Use of Soybean Oil Fry Waste for Economical Biosurfactant Production by Isolated Pseudomonas aeruginosa
By - 07/12/2009
(1 vote)
C. J. B. de Lima and J. Contiero*
Department of Biochemistry and Microbiology, Universidade Estadual de São Paulo Unesp, Rio Claro, São Paulo, Brazil. Biological Sciences Institute CEP 13506-900
*For correspondence - jconti@rc.unesp.br
Current Trends in Biotechnology and Pharmacy , Volume 3 (2) April - 2009
Abstract
The present study sought biotensoactive production from soybean oil fry waste using Pseudomonas aeruginosa ATCC 10145 and Pseudomonas aeruginosa isolated from the soil of a petroleum station having undergone gasoline and diesel oil spills. The results of the experiments were analyzed using a complete factorial experimental design, investigating the concentration of soybean oil waste, ammonia sulfate and residual brewery yeast. Assays were performed in 250-mL Erlenmeyer beakers containing 50 mL of production medium, maintained on a rotary shaker at 200 rpm and a temperature of 30±1 °C for a 48-hour fermentation period. Biosurfactant production was monitored through the determination of rhamnose, surface tension and emulsification activity. The Pseudomonas aeruginosa ATCC 10145 strain and isolated Pseudomonas aeruginosa were able to reduce the surface tension of the initial medium from 61 mN/m to 32.5 mN/m and 30.0 mN/m as well as produce rhamnose at concentrations of 1.96 and 2.89 g/L with emulsification indices of 96% and 100%, respectively.
Keywords: Pseudomonas aeruginosa, Biosurfactant, Rhamnose, Surface-active, Emulsification index, Soybean oil.
Introduction
Surfactants are an important c nlass of chemical compounds widely used in different industries, acting as dispersants and/or solubilizers of organic compounds. The vast majority of commercially employed surfactants are synthesized from petroleum derivatives (1). In the past few decades, however, the interest in surfactants of a microbial origin has increased significantly, above all, due to their biodegradability (2,3,4).
Compounds of a microbial origin that exhibit surfactant properties (reduction of surface tension and/or high emulsifying capacity) are denominated biosurfactants and are metabolic byproducts of bacteria and fungi (5). Glycolipids are the best known microbial surfactants. These compounds are made up of carbohydrates associated to a long chain of aliphatic or hydroxy-aliphatic acids. Rhamnolipids are among the most-studied glycolipids and are compounds that have one or two rhamnose molecules linked to one or two β-hydroxydecanoic acid molecules (6).
Surfactants produced microbiologically offer a number of advantages over their chemical equivalents, such as low toxicity, tolerance to temperature, pH and ionic strength as well as the possibility of being produced from renewable substrates (7,8,9). Biosurfactants can be applied in fields such as agriculture for the formulation of pesticides and herbicides; the food industry as additives in condiments; and in pharmaceutical, textile, cosmetic and petroleum industries, where there are employed for the secondary recovery of petroleum, such as in the removal and mobilization of oil residuals and bioremediation (10).
Despite their advantages, biosurfactants are not widely used by industries due to the high production costs associated to low productivity and the use of expensive substrates. One possible strategy for reducing production costs is the use of alternative substrates, such as agricultural or food industry wastes, which generally contain the high levels of carbohydrates and lipids necessary for the biosynthesis of biosurfactants (11). Moreover, the use of wastes contributes toward a reduction in environmental pollution and the economic valuation of such products. Alternative substrates, such as oil dregs, used oils, molasses and wastes from the processing of cheese, potatoes and cassava, are examples of byproducts with potential for the production of biosurfactants (12,13,14,15).
The aim of the present study was to determine suitable replacements for chemical surfactants byproducts with either low or no toxicity using wastes as raw materials to reduce the cost of these byproducts. The main objectives of the study were to determine the potentiality of an isolated strain of Pseudomonas aeruginosa in producing biosurfactant from soybean oil waste used in the frying of different foods, ammonia nitrate and residue from an autolyzed brewery biomass; and compare its performance to that of Pseudomonas aeruginosa ATCC 10145, using a complete factorial experimental design.
Materials and Methods
Microrganism
P. aeruginosa ATCC 10145 was kindly donated by Dr. Ivano de Fillipis from the Instituto Nacional de Controle de Qualidade em Saúde (INCQS/FIOCRUZ) – Rio de Janeiro, Brazil. P. aeruginosa was isolated from the soil of a petroleum station having undergone gasoline and diesel oil spills located in the city of Uberlândia, Minas Gerais, Brazil. The bacterial strain was identified as P. aeruginosa called strain UFU. The cultures were maintained at 4°C in a bacto nutrient broth (BD, cod. 234000) supplied by the Becton Dickinson and Company, USA.
Culture Isolation
The medium proposed by Vecchioli (16), added with 0.5% (v/v) of soybean oil fry waste as the sole carbon source, was used for the bacterial cultures using the pour-plate technique. Among the isolated microorganisms, the one that demonstrated the best surface tension reduction of the culture medium after fermentation was selected and identified. The isolated microorganism was identified at the Enterobacteria Laboratory of the Oswaldo Cruz Institute (Rio de Janeiro, Brazil), following traditional procedures based on bacterial cytomorphology, biochemistry and physiology.
Growth Medium and Conditions
Growth of the bacterial culture was performed on the medium proposed by Santos (17), consisting of (g/L) NH4NO3 (1.7), Na2HPO4 (7.0), KH2PO4 (3.0), MgSO4.7H2O (0.2), yeast extract (5.0) and glucose (10.0). Biosurfactant production assays were conducted on the same mineral medium used for microbial growth, with the addition of soybean oil fry waste (g/L between 5 and 15), residual brewery yeast (g/L between 0 and 10), NH4SO4 (g/L between 1 and 13). The residual brewery yeast, consisting of 100% inactivated, dried cells of Saccharomyces cerevisiae was provided by a local brewery. The product composition was 8.0% moisture, 40.0% protein, 3.0% fibrous matter, 8.0% mineral matter and aflatoxin (50 ppb). All media were autoclaved at 121°C for 15 min after adjusting the pH to 7.0 with 0.1 N HCl.
The inoculum was prepared by adding three loopfuls of cells from the stock culture to a 500 mL Erlenmeyer beaker containing 100 mL of the growth medium. The inoculated medium was incubated at 30 ± 1°C for 24 hours on a rotary shaker (New Brunswick, USA) at 170 rpm. Afterwards, optical density (600 nm) of bacterial suspension was adjusted to 0.4 and an aliquot of 1 mL of inoculum (2%) was transferred to a 250-mL Erlenmeyer beaker containing 50 mL of medium and incubated at 30°C for 48 hours on a rotary shaker at 170 rpm. Samples were collected at defined time intervals and submitted to analysis.
Complete Factorial Experimental Design
The literature indicates that carbon and nitrogen sources play a critical role in the performance of rhamnose production by P. aeruginosa strains (17). To investigate the effects of soybean oil fry waste (FSOW), ammonium sulfate (AS) and residual brewery yeast (RBY) on the selected dependent variables (rhamnose synthesis, emulsification index and surface tension), a complete factorial experimental design (CFED) was used on two levels (18). Statistical calculations were performed using the Statistic 5.1 software program (State Ease Inc., Minneapolis, MN, USA). Using the CFED method, a total of 16 experiments were conducted with combinations of FSOW, AS, RBY and the two Pseudomonas aeruginosa strains. Table 1 displays the range and levels of the variables investigated.
Table 1. Real values of variables used in complete factorial experimental design
Analytical Methods
Cell growth was determined by measuring the optical density of samples, using a UV-160A visible spectrophotometer (Shimadzu, Co., Tokyo, Japan) at 540 nm. Cell concentration was determined by dry weight filtering through a 0.45 µm previously weighed Millipore membrane (19).
Surface tension (ST) was measured at 25°C using a Tensiometer (Fisher Scientific, USA), which was previously calibrated with surveyor weights. A decrease in surface tension was used as a qualitative measurement of surfactant concentration and a quantitative indicator of efficiency.
The biosurfactant emulsification index (EI) was determined according to Cooper and Goldenberg (20). Cell-free culture samples and kerosene (at a ratio of 4:6) were vigorously mixed for 2 min using a vortex (Phoenix, Brazil, model AP-56) and left undisturbed for 24 h at room temperature. EI 24 is the percentage of the height of the emulsified layer (cm) relative to the total height of the liquid column, determined at the 24-h time point.
The rhamnose concentration was determined according to the methodology described by Rahman et al. (21).
Results and Discussion
Effects of carbon and nitrogen concentrations on rhamnose production
Table 2 displays the results of the complete factorial designs from the FSOW, AS and RBY concentrations.
Table 2. Results of rhamnose production (RM), EI (E24) and ST obtained in the Experiments with P. aeruginosa ATCC 10145 and isolated P. aeruginosa UFU
Table 2 displays the statistical delineation used in the production of rhamnose by P. aeruginosa ATCC 10145 and isolated P. aeruginosa UFU under the different conditions. Both strains were able to use the residue tested (FSOW) and synthesize the biosurfactant. Experiments 11 and 12 employed extreme FSOW, RBY values and a minimal concentration of AS, obtaining the highest production of rhamnose and emulsification index as well as the lowest surface tension value. According to Lang and Wullbrant (22), high concentrations of carbon and nitrogen in the fermented medium are needed for the obtainment of high concentrations of rhamnose. In the present study, the highest amount of rhamnose (2.81 g/L), highest emulsification index (100%) and lowest surface tension (30.5 mN/m) were obtained from the isolated P. aeruginosa UFU, whereas the least adequate condition occurred for P. aeruginosa ATCC 10145, with the lowest production of rhamnose (0.25 g/L), lowest emulsification index (40%) and highest surface tension (41.1 mN/m).
Haba et al. (14) selected the P. aeruginosa 47T2 NCIB 40044 strain from 36 screened strains due to its capacity to produce 2.7 g/l of rhamnose from FSOW. Previous studies found that this strain produced only 6.4 g/l of rhamnose through cultivation in olive oil waste (23). De Lima et al. (24) obtained a final concentration of 2.3 g/L of rhamnose when P. aeruginosa PACL was cultivated in FSOW.
As Table 2 shows, both strains are able to reduce surface tension to below 35 mN/m. According to Cooper (25), an organism is considered a promising biosurfactant producer when it produces tensoactive compounds with a surface tension below 40 mN/m. In order for a biosurfactant to be considered efficient, however, this value must be below 35 mN/m. Studies on rhamnolipid homologues extracted and purified from the fermentation broth by Pseudomonas aeruginosa 47T2 cultivated in oil dregs have described a value of 32.8 mN/M for surface tension (26). In a study carried out with vegetable oils (olive, soybean and sunflower) at a concentration of 20 g/L, Andrés et al. (27) found that the broth fermented by P. aeruginosa 42 A2 achieved surface tension values of 32.0, 34.0 and 335.5 mN/m, respectively.
For all experiments, the biosurfactant produced had intense emulsification properties. The complete kerosene in water emulsions proved stable for 24 hours. This analysis is a practical measurement of biosurfactant use, as it gives the compound the ability to emulsify non-miscible liquids with the formation of a stable emulsion. The results obtained in the present experiment suggest that isolated Pseudomonas aeruginosa UFU has both a better degradation capability regarding soybean oil waste in fermented media as well as a greater potential for producing biosurfactant.
Statistical analysis of the data
From the CFED, the operational conditions of the FSOW (X1), AS (X2), RBY (X3) and P. aeruginosa strains (X4) were determined. As X4 is a qualitative variable and the equation of the empirical model must be in function of the quantitative variables, it was necessary to perform a correction of the adjusted equation, replacing the X4 variation with either –1 or 1 in order to maximize the response. Thus, the adjusted empirical model for rhamnose synthesis containing only significant parameters (p≤0.05; Student’s t test) is represented by Equation 1. Table 3 displays the parameters and significance levels of the model variables.
RM = 1.185 + 0.48X1 – 0.33X2 + 0.187X3 - 0.187X1X2
The results show that X1 and X3 are highly significant among the independent variables, as they have positive coefficients (Eq. 1), according to which an increase in their concentration increases the production yield. The X2 variable and the X1X2, interaction also have a significant effect, as their negative signs initiate when their concentrations are lower in the system, thereby promoting an increase in the response (RM).
Table 3. Results of the regression for rhamnose synthesis
The goodness-of-fit of the model was checked by determining the coefficient (R2) and the multiple correlation coefficient (R). The R2 value (0.9832) for the complete equation (data not shown) indicates that the sample variation of 98.32% for rhamnose was attributed to the independent variables and only 1.68% of the total variation cannot be explained by the model. The value of the adjusted determination coefficient (adjusted R2 = 0.9414) for Equation (1) is also high, which demonstrates the high significance of the model. The high R value (0.9916) demonstrates high agreement between the experimental observations and predicted values. This correlation is also evident on the plot of predicted versus experimental rhamnose values in Figure 1, as all points cluster around the diagonal line, meaning that no significant violations of the model were found.
Figure 1. Predicted vs. experimental values plotted for rhamnose
The 3D response surface (Fig. 2) represents the empirically adjusted equation (Eq. 1) and is plotted to visualize the interactions of the independent variables (FSOW, AS) as well as locate the optimum level of each variable for maximum response (rhamnose synthesis).
Fig 2. Response surface for rhamnose in relation to WFSO and AS.
The response surface in Figure 2 reveals that an increase in FSOW concentration and a decrease in AS concentration cause an increase in rhamnose synthesis.
Kinetics of rhamnose production by isolated P. aeruginosa UFU and P. aeruginosa ATCC 10145
Figure 3 illustrates the kinetics of rhamnose production from the best results obtained by P. aeruginosa ATCC 10145 (Experiment 11) and isolated P. aeruginosa UFU (Experiment 12) using soybean oil waste from fried food preparation as the carbon source.
There was intense cell growth up to 36 h of fermentation, when the stationary phase was established, corresponding to biosurfactant production (Yp/x) of 0.54 for the isolated strain (Fig.3A) and 0.36 g rhamnose/g cells for the ATCC 10145 strain (Fig.3B). At 72 h, the isolated strain a achieved rhamnose synthesis level of 2.81 g/L, while the ATCC 10145 reached 1.96 g/L of rhamnose at 54 h. In previously published studies, Guerra-Santos et al. (28), Haba et al. (14) and Dubey et al. (29) achieved 0.97-2.7 g/L of biosurfactant production with different strains of P. aeruginosa using glucose and fry oil waste as carbon sources.
As Figure 3 shows, biosurfactant production initially follows an exponential growth phase, but when microbial growth ceases and a stationary phase is achieved, rhamnose synthesis continues, which suggests biotensoactive production partially associated with microbial growth. These observations were also described by Mayer et al. (30), Benincasa et al. (31) and Yu-Hong et al. (32). Perhaps the production of biosurfactant can be classified as a secondary metabolite. Biossurfactant production accompanies bacterial growth in fry oil waste, which may help in the adherence of cells to the substrate molecules and their metabolism (33,34).
Due to the biotensoactive accumulation in the medium, there was also a drop in surface tension (Fig.3). Regarding pH, there was a variation ranging from 7.01 to 8.5 and a tendency toward final values greater than 7.2.
The type of carbon source affects the properties (surface tension and emulsification activity) and final concentration of the rhamnose produced. These differences may be associated to the composition of triglycerides in the substrates used (35) as well as the activity of the microbial lipase on these substrates (36).
Differences were found between the two microorganisms tested in the present study with regard to rhamnose production when the same substrate was used. Differences in rhamnose number in the composition of the rhamnolipids may also result in differences in biotensoactive properties (15).
Conclusion
With the complete factorial experimental design , it was possible to determine the behavior of the independent variables on rhamnose production, the emulsification index and surface tension. The present study demonstrated the biosurfactant-producing potential from the re-use of a fried soybean oil substrate by P. aeruginosa ATCC10145 and a new isolated strain, which obtained the best results regarding rhamnose production (2.89 g/L), surface tension (30 mN/m) and emulsification index (100%).
Acknowledgements
The study was supported by Brazilian agency Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) of the Ministério da Educação.
References
1. Fiechter, D. (1992). Biosurfactants: moving towards industrial application. Trends in Biotechnology, 10: 208-216.
2. Ishigami Y., Gama Y., Nagahara H., Motomiya T. and Yamaguchi M. (1988). Liposome containing rhamnolipids. Japanese Patent Kokai, 29: 63–182.
3. Makkar, R.S. and Cameotra, S.S. (1999a). Biosurfactant production by microorganisms on unconventional carbon sources – a review. Journal of Surfactants and Detergents 2: 237–241.
4. Makkar, R.S. and Cameotra, S.S. (1999b). Biochemical and structural characterization of biosurfactant produced by Bacillus subtilis at thermophilic conditions. ournal of Surfactants and Detergents, 2: 371–376.
5. Nitschke, M and Pastore, G.M. (2002). Biosurfactantes: Propriedades e aplicações. Química Nova, l25: 772-776.
6. Desai, J.D. and Banat, I.M. (1997). Microbial production of surfactants and their commercial potential. Microbiology and Molecular Biology Reviews, 61: 47-64.
7. Banat, I.M., Rhaman, K.S.M., Rhaman, T.J., Marchant, R. and Mcclean, S. (2002). Rhamnolipid biosurfactant production by strains of Pseudomonas aeruginosa using low-cost raw materials. Biotechnology Progress, 18: 1277-1281.
8. Kim, S.H., Lim, E.J., Lee, J.D. and Lee, T.H. (2000). Purification and characterization of biosurfactants from Nocardia sp. L-417. Biotechnology Applied Biochemistry, 31: 249-253.
9. Mulligan, C.N., Mahmourides, G. and Gibbs, B.F. (1989). Biosurfactant production by chloramphenicol tolerant strain of Pseudomonas aeruginosa. Journal of Biotechnology, 12: 37-44.
10. Ron, E.Z. and Rosenberg, E. Biosurfactants and oil bioremediation. (20002). In: Current Opinion in Biotechnology. Editora Elsevier, 13: 249-252.
11. Cameotra, S.S. and Makkar, R.S. (1998). Synthesis of biosurfactants in extreme conditions. Applied microbiology and Biotechnology, 50: 520-529.
12. Abalos, A., Pinazo, A., Infante, M.R., Casals, M., Garcia, F. and Manresa, A. (2001). Physicochemical and antimicrobial properties of new rhamnolipid produced by Pseudomonas aeruginosa AT10 from oil refinery wastes. Langmuir, 17: 1367-1371.
13. Siddhartha, G.V.A.O., Nitschke, M., Haddad, R., Eberlin, M.N. and Contiero, J. (2006). Production of Pseudomonas aeruginosa LBI rhamnolipids following growth on Brazilian native oils. Process Biochemistry, 41: 483-488.
14. Haba, E., Espuny, M.J., Busquets, M. and Manresa, A. (2000). Screening and production if rhamnolipids by Pseudomonas aeruginosa 47T2 NCIB 40044 from waste frying oil. Journal of Applied Microbiology, 88: 379-387.
15. Nitschke, M., Siddhartha, G.V.A.O., Gonçalves, L.A., Haddad, R., Eberlin, M.N. and Contiero, J. (2005). Oil wastes as unconventional substrates for rhamnolipid biosurfactant production by Pseudomonas aeruginosa LBI. Biotechnology Program, 21: 1562-1566.
16. Vecchioli, G.I., Del Panno, M.T. and Painceira, M.T. (1990). Use of selected autochthonous soil bacteria to enhance degradation of hydrocarbons in soil. Environmental Pollution 67: 249-258.
17. Santos, A.S., Sampaio, A.P., Vasquez, G.S., Santa Anna, L.M., Pereira, N. and Freire, D.M.G. (2002), Applied Biochemimestry Biotechnology 98: 1025-1035.
18. Jeff Wu, C.F. and Hamada, M. (2000). Experiments: Planning, analysis, and parameter design optimization. New York: Wiley Series in Probability and Statistics.
19. Reis, F.A.S.L., Servulo, E.F.C. and França, F.P. (2004). Lipopeptide surfactant production by Bacillus subtilis grown on low-cost raw materials. Applied Microbiology and Biotechnology, 115: 899-912.
20. Cooper, D.G. and Goldenberg, B.G. (1987). Surface-Active Agents from Two Bacillus Species. Applied Environment Microbiology, 53: 224-229.
21. Rahman, K.S.M., Banat, I.M., Thahira-Rahaman, J., Thayumanavan, T. and Lakshmanaperumalsamy, P. (2002). Bioremediation of gasoline contaminated soil by a bacterial consortium amended whit poultry litter, coir pith and rhamnolipids biosurfactant. Bioresource Technology, 81: 25-32.
22. Lang, S. and Wullbrandt, D. (1999). Rhamnose lipid – Biosynthesis, Microbial Production and Application Potential. Applied Microbiology Biotechnology, 51: 22-32.
23. Mercadé, M.E., Manresa, M. A., Robert, M., Espuny, M. J., Andrés C. and Guinea, J. (1993). Olive oil mill effluent (OOME): New substrate for biosurfactant production. Bioresource Technology, 43: 1-6.
24. De Lima, C.J.B., De França, F.P., Sérvulo, E.F.C., Resende, M.M. and Cardoso, V. L. (2007). Enhancement of rhamnolipids production in residual soybean oil by an isolated strain of Pseudomonas aeruginosa. Applied Biochemistry and Biotechnology, 137: 463–470.
25. Cooper, D.G. and Paddock, D.A. (1984). Production of a biosurfactant from Torulopsis bombicola. Applied and Environmental, 47: 173-176.
26. Haba E., Pinazo, A.O., Jauregui M.J., Espuny M.R. and Manresa, A. (2002). Physicochemical characterization and antimicrobial properties of rhamnolipids produced by Pseudomonas aeruginosa 47T2 NCBIM 40044. Biotechnology and bioengineering, 81: 316-322.
27. Andrés, C., Mercadé, E., Guinea, J. and Manresa, A. (1994). 7,10-Dihydroxy-8 (E) –octadecanoic acid produced by Pseudomonas 42 A2: Evaluation of different cultural parameters of the fermentation. World Journal of Microbiology and Biotechnology, 10: 106-109.
28. Guerra-Santos, L.H., Kappeli, O. and Fiechter, A. (1986). Dependence of Pseudomonas aeruginosa continuous culture biosurfactant production on nutritional and environmental factors. Applied Microbiology and Biotechnology, 24: 443-448.
29. Dubey, K. and Juwakar, A. (2001). Distillery and curd whey wastes as viable alternative sources for biosurfactant production. World Journal Microbiology Biotechnology, 17: 61-69.
30. Mayer, R.M. and Soberón-Chavez, G. (2000). Pseudomonas aeruginosa rhamnolipids: Biosynthesis and potential applications. Applied Microbiology and Biotechnology, 54: 625-633.
31. Benincasa, M., Contiero, J., Manresa, M.A., and Moraes, I.O. (2002). Rhamnolipid production by Pseudomonas aeruginosa LBI growing on soapstock as the sole carbon source. Journal of Food Engineering, 54: 283-288.
32. Yu-Hong, W., Chien-Liang, C. and Jo-Shu, C. (2005). Rhamnolipid production by indigenous Pseudomonas aeruginosa J4 originating from petrochemical wastewater. Biochemical Engineering Journal, 27: 146–154.
33. Baldi, F., Ivosevic, N., Minaui, A., Pepi, M., Fani, R., Svetlicii, V. and Zutii V. (1999). Adhesion of Acinetobacter venetians to diesel fuel droplets studied with in situ electrochemical and molecular probes. Applied Environmen Microbiology, 65: 2041-2048.
34. Rosenberg, M. and Rosenberg, E. (1981). Role of adherence in growth of Acinetobacter calcoaceticus RAG-1 on hexadecane. Journal Bacteriology, 148: 51-57.
35. Kosaric, N. (1993). Biosurfactants: Production, Properties, Applications (1th edition), Marcel Dekker, INC., New York, USA, pp.504.
36. Shabtai, Y. and Daya-Mishne, N. (1992). Production, purification, and properties of a lipase from a bacterium (Pseudomonas aeruginosa YS-7) capable of growing in water-restricted environments. Applied Environment Microbiology, 58: 174-180.