson (1997) reported that PAH mineralization was not correlated with decreased surface tension and emulsification capacity. The response surface plot revealed (Fig. 1a) a maximum biosurfactant production, with the high ammonium chloride concentration and the low glucose concentration. Lang and Wullbrandt (1999) reported that P. putida produces rhamnolipids under limited nitrogen conditions. Our present findings suggest that P. putida behaved differently in the liquid culture because of the toxic effects of phenanthrene. Nevertheless, we observed that nitrogen had a greater effect on P. putida biosurfactant production than the other nutrients, just as in other microorganisms, e.g., Pseudomonas aeruginosa, Rhodococcus erythropolis, Acinetobacter calcoaceticus, and Bacillus subtilis (Guerra-Santos et al. 1984; Desai and Banat 1997; Davis

et al. 1999). Therefore, P. putida not only consumed some nutrients to support the toxic media (through biosurfactant production) but also used phenanthrene as a carbon source, despite the sufficient concentration of glucose. Ammonium and yeast extract were the nutrients preferred by P. putida to produce biosurfactant. The surface plot (Fig. 1b) shows the effect of the interaction ammonium chloride and yeast extract on phenanthrene removal. Maximal phenanthrene removal was obtained at high and low amounts of yeast extract and ammonium chloride respectively (p <0.02) according with the analysis of variance (Table 4). The quadratic model fitted (with coded values) is given by the follow equation:
YPR 56:13 2:62X1 3:8X2 2:0X3 15:65X3 1:77X1 X2 14:83X1 X3 15:95X2 X3 12:54X1 2 3:97X2 2 4:57X3 2 3 11:28X1 2 X2 25:98X1 2 X3 6:25X1 X2 2

In this case X3, X1X3, X2X3, X12, X12X3 are significant model terms. YPR predicted values and the YPR observed values were found to be in good agreement. In the experimental units obtained from the experimental design, the C:N ratio ranged from 6:1 to 3:1 and the C:P ratio ranged from 7:1 to 13:1. The best formulations for phenanthrene removal were: a) treatment 1, with a C: N ratio of 3:1 and a C:P ratio of 7:1; b) treatment 11 with a C:N ratio of 5:1 and a C:P ratio of 10:1; and c) treatment 13, with a C:N ratio of 4:1 and a C:P ratio of 10:1. The C:P ratio of formulations 11 and 13 was similar to that used by Guerra-Santos et al. (1984), who reported this ratio as the best condition for the production of rhamnolipids by P. aeruginosa; P. putida exhibited a similar behavior in the present study. Nevertheless, the conditions required for biosurfactant production were different from those needed for pollutant removal by this microorganism, according to (Chayabutra and Ju 2001). Our results showed that P. putida required only a low concentration of glucose, but needed the total phosphorus concentration to enhance phenanthrene removal. Analyses of polar and non-polar fractions of the biosurfactant The non-polar fraction of the biosurfactant produced with all the medium formulations of the experimental design was composed of fatty acids (Table 5), the highest concentrations were palmitic acid (from 2 to 16.9%), arachidic acid (from 12.2 to 63.8%), and behenic acid (from 9.2 to 78.3%); fatty acids with a higher molecular weight were present in higher concentrations than those with a lower molecular weight. Only formulation 9 produced lauric acid (22.7%). We observed an increase in high-molecular weight

Fig. 1 Response surface plots (coded values) showing the effect of interaction a glucoseammonium chloride on biosurfactant production, and b ammonium chlorideyeast extract on phenanthrene removal

610 Table 4 ANOVA results of the Box-Behnken experimental design to improve Phenanthrene removal (PR) Source Model X1 X2 X3 X1X2 X1X3 X2X3 X1X1 X2X2 X3X3 X12X2 X12X3 X1X22 Pure error Corrected total df 12 1 1 1 1 1 1 1 1 1 1 1 1 2 14 Sum of squares 4,573.74 27.56 57.76 906.01 12.6 879.12 1,017.61 580.78 58.10 77.0 254.25 1,349.40 78.13 51.18 4,625.55

Ann Microbiol (2011) 61:605613 Mean square 381.15 27.56 57.76 906.01 12.6 879.12 1,017.61 580.78 58.10 77.0 254.25 1,349.40 78.13 25.9 F value 14.71 1.06 2.23 34.98 0.49 33.94 39.28 22.42 2.24 2.97 9.82 52.09 3.02 Pr > F 0.06 0.41 0.27 0.02a 0.55 0.02a 0.02a 0.04a 0.27 0.22 0.09 0.02a 0.22

a Significant at 5% level, R2 =0.9888, adj-R2 =0.9216, C.V=11.34, PR Mean=44.89

fatty acids in this study as compared with those obtained in previous reports (Amzcua-Vega et al. 2004; MartnezToledo et al. 2006). The fatty acid composition of the biosurfactant produced in formulation 11 (the best for phenanthrene removal) was: 6.2% palmitic acid, 8.5% stearic acid, 7.2% linoleic acid, 31.4% arachidic acid, 31.6% behenic acid, and 15.1% unidentified fatty acids. These concentrations did not correlate with the results of the response variables, nor with the nutrients or their concentrations. Nevertheless, the relative concentrations (obtained from the chromatogram area) of rhamnose were higher than those obtained in previous reports (AmzcuaTable 5 Relative abundance of the polar and non-polar fractions of the rhamnolipids produced by P. putida in different medium formulations obtained in Box-Behnken experimental design

Vega et al. 2004; Martnez-Toledo et al. 2006). The relative concentration observed in the biosurfactant ranged from 7.6 to 97.7%. The highest relative concentration of rhamnose was observed in the biosurfactant obtained with treatment 6 (Table 5). Thus, the culture formulation induced P. putida to produce rhamnolipids with different molecular structures in the non-polar fraction as well as differences in the quantity; due to, the ratio between the carbon and phosphorus in the culture formulation. The media with P. putida, could be applied for bioremediation tests like biopiles with bioaugmentation and biostimulation treatments.

Formulation

Rhamnose (% chromatogram area)

Fatty acids (% chromatogram area) C16:0 C18:0 0.8 nd nd 16.1 7.8 3.7 6.2 3.4 nd nd 8.5 nd 3.5 2.7 3.9 1.5 C18:2n6c nd 4.5 nd 2.6 3.2 4.6 12.6 nd nd nd 7.2 nd nd nd nd C20:0 59.5 12.7 59.4 11.8 35.9 33.6 42.3 63.8 31.6 12.2 31.4 36.0 22.2 19.7 9.8 6.5 C22:0 15.2 Nd 21.3 16.9 15.2 9.2 9.6 24.5 nd 78.3 31.6 50.4 8.64 13.9 17.6 4.5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 SDT

90.2 73.7 52.8 91.6 16.2 97.7 7.6 36.4 94.4 87.5 83.6 42.8 66.9 61.1 92.9 16.9

4.9 16.9 3.5 8.9 6.7 13.1 15.5 5.3 10.2 4.6 6.2 2.0 3.9 3.5 2.7 0.66

C16:0 = Palmitic acid, C18:0 = Stearic acid, C18:2n6c = Linoleic acid, C20:0 = Arachidic acid, C22:0 = Behenic acid nd Not detected, SDT standard deviation

Ann Microbiol (2011) 61:605613 Fig. 2 Kinetic growth of P. putida

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Time course of cell growth in the kinetic study In the kinetics study, the biomass concentration was 0.33 mg/l at 2 h, maximum cellular growth occurred at 6 h (0.46 mg/l), and then it stabilized at 0.26 mg/l from 34 h until the end of the study. In liquid culture with PAHs in crystal form, bacterial growth fits a first-order exponential, then becomes linear, with the dissolution of the PAH crystals being the limiting growth factor (Volkering et al. 1992) our findings were consistent with this behavior. Fast phenanthrene removal was observed for up to 10 h (Fig. 2), then no relevant changes were observed until 70 h. The general linear model analysis showed that phenanthrene removal was significantly (p<0.0001) affected by time; the least significant difference results revealed no significant differences in phenanthrene removal among the last five time points. The maximum phenanthrene removal was 56.9% at 70 h. PAHs can be trapped in synthetic surfactants when the CMC in the medium is exceeded, which reduces

their bioavailability to the microorganisms, thereby stopping PAH removal as well as biomass production (Volkering et al. 1995). It is possible that the CMC in the liquid culture was exceeded at 46 h, because after that time, the change in variable response was no longer significant. At the initial time of the kinetic studies, glucose consumption was slow, but at 14 h, its consumption increased to 17.5%. At 70 h, 39% of the glucose was consumed (Fig. 2). Glucose consumption positively correlated with phenanthrene removal and negatively correlated with the biomass (R=0.75, p<0.001; R=0.43, p<0.02, respectively). Therefore, phenanthrene is co-metabolically consumed with glucose by P. putida; this result is consistent with that of Madigan et al. (2004). Production of surface tension agents during kinetic growth The maximal biosurfactant production (50 mg/l) was reached at 10 h, then it decreased, and finally, at 70 h, the production was constant (Fig. 2). When phenanthrene removal was high, biosurfactant production was low; this result is similar to that reported by Martnez-Toledo et al. (2006). The biosurfactant appeared to be consumed as a cosubstrate by P. putida, which allowed for continued growth and biosurfactant production. Other authors (Noordman et al. 1998; Volkering et al. 1998) observed that the biosurfactant was consumed as the primary substrate with subsequent co-metabolic biodegradation of the pollutants. The results also indicated a maximum surface tension decrease at 10 h (23.1 mN/m), after that, the surface tension continued to decrease; at 46 h, the surface tension decreased to 18.7 mN/m (Fig. 2). The final surface tension correlated with the biomass (R=0.45, p<0.02). Phenanthrene removal and cellular growth were stopped when the surface tension and the biosurfactant production remained the same. Similar results were obtained by Amzcua-Vega et al. (2004), who reported that when the surface tension

Fig. 3 Spectrum of the molecular structure of biosurfactant produced by P. putida

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had its maximum change, a subsequent phase without change occurred. If the media inoculated with P. putida was applied in bioremediation tests, the results could be observed after 10 h, and re-inoculation might not be necessary until 46 h. The CMC was determined in liquid culture medium that presented the maximum surface tension decrease (18.7 mN/m); 27 mg/l biosurfactant was produced in this culture medium. For this purpose, a 1:2,000 dilution (culture medium:sterile deionized water) was used with a maximum absorbance at 198 nm; with this dilution, a standard calibration was performed. A graph was drawn, as reported by Sustersick-Caas (2004), the value obtained from the graph was 0.219 l/ml. The biosurfactant density (at that dilution) was the same as the water density; therefore, the CMC of the biosurfactant was 430 mg/l. This result indicates that the CMC is higher than the CMC of the biosurfactants produced by other microorganisms (103 to 10 mg/l) (Haferburg et al. 1986; Mulligan et al. 1989; Bongolo 1999) nevertheless, this CMC is lower than those obtained with synthetic surfactants (590 mg/l of linear alkyl benzene sulphonate), and, in general, lower than that of other synthetic surfactants: BRIJ-35, Tergitol NP 10, and Genapol X-100 (90,000, 36,000, and 150,000 mg/l, respectively) used for soil bioremediation (Volke-Seplveda and Velasco-Trejo 2003). Therefore, the biosurfactant produced by P. putida could enhance bioremediation of soils contaminated with PAHs. The biosurfactant produced by P. putida in the kinetic study at 10 h (50 mg/l), 14 h (16 mg/l), and 34 h (30 mg/l) was analyzed by HPLC. The results showed a peak of a compound that eluted at 12 min on the chromatogram. The diode-array detector allowed us to observe a spectrum similar (Fig. 3) to that obtained with the rhamnolipids produced by P. aeruginosa (Schenk et al. 1995). Therefore, P. putida followed a metabolic pathway to produce rhamnolipids that was similar to that of P. aeruginosa and produced a biosurfactant with similar molecular structure in the presence of phenanthrene, with low cellular growth. In conclusion, the Box-Behnken experimental design was an excellent tool to improve the culture media formulation in order to produce biosurfactant and removal phenanthrene by P. putida. Besides, it was confirmed that P. putida produce rhamnolipids. Therefore, due to its metabolic capability, P. putida has advantages over other microorganisms due to their capacity to produce biosurfactants and the enhance phenanthrene removal under the selected conditions using an experimental design.
Acknowledgments We are grateful to CONACYT (Consejo Nacional de Ciencia y Tecnologa) for the scholarship given to Angeles Martnez Toledo that supported this work. Thanks to Elvira Ros-Leal, Juan Corona, and Cirino Chavez for their technical assistance.

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