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Brazilian Journal of Chemical Engineering

Print version ISSN 0104-6632On-line version ISSN 1678-4383

Braz. J. Chem. Eng. vol.25 no.2 São Paulo Apr./June 2008 



Use of weathered diesel oil as a low-cost raw material for biosurfactant production



A. P. MarianoI, II, *; D. M. BonottoI; D. F. AngelisII; M. P. S. PirôlloII; J. ContieroII

IInstituto de Geociências e Ciências Exatas (IGCE), Universidade Estadual Paulista (Unesp)
IIDepartamento de Bioquímica e Microbiologia, Instituto de Biociências (IB), Universidade Estadual Paulista (Unesp), Av. 24-A, 1515, Cx. P. 199, CEP: 13506-900, Rio Claro - SP, Brasil. E-mail:




This work aimed to investigate the capability of biosurfactant production by Staphylococcus hominis, Kocuria palustris and Pseudomonas aeruginosa LBI, using weathered diesel oil from a long-standing spillage as raw material. The effect of the culture media (Robert or Bushnell-Haas) and of the carbon source (spilled diesel oil or commercial diesel oil) on biosurfactant production was evaluated. Erlenmeyer flasks (250 mL) containing the cell broth were agitated (240 rpm) for 144 h at 27±2ºC. Biosurfactant production was monitored according to the De Nöuy ring method using a Krüss K6 tensiometer. Considering the possibility of intracellular storage of biosurfactant in the cell wall of the cultures S. hominis and K. palustris, experiments were also done applying ultrasound as a way to rupture the cells. For the conditions studied, the cultures did not indicate production of biosurfactants. Results obtained with a hydrocarbon biodegradability test based on the redox indicator 2,6-dichlorophenol indophenol showed that only the commercial diesel was biodegraded by the cultures.

Keywords: Biosurfactant; Diesel oil; Raw material; Weathered.




Commercial viability of biosurfactants is still limited by their high production costs, associated with inefficient recovery methods and with the use of expensive raw materials (Nitschke and Pastore, 2002). These costs can be significantly reduced with the development of cheaper processes and the use of low-cost raw materials, which account for 10-30% of the overall cost (Cameotra and Makkar, 1998). Biosurfactants can be commercially produced at levels of up to 100 g/L, as reported for rhamnolipids from Pseudomonas (Maier and Soberon-Chavez, 2000). This production level, combined with the use of cheap renewable substrates as organic wastes, makes the cost of biosurfactants competitive with the cost of synthetic surfactants (Makkar and Cameotra, 2002). Alternative substrates have been suggested for biosurfactant production, especially water-miscible agro-industrial wastes: molasses, whey, cassava wastewater and distillery wastes (Babu et al., 1996; Nitschke and Pastore, 2006). However, there are few examples of the use of hydrophobic wastes as cheap substrates, for instance, waste frying oils, used lubricant oils and oily sludge from petroleum refineries (Mercadé et al., 1996; Haba et al., 2000; Pirôllo, 2006). Thus, this work aimed to verify the capability of three bacterial cultures to produce biosurfactants using weathered diesel oil recovered from the groundwater at a petrol station as raw material.



Growth Conditions

Experiments were carried out in 250 mL Erlenmeyer flasks containing 50 mL liquid medium. Different combinations of culture media and carbon sources were evaluated. Thus, the effect of two media on biosurfactant production was tested: a) the medium described by Robert et al. (1989) and the Bushnell-Hass (BH) medium (Difco, 1984). The former medium has been employed in biosurfactant production experiments using different strains of P. aeruginosa (Robert et al., 1989; Benincasa et al., 2002; Silva, 2005; Pirôllo, 2006), while the BH medium is normally used in studies where the biodegradation of hydrocarbons is evaluated (Hanson et al., 1993). As carbon sources, we tested the diesel oil (W-D) recovered from the groundwater at a petrol station [whose soil was the source of the cultures Staphylococcus hominis and Kocuria palustris (Mariano et al., 2007)] and, comparatively, a commercial diesel oil (C-D) purchased from a local petrol station. The mineral media plus the carbon source [different concentrations of diesel oil (Table 1)] were sterilised together in an autoclave at 121ºC and 1 atm for 15 minutes. Then, inoculum (1 mL; O.D. of 0.65 at 610 nm SHIMADZU UV-1601PC) was added to the flasks, which were kept under agitation (240 rpm) for 144 hours at 27±2ºC. Experiments including the strain Pseudomonas aeruginosa LBI [isolated by Benincasa et al. (2002) from a hydrocarbon contaminated area] and the Robert media with the commercial diesel were not carried out because this strain had previously proved to be an efficient biosurfactant producer under these conditions (Pirôllo, 2006).



The spillage at the petrol station that released diesel oil into the groundwater occurred approximately ten years ago. Analyses of BTEX (benzene, toluene, ethylbenzene and xylenes), PAH (polyaromatic compounds) and TPH (total petroleum hydrocarbons) showed that the composition of the diesel oil had been altered probably due to both biological and physico-chemical processes (Mariano, 2006).

Surface Tension Measurement

After the incubation period, the surface tension of the cell-free culture was measured employing the De Nöuy ring method with a Krüss K6 tensiometer.

Verification of Bioemulsifiers

For experiments 1, 4, 5, 11, 14 and 15 (Table 1), the emulsion index (E24) was determined in accordance with Iqbal et al. (1995) for the weathered diesel oil (W-D) or kerosene.

Application of Ultrasound

Considering the possibility of intracellular storage of biosurfactant in the cell wall (Gomes et al., 2004), in some experiments (Table 1) ultrasound was also applied as a way to rupture the cells to obtain the release of biosurfactant into the broth. The ultrasound was irradiated in the culture broth (with the cells) before measuring the superficial tension. A cells disrupter (UNIQUE) was employed (ultrasonic frequency of 20 KHz, 306 W). For experiments 10 and 20, the ultrasound was irradiated for 20 minutes and for experiments 6, 8, 16 and 18, for 10 minutes. The ultrasound application was only employed in experiments with the cultures S. hominis and K. palustris because the strain P. aeruginosa LBI is an extracellular biosurfactant producer (Benincasa et al., 2002).

Diesel oil Biodegradability Test

The diesel oil biodegradability test was done using the redox indicator 2,6-dichlorophenol indophenol (DCPIP) (Hanson et al., 1993). During the microbial oxidation of hydrocarbons, electrons are transferred to electron acceptors. By incorporating an electron acceptor such as DCPIP into the culture medium, it is possible to ascertain the ability of the microorganism to utilise hydrocarbon substrate by observing the colour change of DCPIP from blue (oxidised) to colourless (reduced). The capability of the cultures S. hominis, K. palustris and P. aeruginosa LBI to degrade both diesel oils (W-D and C-D) was verified. Inoculum was added (125 µL, O.D. = 0.55) to test tubes (duplicates) that contained sterile BH medium (7.5 mL) and 50 µL of diesel oil. The concentration of DCPIP was 27 mg/mL. Test tubes were kept under agitation (240 rpm) at 27±2ºC.



Table 1 shows the results of superficial tension measures of the culture broth without inoculum (initial superficial tension) and after incubation time (final superficial tension) in accordance with the experimental conditions. Results demonstrate that no significant decrease in superficial tension was verified in any of the experiments. According to Haba et al. (2000), good biosurfactant producers were considered to be those that decrease the surface tension to 40 mN/m or less. Thus no biosurfactant production was obtained in the experiments. In other works, the strain P.aeruginosa LBI was capable of producing the rhamnolipid biosurfactant using soapstock (Moraes et al., 2002), mannitol and glycerol (Silva, 2005) and kerosene, diesel oil, crude oil and oily sludge (Pirôllo, 2006).

Emulsification tests in experiments 1, 4, 5, 11, 14 and 15 demonstrated that no bioemulsifier was produced by the cultures S. hominis and K. palustris, since the E24 for both diesel oil and kerosene was zero. Carrying out this test is important because not all biosurfactants show the property of reducing the surface tension. However, some biosurfactants, such as emulsan, are good emulsifier agents of hydrocarbons (Kim et al., 1997). According to Ron and Rosemberg (2001), biosurfactants with low-molecular-weight molecules lower surface and interfacial tensions efficiently, while high-molecular-weight polymers are good emulsifiers. The rhamnolipid biosurfactant produced by the strain P. aeruginosa LBI from commercial diesel (Pirôllo, 2006), was capable of emulsifying different compounds, such as benzene, kerosene, diesel oil and crude oil.

The results obtained with the biodegradability test are presented in Table 2. Examining the results for P. aeruginosa LBI, it can be seen that they agree with those obtained in the biosurfactant production, i.e., this culture was only capable of producing biosurfactant using a carbon source (C-D) that could be biodegraded (Pirôllo, 2006).



Both cultures S. hominis and K. palustris demonstrated the capability to degrade the commercial diesel oil. However, it is known that not all hydrocarbonoclastic microorganisms are able to produce biosurfactants. Some microbial cells show high superficial hydrophobicity and are considered a biosurfactant, such as hydrocarbonoclastic bacteria, some species of Cyanobacteria and some pathogens such as S. aureus and Serratia sp. (Nitschke and Pastore, 2002). No study relating the cultures S. hominis and K. palustris to hydrocarbon biodegradation and biosurfactant production was found. Nevertheless, Gomes et al. (2004) describe the biosurfactant storage in the cell wall of a strain of S. aureus.

Analyzing the effect of culture medium on biosurfactant production, in Pirôllo (2006) the culture P. aeruginosa LBI was capable of producing biosurfactant in Robert medium and using commercial diesel oil as carbon source. This suggests that the BH medium (as employed in experiment 23) can not be suitable for biosurfactant production, especially in cases where hydrocarbons are used as raw material. This medium does not have any easily biodegradable carbon source, such as yeast extract, which can be used as a start-up for the microorganisms. Moreover, BH medium has a high iron concentration. Ramana and Karanth (1989) observed that the presence of iron in the medium inhibited the production of glycolipids by P. aeruginosa CFTR-6, mainly in concentrations of FeSO4.7H2O higher than 0.01 g/L. This fact was also verified by Silva (2005) with the strain P. aeruginosa LBI. Further discussions about the effect of culture medium on biosurfactant production can be found in Guerra-Santos et al. (1984) and Kim et al. (2003).

Another question to be considered is that even cultures with proved capability to produce biosurfactant, for instance P. aeruginosa LBI, can not show an immediate positive response to raw materials whose characteristics were altered by physico-chemical and biological mechanisms due to a long period these compounds were under environmental conditions, as the weathered diesel oil used in this work, in which there are more recalcitrant fractions (Mariano, 2006). This fact may in some cases lower the in-situ production of biosurfactants in bioremediation processes of long-standing contaminated areas based on the addition of these microorganisms.

Attempts to use hydrophobic wastes as cheap substrates must not be disregarded, since other studies have proved that it is possible. Moreover, the use of low-cost raw material in production of biosurfactants may not only minimise industrial pollutants, but also simultaneously generate extremely useful and value-added products.



The authors acknowledge the support received from the Agência Nacional do Petróleo, Gás Natural e Biocombustíveis (ANP) (PRH-05) and the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP).



Babu, P.S., Vaidya, A.N., Bal, A.S., Kapur, R., Juwarkar, A. and Khanna, P., Kinetics of biosurfactant production by Pseudomonas aeruginosa strain BS2 from industrial wastes, Biotechnology Letters, 18, 263 (1996).         [ Links ]

Benincasa, M., Contiero, J., Manresa, M.A. and Moraes, I.O., Rhamnolipid production by Pseudomonas aeruginosa LBI growing on soapstock as the carbon source, Journal of Food Engineering, 54, 283 (2002).         [ Links ]

Cameotra, S.S. and Makkar, R.S., Synthesis of biosurfactants in extreme conditions, Applied Microbiology and Biotechnology, 50, 520 (1998).         [ Links ]

Difco, Manual 10th ed. Detroit: Difco Laboratories (1984).         [ Links ]

Gomes, R.V., Martins, S.C.S. and Melo, V.M.M., Produção de biossurfactante por Staphylococcus aureus isolado de uma amostra de petróleo pesado. IX ENAMA – Encontro Nacional de Microbiologia Ambiental, Curitiba (2004).         [ Links ]

Guerra-Santos, L.H., Käppeli, O. and Fiechter, A., Pseudomonas aeruginosa biosurfactant production in continuous culture with glucose as carbon source, Appl. Environ. Microbiol., 48, 301 (1984).         [ Links ]

Haba, E., Espuny, M.J., Busquets, M. and Manresa, A., Screening and production of rhamnolipids by Pseudomonas aeruginosa 47T2 NCIB 40044 from waste frying oils, Journal of Applied Microbiology, 88, 379 (2000).         [ Links ]

Hanson, K.G., Desai, J.D. and Desai, A.J., A rapid and simple screening technique for potential crude oil degrading microorganisms, Biotechnology Techniques, 7, 745 (1993).         [ Links ]

Iqbal, S., Khalid, Z.M. and Malik, K.A., Enhanced biodegradation and emulsification of crude oil and hyperproduction of biosurfactants by a gamma ray-induced mutant of Pseudomonas aeruginosa, Letters in Applied Microbiology, 21, 176 (1995).         [ Links ]

Kim, P., Oh, D., Kim, S. and Kim, J., Relationship between emulsifying activity and carbohydrate backbone structure of emulsan from Acinetobacter calcoaceticus RAG-1, Biotechnology Letters, 19, 457 (1997).         [ Links ]

Kim, E.J., Sabra, W. and Zeng, A.P., Iron deficiency leads to inhibition of oxygen transfer and enhanced formation of virulence factors in cultures of Pseudomonas aeruginosa PAO1. Microbiol., 149, 2627 (2003).         [ Links ]

Maier, R.M. and Soberon-Chavez, G., Pseudomonas aeruginosa rhamnolipids: Biosynthesis and potential applications, Appl. Microbiol. Biotechnol., 54, 625 (2000).         [ Links ]

Makkar, R.S. and Cameotra, S.S., An update on the use of unconventional substrates for biosurfactant production and their new applications, Appl. Microbiol. Biotechnol., 58, 428 (2002).         [ Links ]

Mariano, A.P., Avaliação do potencial de biorremediação de solos e de águas subterrâneas contaminados com óleo diesel. Ph.D. diss., Instituto de Geociências e Ciências Exatas (IGCE)-Universidade Estadual Paulista (Unesp) (2006).         [ Links ]

Mariano, A.P., Kataoka, A.P.A.G., Angelis, D.F. and Bonotto, D.M., Laboratory study on the bioremediation of diesel oil contaminated soil from a petrol station, Brazilian Journal of Microbiology, 38 (2), 346 (2007).         [ Links ]

Mercadé, M.E., Monleon, L., de Andres, C., Rodon, I., Martinez, E., Espuny, M.J. and Manresa, A., Screening and selection of surfactant-producing bacteria from waste lubricating oil, J. Appl. Bacteriol., 81, 161 (1996).         [ Links ]

Moraes, I.O., Benincasa, M. and Monte Alegre, R., Production and characterization of rhamnolipids produced by a newly isolated strain of Pseudomonas aeruginosa, Braz. J. Food Technol., 5, 145 (2002).         [ Links ]

Nitschke, M. and Pastore, G.M., Biossurfactantes: propriedades e aplicações, Química Nova, 25, 772 (2002).         [ Links ]

Nitschke, M. and Pastore, G.M., Production and properties of a surfactant obtained from Bacillus subtilis grown on cassava wastewater, Bioresource Technology, 97, 336 (2006).         [ Links ]

Pirôllo, M.P.S., Estudo da produção de biossurfactantes utilizando hidrocarbonetos. Master's thesis, Instituto de Biociências-Universidade Estadual Paulista (Unesp) (2006).         [ Links ]

Ramana, K.V. and Karanth, N.G., Factors affecting biosurfactant production using Pseudomonas aeruginosa CFTR-6 under submerged conditions, Journal of Chemical Technology and Biotechnology, 45, 249 (1989).         [ Links ]

Robert, M., Mercadé, M.E., Bosh, M.P., Parra, J.L., Espuny, M.J., Manresa, M.A. and Guinea, J., Effect of the carbon source on biosurfactant production by Pseudomonas aeruginosa 44T1, Biotechnology Letters, 11, 871 (1989).         [ Links ]

Ron, E.Z. and Rosemberg, E., Natural role of biosurfactants, Environmental Microbiology, 3, 229 (2001).         [ Links ]

Silva, B.H., Influência das fontes: carbono e nitrogêneo na produção de biossurfactantes por Pseudomonas aeruginosa ATCC 31479 e Pseudomonas aeruginosa LBI. Final term paper (Biological Sciences), Instituto de Biociências-Universidade Estadual Paulista (Unesp) (2005).         [ Links ]



(Received: February 01, 2007 ; Accepted: February 25, 2008)



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