- Email: [email protected]
Use of response surface optimization for the production of biosurfactant from Rhodococcus spp. MTCC 2574
Available online at www.sciencedirect.com
Bioresource Technology 99 (2008) 7875–7880
Short Communication
Use of response surface optimization for the production of biosurfactant from Rhodococcus spp. MTCC 2574 Snehal R. Mutalik a, Bhalchandra K. Vaidya a, Renuka M. Joshi a, Kiran M. Desai b, Sanjay N. Nene a,* a
Biochemical Engineering Department, Chemical Engineering and Process Development Division, National Chemical Laboratory, Pune 411008, India b Food Engineering and Technology Department, Institute of Chemical Technology, Matunga, Mumbai 400019, India Received 21 September 2007; received in revised form 21 February 2008; accepted 21 February 2008 Available online 3 June 2008
Abstract The production of biosurfactant from Rhodococcus spp. MTCC 2574 was effectively enhanced by response surface methodology (RSM). Rhodococcus spp. MTCC 2574 was selected through screening of seven different Rhodococcus strains. The preliminary screening experiments (one-factor at a time) suggested that carbon source: mannitol, nitrogen source: yeast extract and meat peptone and inducer: n-hexadecane are the critical medium components. The concentrations of these four media components were optimized by using central composite rotatable design (CCRD) of RSM. The adequately high R2 value (0.947) and F score 19.11 indicated the statistical significance of the model. The optimum medium composition for biosurfactant production was found to contain mannitol (1.6 g/L), yeast extract (6.92 g/L), meat peptone (19.65 g/L), n-hexadecane (63.8 g/L). The crude biosurfactant was obtained from methyl tert-butyl ether extraction. The yield of biosurfactant before and after optimization was 3.2 g/L of and 10.9 g/L, respectively. Thus, RSM has increased the yield of biosurfactant to 3.4-fold. The crude biosurfactant decreased the surface tension of water from 72 mN/m to 30.8 mN/m (at 120 mg L1) and achieved a critical micelle concentration (CMC) value of 120 mg L1. Ó 2008 Published by Elsevier Ltd. Keywords: Biosurfactant; Rhodococcus spp.; Response surface methodology; Medium optimization
1. Introduction Biosurfactants or microbial surfactants are surfaceactive biomolecules produced by a variety of microorganisms. Biosurfactants are receiving considerable attention due to their unique properties such as higher biodegradability, lower toxicity, and greater stability towards temperature and pH (Mukherjee et al., 2007). In the past few decades, biosurfactants have been identified for commercial importance, specifically, in the field of oil recovery (Ivshina et al., 1998); secondary or tertiary environmental bioremediation (Christofi and Ivshina, 2002); pharmaceuticals (Rodrigues et al., 2006a); and food processing and cosmetics (Banat et al., 2000). *
Corresponding author. Tel.: +91 20 25902347; fax: +91 20 25902612. E-mail address: [email protected] (S.N. Nene).
0960-8524/$ - see front matter Ó 2008 Published by Elsevier Ltd. doi:10.1016/j.biortech.2008.02.027
Members of the genus Rhodococcus are known to produce surface-active trehalose-lipids. Bell et al. (1998) have reported that some biosurfactants including those from rhodococci are more effective and efficient than many existing synthetic surface-active agents. The production of biosurfactant from different strains of Rhodococcus has been reported in the literature (Lang and Philp, 1998; Kuyukina et al., 2001; Pirog et al., 2004). The yield of biosurfactant varies considerably with the hydrocarbon which is used to induce the production of biosurfactant. The potential of Rhodococcus biosurfactant in a variety of industrial applications has been proposed, but like other biosurfactants it is yet to achieve market penetration (Mukherjee et al., 2007). Rhodococcus biosurfactants have not achieved significant market share because of their high cost as compared to synthetic surfactants. Thus, to compete with the large-scale production of synthetic surfactants from
7876
S.R. Mutalik et al. / Bioresource Technology 99 (2008) 7875–7880
hydrocarbon feedstocks, it is desirable to enhance the production of biosurfactant by Rhodococcus. One of the methods of achieving the above objective is the selection of appropriate media components and optimal culture conditions for maximum biosurfactant productivity. The limitations of classical method of media optimization can be overcome by the application of statistical based approach (Lotfy et al., 2007; Tanyildizi et al., 2005). RSM, an extensively used statistical technique for media optimization, is a collection of statistical techniques which uses design of experiments (DoE) for building models, evaluating the effects of factors and searching for the optimum conditions. (Rodrigues et al., 2006b). In the present work, we have used a central composite rotatable design (CCRD) of response surface methodology for media optimization to enhance biosurfactant production by Rhodococcus spp. MTCC 2574. 2. Methods 2.1. Materials All bacterial growth media components were purchased from Hi-Media, India. All other chemicals were of analytical grade procured from S.D. Fine Chemicals, India. 2.2. Microorganism and growth conditions The seven microbial strains namely Rhodococcus spp. MTCC 2574, 2678, 2683 and Rhodococcus erythropolis MTCC 1526, 1548, 2794, 3951 were procured from MTCC-Chandigarh (India). All Rhodococcus strains were maintained on nutrient agar (beef extract 10 g/L, NaCl 5 g/L, peptone 10 g/L, agar 20 g/L, pH 7.0–7.5). The liquid fermentation medium used for batch culture experiments contained (g L1) glucose (10), yeast extract (3), meat peptone (7.5), Na2HPO4 (4.0), KH2PO4 (2.0), MgSO4 7H2O (0.2), CaCl2 2H2O (0.02), ammonium ferric citrate (0.05), trace mineral solution (1 mL/L) [termed as medium A]. The composition of trace mineral medium was (g L1) H3BO3 (0.1), MnCl2 4H2O (0.1), ZnSO4 H2O (0.1), FeCl3 6H2O (0.1), CaCl2 2H2O (1), CuCl2 2H2O (0.05). Five milliliter of inoculum was transferred to 45 mL of liquid fermentation medium contained in a 250 mL Erlenmeyer flask and incubated for 36 h at 30 °C on rotary shaker at 200 rpm. 2.3. Selection of optimum nitrogen source, carbon source and inducer The organic nitrogen source (yeast extract and meat peptone) from Medium A was replaced by inorganic nitrogen sources (namely urea, ammonium sulphate and ammo-
% EI24 ¼
nium phosphate) at equivalent nitrogen level. To evaluate the optimum carbon source, glucose was replaced by an equivalent amount of different carbon sources namely glycerol, sucrose, sorbitol and mannitol. Seven inducers (3% v/ v each) were screened to evaluate the corresponding enhancement in biosurfactant production. Biosurfactant production was calculated in terms of emulsification index (% EI24) as described in Section 2.7.1. 2.4. Biosurfactant profile The cell growth and biosurfactant production were simultaneously studied to establish the biosurfactant profile. Cells of Rhodococcus spp. MTCC 2574 were grown on liquid medium containing (g L1) mannitol (10.12), yeast extract (3), meat peptone (7.5), Na2HPO4 (4.0), KH2PO4 (2.0), MgSO4 7H2O (0.2), CaCl2 2H2O (0.02), ammonium ferric citrate (0.05), trace mineral solution (1 mL/L) and n-hexadecane (3% v/v) (termed as medium B). The samples were aseptically removed at a regular interval of 4 h up to 48 h and analyzed for optical density (600 nm), % EI24 and % hydrophobicity. 2.5. Experimental design and data analysis To examine the combined effect of four different medium components (mannitol, yeast extract, meat peptone and n-hexadecane) on biosurfactant production by Rhodococcus spp. MTCC 2574, 30 experiments were performed in duplicate. The value of the dependent response (% EI24) was the mean of two replications. The second-order polynomial coefficients were calculated and analyzed using the trial version of ‘Design Expert’ software (Version 6.0, Stat-Ease Inc., USA). 2.6. Extraction of biosurfactant The extraction of biosurfactant was done by using methyl tert-butyl ether (MTBE) as described earlier by Kuyukina et al. (2001). The product was thoroughly washed thrice with petroleum ether to remove residual n-hexadecane to obtain crude biosurfactant. The crude biosurfactant was finally dried by lyophilization. 2.7. Analytical methods 2.7.1. Emulsification Index (% EI24) Emulsification Index (% EI24) was used to quantify the biosurfactant produced by Rhodococcus cells (Fleck et al., 2000). The values of % EI24 were determined as described earlier by Nitschke and Pastore (2006). The percent ratio of the height of emulsified zone to total height after 24 h gives ‘emulsification index’ (% EI24) as given in Eq. (1)
Height of emulsified zone 100 Total height of liquidðsum of aqueous; oil and emulsified zoneÞ
ð1Þ
S.R. Mutalik et al. / Bioresource Technology 99 (2008) 7875–7880
2.7.2. % Cellular hydrophobicity Pruthi and Cameotra (1997) reported an increase in the cell surface hydrophobicity with the biosynthesis of biosurfactant. % Cellular hydrophobicity was determined as described earlier (Lin et al., 2005). The % cellular hydrophobicity was calculated as expressed in Eq. (2)
7877
not shown) for all seven Rhodococcus strains and therefore organic nitrogen sources were selected for further studies. The effect of carbon source on cell density for seven Rhodococcus strains is given in Fig. 1. The optimum carbon source was found to vary depending upon the Rhodococcus strain. Among the different Rhodococcus strains, MTCC
OD400 of aqueous phase of culture after the addition of n-dodecane % Hydrophobicity ¼ 1 100 OD400 of culture before the addition of n-dodecane
2.7.3. Characterization of crude biosurfactant The type of emulsion was determined by using methyl orange and Sudan red III as described by Tian et al. (1998). Total carbohydrate and protein content of crude biosurfactant were estimated by phenol sulphuric acid method (Dubois et al., 1956) and Folin Lowry method (Lowry et al., 1951), respectively. Surface tension measurement and critical micelle concentration were detected using Kruss K-11 Tensiometer (accuracy ± 0.1 mN/m). 3. Results and discussion The biosurfactant production by Rhodococcus spp. is reported to be primarily cell-growth associated (Ghurye and Vipulanandan, 1994; Rodrigues et al., 2006b). The initial attempts therefore were made to get high cell density, based on a one-factor-at-a-time strategy.
ð2Þ
2574 gave a maximum cell density when grown on mannitol as carbon source. Here, seven hydrocarbons were evaluated for the enhancement of biosurfactant production in MTCC 2574 (results not shown). The production of biosurfactant was expressed in terms of emulsification index (% EI24). The fermentation broth was subjected for centrifugation and the cell-free supernatant and sonicated cell-suspension were analyzed for % EI24. The % EI24 values of cell-free supernatant were very low (in the range of 0–7%) as compared with sonicated cell-suspension. This indicates that a negligible amount of biosurfactant is released from the cell surface to the fermentation broth and confirms the cell-wall associated nature of Rhodococcus biosurfactant as described by Bicca et al. (1999). n-Hexadecane gave maximum EI24 (59.2%) and hence selected for further experiments. 3.2. Biosurfactant profile
3.1. Selection of optimum nitrogen source, carbon source and inducer The complete replacement of organic nitrogen by inorganic nitrogen resulted in a very low cell growth (results
The cell density, % EI24 and % cellular hydrophobicity were studied with respect to fermentation time, and the biosurfactant profile was established (Fig. 2). During the course of fermentation, % EI24 was found to remain almost
25
O.D. (600 nm) at 36 h
20
15
10
5
0 1548 Glucose
2678
3951 Sucose
2683 Sorbitol
2794
2574
1526
Mannitol
Glycerol
Fig. 1. Effect of carbon source on the biomass of different Rhodococcus strains.
7878
S.R. Mutalik et al. / Bioresource Technology 99 (2008) 7875–7880
Fig. 2. Biosurfactant profile (i.e. the effect of fermentation time on cell density, % EI24 and % hydrophobicity).
constant up to 20 h % EI24 was found to increase after 20 h, reached a maximum at 28 h and thereafter remained constant till 36 h % EI24 was found to decline along with cell density from 36 h to 48 h. Thus, the biosurfactant content of the growing Rhodococcus spp. MTCC 2574 was found to reach a maximum at 28 h. Therefore, all further fermentations were terminated at 28 h. The % hydrophobicity of each sample was carried out in order to confirm the biosurfactant production. The % hydrophobicity increased with increase in cell density, reached a maximum at 28 h and was found to decline from 36 h to 48 h. 3.3. Optimization of biosurfactant production The coded values of independent variables are given in Table 1. The design of experiments and the respective experimental and predicted % EI24 are given in Table 2. After regression analysis, the second-order response model was obtained which is given in Eq. (3) % EI24 ¼ 33:21 þ 11:55A þ 0:455B 5:20583C þ 2:89833D þ 4:97708A2 þ 4:99583B2 þ 4:98458C2 1:43667D2 2:86625A B 3:35000A C þ 1:41250A D þ 8:53125B C þ 1:94125B D þ 1:56750C D
ð3Þ
(where A: mannitol, B: yeast peptone, C: meat peptone, D: n-hexadecane and A, C, D, A2, B2, C2, AB, AC, BC were identified as significant terms). A low value of the coefficient of variation (12.05%) indicates the very high degree of precision and a good reliability of the experimental val-
Table 1 Coded values of independent variables* No.
1 2 3 4
Variable
Mannitol Yeast extract Meat peptone n-Hexadecane
Coded values 2
1
0
1
2
0 0 0 0
10 2 5 20
20 4 10 40
30 6 15 60
40 8 20 80
* Concentrations in g/L.
ues. The fit of the model was also expressed by the coefficient of determination R2, which was found to be 0.947, indicating that 94.7% of the variability in the response could be explained by the model. The solution was obtained by substituting the levels of the factors into the regression equation. The optimal concentrations for the four components were found as mannitol (1.6 g/L), yeast extract (6.92 g/L), meat peptone (19.65 g/L), n-hexadecane (63.8 g/L) (termed as medium C, all remaining components are in the same concentration as medium B). The predicted response (95.3%) was experimentally verified (93.9%). The agreement between predicted value and experimental value of % EI24 confirms the significance of the model. In the optimized medium, the concentration of n-hexadecane (63.8 g/L) was relatively high as compared to mannitol (1.6 g/L). This indicates that the elevated hydrocarbon contents are necessary for higher biosurfactant production. Here, it can be speculated that the mannitol acts as a primary carbon source during the initial growth phase, while in the latter stages of fermentation, Rhodococcus cells probably use n-hexadecane as a carbon source. Thus, an appropriate combination of a simple carbon source and an inducer enhanced the production of biosurfactant.
S.R. Mutalik et al. / Bioresource Technology 99 (2008) 7875–7880
7879
Table 2 Central composite rotatable design (CCRD) matrix of independent variables and their corresponding experimental and predicted yields of biosurfactant No.
Media components (coded values) Mannitol
Yeast extract
1 1 1 2 1 1 3 1 1 4 1 1 5 1 1 6 1 1 7 1 1 8 1 1 9 1 1 10 1 1 11 1 1 12 1 1 13 1 1 14 1 1 15 1 1 16 1 1 17 2 0 18 2 0 19 0 2 20 0 2 21 0 0 22 0 0 23 0 0 24 0 0 25 0 0 26 0 0 27 0 0 28 0 0 29 0 0 30 0 0 * Values indicate mean of duplicate observations.
% EI24 Meat peptone
n-Hexadecane
Experimental*
Predicted
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 2 2 0 0 0 0 0 0 0 0
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 2 2 0 0 0 0 0 0
35.21 78.9 32.9 50.0 24.9 35.4 39.6 50.0 36.2 78.9 37.8 54.0 23.6 42.6 46.1 72.5 31.5 77.0 58.4 50.3 68.2 40.3 22.4 34.8 33.9 32.7 33.3 32.7 33.9 32.7
44.3 76.9 29.9 51.2 20.4 39.7 40.2 48.0 40.2 78.6 33.7 60.6 22.6 47.6 50.2 63.7 30.0 76.2 52.3 54.1 63.6 42.7 21.7 33.3 33.2 33.2 33.2 33.2 33.2 33.2
3.4. MTBE extraction of biosurfactant The MTBE extraction method described by Kuyukina et al. (2001) was used for the isolation of biosurfactant from Rhodococcus spp. 2574 grown on optimum medium (Medium C, described in Section 3.3). However, the product obtained from this method is known to contain a large amount (approximately 40–50%) of residual n-hexadecane (Kuyukina et al., 2001). Due to the presence of residual n-hexadecane, the accurate yield of the product cannot be determined. In order to remove residual n-hexadecane, the extract was treated with petroleum ether (around 3–4 times) repeatedly and freeze-dried. This method effectively removed the residual n-hexadecane. Here, to the best of our knowledge, we report for the first time an essential step in the extraction of Rhodococcus biosurfactant. The crude biosurfactant appeared as a light brown coloured powder. The yield of crude biosurfactant was expressed in gram per liter of fermentation broth. The yields of crude biosurfactant before and after optimization were 3.2 g/L and 10.9 g/L, respectively. The optimization by RSM effectively enhanced the yield of biosurfactant to 3.4 times. The yield obtained from the optimized medium is considerably higher as compared to the reported yields from the previ-
ous reports (Ivshina et al., 1998; Kuyukina et al., 2001; Pirog et al., 2004). 3.5. Characterization of crude biosurfactant The crude biosurfactant was observed to form an o/w emulsion. The crude biosurfactant was found to contain 18.5% protein and 51.2% total carbohydrates. The crude biosurfactant decreased the surface tension of water from 72 mN/m to 30.8 mN/m (at 120 mg L1) and achieved a CMC value of 120 mg L1. 4. Conclusions The use of an organic nitrogen source gave higher cell density than inorganic nitrogen. Among the seven Rhodococcus strains selected for the work, MTCC 2574 was found to give the maximum cell density when grown on a medium containing mannitol as the carbon source. Among the seven different inducers studied for MTCC 2574, n-hexadecane gave the best results. In conclusion, the production of biosurfactant was found to depend greatly on the media components. Using the RSM, it was possible to model the individual and interactive effects of media
7880
S.R. Mutalik et al. / Bioresource Technology 99 (2008) 7875–7880
the components on biosurfactant yield. The validity of the model was confirmed by the close agreement between the experimental the and predicted values. The medium optimization by RSM effectively enhanced the biosurfactant yield by 3.4-folds. Acknowledgements The authors would like to thank Dr. S.S. Bhagwat (ICT, Mumbai) for his help in the measurement of surface tension and CMC. BKV would like to acknowledge the SRF grant from CSIR-India. RMJ would like to thank the financial support from DST-India. References Banat, I.M., Makkar, R.S., Cameotra, S.S., 2000. Potential commercial applications of microbial surfactants. Appl. Microbiol. Biotechnol. 53, 495–508. Bell, K.S., Philp, J.C., Aw, D.W., Christofi, N., 1998. The genus Rhodococcus. J. Appl. Microbiol. 85, 195–210. Bicca, F.C., Fleck, L.C., Ayub, M.A.Z., 1999. Production of biosurfactant by hydrocarbon degrading Rhodococcus ruber and Rhodococcus erythropolis. Rev. Microbiol. 30, 231–236. Christofi, N., Ivshina, I., 2002. Microbial surfactants and their use in field studies of soil remediation. J. Appl. Microbiol. 93, 915–929. Dubois, M., Gilles, K., Hamilton, J., Rebers, P., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356. Fleck, L.C., Bicca, F.C., Ayub, M.A.Z., 2000. Physiological aspects of hydrocarbon emulsification, metal resistance and DNA profile of biodegrading bacteria isolated from oil polluted sites. Biotechnol. Lett. 22, 285–289. Ghurye, G.L., Vipulanandan, C., 1994. A practical approach to biosurfactant production using nonaseptic fermentation of mixed cultures. Biotechnol. Bioeng. 44, 661–666. Ivshina, I.B., Kuyukina, M.S., Philp, J.C., Christofi, N., 1998. Oil desorption from mineral and organic materials using biosurfactant
complexes produced by Rhodococcus species. World J. Microbiol. Biotechnol. 14, 711–717. Kuyukina, M.S., Ivshina, I., Philip, J., Christofi, N., Dunbar, S., Ritchkova, M., 2001. Recovery of Rhodococcus biosurfactants using methyl tertiary-butyl ether extraction. J. Microbiol. 46, 149–156. Lang, S., Philp, J.C., 1998. Surface-active lipids in rhodococci. Anton van Leeuwen 74, 59–70. Lin, T.C., Young, C.C., Ho, M., Yeh, M.S., Chou, C.L., Wei, Y.H., Chang, J.S., 2005. Characterization of floating activity of indigenous diesel-assimilating bacterial isolates. J. Biosci. Bioeng. 99, 466–472. Lotfy, W.A., Ghanem, K.M., El-Helow, E.R., 2007. Citric acid production by a novel Aspergillus niger isolate: II. Optimization of process parameters through statistical experimental designs. Bioresour. Technol. 98, 3470–3477. Lowry, O.H., Roseburgh, N., Farr, A.L., Randall, R., 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265– 275. Mukherjee, S., Das, P., Sen, R., 2007. Towards commercial production of microbial surfactants. Trends Biotechnol. 24, 509–515. Nitschke, N., Pastore, G.M., 2006. Production and properties of a surfactant obtained from Bacillus subtilis grown on cassava wastewater. Bioresour. Technol. 97, 336–341. Pirog, T.P., Shevchuk, T.A., Voloshina, I.N., Karpenko, E.V., 2004. Production of surfactants by Rhodococcus erythropolis strain EK-1, grown on hydrophilic and hydrophobic substrates. Appl. Biochem. Microbiol. 40, 470–475. Pruthi, V., Cameotra, S.S., 1997. Rapid identification of biosurfactant producing bacterial strains using a cell surface hydrophobicity technique. Biotechnol. Tech. 11, 671–674. Rodrigues, L., Banat, I.M., Teixeira, J., Oliveira, R., 2006a. Biosurfactants: potential applications in medicine. J. Antimicrob. Chemother. 57, 609–618. Rodrigues, L., Teixeira, J., Oliveira, R., van der Mei, H.C., 2006b. Response surface optimization of the medium components for the production of biosurfactants by probiotic bacteria. Process Biochem. 41, 1–10. ¨ zer, D., Elibol, M., 2005. Optimization of a-amylase Tanyildizi, M.S., O production by Bacillus sp. using response surface methodology. Process Biochem. 40, 2291–2296. Tian, B., Dong, C., Chen, L., 1998. Preparation of Konjac glucomannan ester of palmitic acid and its emulsification. J. Appl. Polym. Sci. 67, 1035–1038.
Bioresource Technology 99 (2008) 7875–7880
Short Communication
Use of response surface optimization for the production of biosurfactant from Rhodococcus spp. MTCC 2574 Snehal R. Mutalik a, Bhalchandra K. Vaidya a, Renuka M. Joshi a, Kiran M. Desai b, Sanjay N. Nene a,* a
Biochemical Engineering Department, Chemical Engineering and Process Development Division, National Chemical Laboratory, Pune 411008, India b Food Engineering and Technology Department, Institute of Chemical Technology, Matunga, Mumbai 400019, India Received 21 September 2007; received in revised form 21 February 2008; accepted 21 February 2008 Available online 3 June 2008
Abstract The production of biosurfactant from Rhodococcus spp. MTCC 2574 was effectively enhanced by response surface methodology (RSM). Rhodococcus spp. MTCC 2574 was selected through screening of seven different Rhodococcus strains. The preliminary screening experiments (one-factor at a time) suggested that carbon source: mannitol, nitrogen source: yeast extract and meat peptone and inducer: n-hexadecane are the critical medium components. The concentrations of these four media components were optimized by using central composite rotatable design (CCRD) of RSM. The adequately high R2 value (0.947) and F score 19.11 indicated the statistical significance of the model. The optimum medium composition for biosurfactant production was found to contain mannitol (1.6 g/L), yeast extract (6.92 g/L), meat peptone (19.65 g/L), n-hexadecane (63.8 g/L). The crude biosurfactant was obtained from methyl tert-butyl ether extraction. The yield of biosurfactant before and after optimization was 3.2 g/L of and 10.9 g/L, respectively. Thus, RSM has increased the yield of biosurfactant to 3.4-fold. The crude biosurfactant decreased the surface tension of water from 72 mN/m to 30.8 mN/m (at 120 mg L1) and achieved a critical micelle concentration (CMC) value of 120 mg L1. Ó 2008 Published by Elsevier Ltd. Keywords: Biosurfactant; Rhodococcus spp.; Response surface methodology; Medium optimization
1. Introduction Biosurfactants or microbial surfactants are surfaceactive biomolecules produced by a variety of microorganisms. Biosurfactants are receiving considerable attention due to their unique properties such as higher biodegradability, lower toxicity, and greater stability towards temperature and pH (Mukherjee et al., 2007). In the past few decades, biosurfactants have been identified for commercial importance, specifically, in the field of oil recovery (Ivshina et al., 1998); secondary or tertiary environmental bioremediation (Christofi and Ivshina, 2002); pharmaceuticals (Rodrigues et al., 2006a); and food processing and cosmetics (Banat et al., 2000). *
Corresponding author. Tel.: +91 20 25902347; fax: +91 20 25902612. E-mail address: [email protected] (S.N. Nene).
0960-8524/$ - see front matter Ó 2008 Published by Elsevier Ltd. doi:10.1016/j.biortech.2008.02.027
Members of the genus Rhodococcus are known to produce surface-active trehalose-lipids. Bell et al. (1998) have reported that some biosurfactants including those from rhodococci are more effective and efficient than many existing synthetic surface-active agents. The production of biosurfactant from different strains of Rhodococcus has been reported in the literature (Lang and Philp, 1998; Kuyukina et al., 2001; Pirog et al., 2004). The yield of biosurfactant varies considerably with the hydrocarbon which is used to induce the production of biosurfactant. The potential of Rhodococcus biosurfactant in a variety of industrial applications has been proposed, but like other biosurfactants it is yet to achieve market penetration (Mukherjee et al., 2007). Rhodococcus biosurfactants have not achieved significant market share because of their high cost as compared to synthetic surfactants. Thus, to compete with the large-scale production of synthetic surfactants from
7876
S.R. Mutalik et al. / Bioresource Technology 99 (2008) 7875–7880
hydrocarbon feedstocks, it is desirable to enhance the production of biosurfactant by Rhodococcus. One of the methods of achieving the above objective is the selection of appropriate media components and optimal culture conditions for maximum biosurfactant productivity. The limitations of classical method of media optimization can be overcome by the application of statistical based approach (Lotfy et al., 2007; Tanyildizi et al., 2005). RSM, an extensively used statistical technique for media optimization, is a collection of statistical techniques which uses design of experiments (DoE) for building models, evaluating the effects of factors and searching for the optimum conditions. (Rodrigues et al., 2006b). In the present work, we have used a central composite rotatable design (CCRD) of response surface methodology for media optimization to enhance biosurfactant production by Rhodococcus spp. MTCC 2574. 2. Methods 2.1. Materials All bacterial growth media components were purchased from Hi-Media, India. All other chemicals were of analytical grade procured from S.D. Fine Chemicals, India. 2.2. Microorganism and growth conditions The seven microbial strains namely Rhodococcus spp. MTCC 2574, 2678, 2683 and Rhodococcus erythropolis MTCC 1526, 1548, 2794, 3951 were procured from MTCC-Chandigarh (India). All Rhodococcus strains were maintained on nutrient agar (beef extract 10 g/L, NaCl 5 g/L, peptone 10 g/L, agar 20 g/L, pH 7.0–7.5). The liquid fermentation medium used for batch culture experiments contained (g L1) glucose (10), yeast extract (3), meat peptone (7.5), Na2HPO4 (4.0), KH2PO4 (2.0), MgSO4 7H2O (0.2), CaCl2 2H2O (0.02), ammonium ferric citrate (0.05), trace mineral solution (1 mL/L) [termed as medium A]. The composition of trace mineral medium was (g L1) H3BO3 (0.1), MnCl2 4H2O (0.1), ZnSO4 H2O (0.1), FeCl3 6H2O (0.1), CaCl2 2H2O (1), CuCl2 2H2O (0.05). Five milliliter of inoculum was transferred to 45 mL of liquid fermentation medium contained in a 250 mL Erlenmeyer flask and incubated for 36 h at 30 °C on rotary shaker at 200 rpm. 2.3. Selection of optimum nitrogen source, carbon source and inducer The organic nitrogen source (yeast extract and meat peptone) from Medium A was replaced by inorganic nitrogen sources (namely urea, ammonium sulphate and ammo-
% EI24 ¼
nium phosphate) at equivalent nitrogen level. To evaluate the optimum carbon source, glucose was replaced by an equivalent amount of different carbon sources namely glycerol, sucrose, sorbitol and mannitol. Seven inducers (3% v/ v each) were screened to evaluate the corresponding enhancement in biosurfactant production. Biosurfactant production was calculated in terms of emulsification index (% EI24) as described in Section 2.7.1. 2.4. Biosurfactant profile The cell growth and biosurfactant production were simultaneously studied to establish the biosurfactant profile. Cells of Rhodococcus spp. MTCC 2574 were grown on liquid medium containing (g L1) mannitol (10.12), yeast extract (3), meat peptone (7.5), Na2HPO4 (4.0), KH2PO4 (2.0), MgSO4 7H2O (0.2), CaCl2 2H2O (0.02), ammonium ferric citrate (0.05), trace mineral solution (1 mL/L) and n-hexadecane (3% v/v) (termed as medium B). The samples were aseptically removed at a regular interval of 4 h up to 48 h and analyzed for optical density (600 nm), % EI24 and % hydrophobicity. 2.5. Experimental design and data analysis To examine the combined effect of four different medium components (mannitol, yeast extract, meat peptone and n-hexadecane) on biosurfactant production by Rhodococcus spp. MTCC 2574, 30 experiments were performed in duplicate. The value of the dependent response (% EI24) was the mean of two replications. The second-order polynomial coefficients were calculated and analyzed using the trial version of ‘Design Expert’ software (Version 6.0, Stat-Ease Inc., USA). 2.6. Extraction of biosurfactant The extraction of biosurfactant was done by using methyl tert-butyl ether (MTBE) as described earlier by Kuyukina et al. (2001). The product was thoroughly washed thrice with petroleum ether to remove residual n-hexadecane to obtain crude biosurfactant. The crude biosurfactant was finally dried by lyophilization. 2.7. Analytical methods 2.7.1. Emulsification Index (% EI24) Emulsification Index (% EI24) was used to quantify the biosurfactant produced by Rhodococcus cells (Fleck et al., 2000). The values of % EI24 were determined as described earlier by Nitschke and Pastore (2006). The percent ratio of the height of emulsified zone to total height after 24 h gives ‘emulsification index’ (% EI24) as given in Eq. (1)
Height of emulsified zone 100 Total height of liquidðsum of aqueous; oil and emulsified zoneÞ
ð1Þ
S.R. Mutalik et al. / Bioresource Technology 99 (2008) 7875–7880
2.7.2. % Cellular hydrophobicity Pruthi and Cameotra (1997) reported an increase in the cell surface hydrophobicity with the biosynthesis of biosurfactant. % Cellular hydrophobicity was determined as described earlier (Lin et al., 2005). The % cellular hydrophobicity was calculated as expressed in Eq. (2)
7877
not shown) for all seven Rhodococcus strains and therefore organic nitrogen sources were selected for further studies. The effect of carbon source on cell density for seven Rhodococcus strains is given in Fig. 1. The optimum carbon source was found to vary depending upon the Rhodococcus strain. Among the different Rhodococcus strains, MTCC
OD400 of aqueous phase of culture after the addition of n-dodecane % Hydrophobicity ¼ 1 100 OD400 of culture before the addition of n-dodecane
2.7.3. Characterization of crude biosurfactant The type of emulsion was determined by using methyl orange and Sudan red III as described by Tian et al. (1998). Total carbohydrate and protein content of crude biosurfactant were estimated by phenol sulphuric acid method (Dubois et al., 1956) and Folin Lowry method (Lowry et al., 1951), respectively. Surface tension measurement and critical micelle concentration were detected using Kruss K-11 Tensiometer (accuracy ± 0.1 mN/m). 3. Results and discussion The biosurfactant production by Rhodococcus spp. is reported to be primarily cell-growth associated (Ghurye and Vipulanandan, 1994; Rodrigues et al., 2006b). The initial attempts therefore were made to get high cell density, based on a one-factor-at-a-time strategy.
ð2Þ
2574 gave a maximum cell density when grown on mannitol as carbon source. Here, seven hydrocarbons were evaluated for the enhancement of biosurfactant production in MTCC 2574 (results not shown). The production of biosurfactant was expressed in terms of emulsification index (% EI24). The fermentation broth was subjected for centrifugation and the cell-free supernatant and sonicated cell-suspension were analyzed for % EI24. The % EI24 values of cell-free supernatant were very low (in the range of 0–7%) as compared with sonicated cell-suspension. This indicates that a negligible amount of biosurfactant is released from the cell surface to the fermentation broth and confirms the cell-wall associated nature of Rhodococcus biosurfactant as described by Bicca et al. (1999). n-Hexadecane gave maximum EI24 (59.2%) and hence selected for further experiments. 3.2. Biosurfactant profile
3.1. Selection of optimum nitrogen source, carbon source and inducer The complete replacement of organic nitrogen by inorganic nitrogen resulted in a very low cell growth (results
The cell density, % EI24 and % cellular hydrophobicity were studied with respect to fermentation time, and the biosurfactant profile was established (Fig. 2). During the course of fermentation, % EI24 was found to remain almost
25
O.D. (600 nm) at 36 h
20
15
10
5
0 1548 Glucose
2678
3951 Sucose
2683 Sorbitol
2794
2574
1526
Mannitol
Glycerol
Fig. 1. Effect of carbon source on the biomass of different Rhodococcus strains.
7878
S.R. Mutalik et al. / Bioresource Technology 99 (2008) 7875–7880
Fig. 2. Biosurfactant profile (i.e. the effect of fermentation time on cell density, % EI24 and % hydrophobicity).
constant up to 20 h % EI24 was found to increase after 20 h, reached a maximum at 28 h and thereafter remained constant till 36 h % EI24 was found to decline along with cell density from 36 h to 48 h. Thus, the biosurfactant content of the growing Rhodococcus spp. MTCC 2574 was found to reach a maximum at 28 h. Therefore, all further fermentations were terminated at 28 h. The % hydrophobicity of each sample was carried out in order to confirm the biosurfactant production. The % hydrophobicity increased with increase in cell density, reached a maximum at 28 h and was found to decline from 36 h to 48 h. 3.3. Optimization of biosurfactant production The coded values of independent variables are given in Table 1. The design of experiments and the respective experimental and predicted % EI24 are given in Table 2. After regression analysis, the second-order response model was obtained which is given in Eq. (3) % EI24 ¼ 33:21 þ 11:55A þ 0:455B 5:20583C þ 2:89833D þ 4:97708A2 þ 4:99583B2 þ 4:98458C2 1:43667D2 2:86625A B 3:35000A C þ 1:41250A D þ 8:53125B C þ 1:94125B D þ 1:56750C D
ð3Þ
(where A: mannitol, B: yeast peptone, C: meat peptone, D: n-hexadecane and A, C, D, A2, B2, C2, AB, AC, BC were identified as significant terms). A low value of the coefficient of variation (12.05%) indicates the very high degree of precision and a good reliability of the experimental val-
Table 1 Coded values of independent variables* No.
1 2 3 4
Variable
Mannitol Yeast extract Meat peptone n-Hexadecane
Coded values 2
1
0
1
2
0 0 0 0
10 2 5 20
20 4 10 40
30 6 15 60
40 8 20 80
* Concentrations in g/L.
ues. The fit of the model was also expressed by the coefficient of determination R2, which was found to be 0.947, indicating that 94.7% of the variability in the response could be explained by the model. The solution was obtained by substituting the levels of the factors into the regression equation. The optimal concentrations for the four components were found as mannitol (1.6 g/L), yeast extract (6.92 g/L), meat peptone (19.65 g/L), n-hexadecane (63.8 g/L) (termed as medium C, all remaining components are in the same concentration as medium B). The predicted response (95.3%) was experimentally verified (93.9%). The agreement between predicted value and experimental value of % EI24 confirms the significance of the model. In the optimized medium, the concentration of n-hexadecane (63.8 g/L) was relatively high as compared to mannitol (1.6 g/L). This indicates that the elevated hydrocarbon contents are necessary for higher biosurfactant production. Here, it can be speculated that the mannitol acts as a primary carbon source during the initial growth phase, while in the latter stages of fermentation, Rhodococcus cells probably use n-hexadecane as a carbon source. Thus, an appropriate combination of a simple carbon source and an inducer enhanced the production of biosurfactant.
S.R. Mutalik et al. / Bioresource Technology 99 (2008) 7875–7880
7879
Table 2 Central composite rotatable design (CCRD) matrix of independent variables and their corresponding experimental and predicted yields of biosurfactant No.
Media components (coded values) Mannitol
Yeast extract
1 1 1 2 1 1 3 1 1 4 1 1 5 1 1 6 1 1 7 1 1 8 1 1 9 1 1 10 1 1 11 1 1 12 1 1 13 1 1 14 1 1 15 1 1 16 1 1 17 2 0 18 2 0 19 0 2 20 0 2 21 0 0 22 0 0 23 0 0 24 0 0 25 0 0 26 0 0 27 0 0 28 0 0 29 0 0 30 0 0 * Values indicate mean of duplicate observations.
% EI24 Meat peptone
n-Hexadecane
Experimental*
Predicted
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 2 2 0 0 0 0 0 0 0 0
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 2 2 0 0 0 0 0 0
35.21 78.9 32.9 50.0 24.9 35.4 39.6 50.0 36.2 78.9 37.8 54.0 23.6 42.6 46.1 72.5 31.5 77.0 58.4 50.3 68.2 40.3 22.4 34.8 33.9 32.7 33.3 32.7 33.9 32.7
44.3 76.9 29.9 51.2 20.4 39.7 40.2 48.0 40.2 78.6 33.7 60.6 22.6 47.6 50.2 63.7 30.0 76.2 52.3 54.1 63.6 42.7 21.7 33.3 33.2 33.2 33.2 33.2 33.2 33.2
3.4. MTBE extraction of biosurfactant The MTBE extraction method described by Kuyukina et al. (2001) was used for the isolation of biosurfactant from Rhodococcus spp. 2574 grown on optimum medium (Medium C, described in Section 3.3). However, the product obtained from this method is known to contain a large amount (approximately 40–50%) of residual n-hexadecane (Kuyukina et al., 2001). Due to the presence of residual n-hexadecane, the accurate yield of the product cannot be determined. In order to remove residual n-hexadecane, the extract was treated with petroleum ether (around 3–4 times) repeatedly and freeze-dried. This method effectively removed the residual n-hexadecane. Here, to the best of our knowledge, we report for the first time an essential step in the extraction of Rhodococcus biosurfactant. The crude biosurfactant appeared as a light brown coloured powder. The yield of crude biosurfactant was expressed in gram per liter of fermentation broth. The yields of crude biosurfactant before and after optimization were 3.2 g/L and 10.9 g/L, respectively. The optimization by RSM effectively enhanced the yield of biosurfactant to 3.4 times. The yield obtained from the optimized medium is considerably higher as compared to the reported yields from the previ-
ous reports (Ivshina et al., 1998; Kuyukina et al., 2001; Pirog et al., 2004). 3.5. Characterization of crude biosurfactant The crude biosurfactant was observed to form an o/w emulsion. The crude biosurfactant was found to contain 18.5% protein and 51.2% total carbohydrates. The crude biosurfactant decreased the surface tension of water from 72 mN/m to 30.8 mN/m (at 120 mg L1) and achieved a CMC value of 120 mg L1. 4. Conclusions The use of an organic nitrogen source gave higher cell density than inorganic nitrogen. Among the seven Rhodococcus strains selected for the work, MTCC 2574 was found to give the maximum cell density when grown on a medium containing mannitol as the carbon source. Among the seven different inducers studied for MTCC 2574, n-hexadecane gave the best results. In conclusion, the production of biosurfactant was found to depend greatly on the media components. Using the RSM, it was possible to model the individual and interactive effects of media
7880
S.R. Mutalik et al. / Bioresource Technology 99 (2008) 7875–7880
the components on biosurfactant yield. The validity of the model was confirmed by the close agreement between the experimental the and predicted values. The medium optimization by RSM effectively enhanced the biosurfactant yield by 3.4-folds. Acknowledgements The authors would like to thank Dr. S.S. Bhagwat (ICT, Mumbai) for his help in the measurement of surface tension and CMC. BKV would like to acknowledge the SRF grant from CSIR-India. RMJ would like to thank the financial support from DST-India. References Banat, I.M., Makkar, R.S., Cameotra, S.S., 2000. Potential commercial applications of microbial surfactants. Appl. Microbiol. Biotechnol. 53, 495–508. Bell, K.S., Philp, J.C., Aw, D.W., Christofi, N., 1998. The genus Rhodococcus. J. Appl. Microbiol. 85, 195–210. Bicca, F.C., Fleck, L.C., Ayub, M.A.Z., 1999. Production of biosurfactant by hydrocarbon degrading Rhodococcus ruber and Rhodococcus erythropolis. Rev. Microbiol. 30, 231–236. Christofi, N., Ivshina, I., 2002. Microbial surfactants and their use in field studies of soil remediation. J. Appl. Microbiol. 93, 915–929. Dubois, M., Gilles, K., Hamilton, J., Rebers, P., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356. Fleck, L.C., Bicca, F.C., Ayub, M.A.Z., 2000. Physiological aspects of hydrocarbon emulsification, metal resistance and DNA profile of biodegrading bacteria isolated from oil polluted sites. Biotechnol. Lett. 22, 285–289. Ghurye, G.L., Vipulanandan, C., 1994. A practical approach to biosurfactant production using nonaseptic fermentation of mixed cultures. Biotechnol. Bioeng. 44, 661–666. Ivshina, I.B., Kuyukina, M.S., Philp, J.C., Christofi, N., 1998. Oil desorption from mineral and organic materials using biosurfactant
complexes produced by Rhodococcus species. World J. Microbiol. Biotechnol. 14, 711–717. Kuyukina, M.S., Ivshina, I., Philip, J., Christofi, N., Dunbar, S., Ritchkova, M., 2001. Recovery of Rhodococcus biosurfactants using methyl tertiary-butyl ether extraction. J. Microbiol. 46, 149–156. Lang, S., Philp, J.C., 1998. Surface-active lipids in rhodococci. Anton van Leeuwen 74, 59–70. Lin, T.C., Young, C.C., Ho, M., Yeh, M.S., Chou, C.L., Wei, Y.H., Chang, J.S., 2005. Characterization of floating activity of indigenous diesel-assimilating bacterial isolates. J. Biosci. Bioeng. 99, 466–472. Lotfy, W.A., Ghanem, K.M., El-Helow, E.R., 2007. Citric acid production by a novel Aspergillus niger isolate: II. Optimization of process parameters through statistical experimental designs. Bioresour. Technol. 98, 3470–3477. Lowry, O.H., Roseburgh, N., Farr, A.L., Randall, R., 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265– 275. Mukherjee, S., Das, P., Sen, R., 2007. Towards commercial production of microbial surfactants. Trends Biotechnol. 24, 509–515. Nitschke, N., Pastore, G.M., 2006. Production and properties of a surfactant obtained from Bacillus subtilis grown on cassava wastewater. Bioresour. Technol. 97, 336–341. Pirog, T.P., Shevchuk, T.A., Voloshina, I.N., Karpenko, E.V., 2004. Production of surfactants by Rhodococcus erythropolis strain EK-1, grown on hydrophilic and hydrophobic substrates. Appl. Biochem. Microbiol. 40, 470–475. Pruthi, V., Cameotra, S.S., 1997. Rapid identification of biosurfactant producing bacterial strains using a cell surface hydrophobicity technique. Biotechnol. Tech. 11, 671–674. Rodrigues, L., Banat, I.M., Teixeira, J., Oliveira, R., 2006a. Biosurfactants: potential applications in medicine. J. Antimicrob. Chemother. 57, 609–618. Rodrigues, L., Teixeira, J., Oliveira, R., van der Mei, H.C., 2006b. Response surface optimization of the medium components for the production of biosurfactants by probiotic bacteria. Process Biochem. 41, 1–10. ¨ zer, D., Elibol, M., 2005. Optimization of a-amylase Tanyildizi, M.S., O production by Bacillus sp. using response surface methodology. Process Biochem. 40, 2291–2296. Tian, B., Dong, C., Chen, L., 1998. Preparation of Konjac glucomannan ester of palmitic acid and its emulsification. J. Appl. Polym. Sci. 67, 1035–1038.