Comparison of methods to detect biosurfactant production by diverse microorganisms
Introduction
Biosurfactants are a diverse group of surface-active chemical compounds that are produced by a wide variety of microorganisms (Banat, 1995). The types of biosurfactants include lipopeptides synthesized by many bacilli and other species, glycolipids synthesized by Pseudomonas species and Candida species, phospholipids synthesized by Thiobacillus thiooxidans, polysaccharide–lipid complexes synthesized by Acinetobacter species, or even the microbial cell surface itself Van Dyke et al., 1991, Bodour and Miller-Maier, 2002. Having both polar and nonpolar domains, biosurfactants are able to partition at water–oil or water–air interfaces and thus reduce the interfacial or surface tension Banat, 1995, Banat et al., 2002, Georgiou et al., 1992. Such surface properties made them good candidates for enhanced oil recovery (EOR) Ron and Rosenberg, 2001, Van Dyke et al., 1991. Some biosurfactants are known to have therapeutic applications as antibiotics and antifungal or antiviral compounds. Biosurfactants can also be used in bioremediation of soil or sand (Van Dyke et al., 1991) or in the cleanup of hydrocarbon contamination in groundwater (Ron and Rosenberg, 2001). These diverse applications necessitate an easy, rapid, and reliable method to detect biosurfactant production with a minimum number of false positives and/or negatives.
Biosurfactant production is sometimes detected by measuring emulsification Van Dyke et al., 1993, Makkar and Cameotra, 1997, Makkar and Cameotra, 1998, hemolytic activity Carrillo et al., 1996, Banat, 1993, Yonebayashi et al., 2000, Mulligan et al., 1984, or cell surface hydrophobicity vander Mei et al., 1987, Mozes and Rouxhet, 1987, Neu and Poralla, 1990, Pruthi and Cameotra, 1997, Dillon et al., 1986. The use of methods that measure properties other than the surface activity can be problematic. Although a direct correlation was found between surface activity and emulsification activity Cooper and Goldenberg, 1987, Denger and Schink, 1995, and the emulsification index has been used as a screening method Cooper and Goldenberg, 1987, Denger and Schink, 1995, Makkar and Cameotra, 1997, Makkar and Cameotra, 1998, the ability of a molecule to form a stable emulsion is not always associated with surface tension lowering activity Van Dyke et al., 1993, Willumsen and Karlson, 1997, Trebbau de Acevedo and McInerney, 1996, Bosch et al., 1988. Cell surface hydrophobicity is an important aspect in bacterial cell adhesion to surfaces (vander Mei et al., 1987). Since hydrophobic surfaces are usually associated with molecules with low surface energy (Mozes and Rouxhet, 1987), Neu and Poralla (1990) used this property to screen for biosurfactant production. Pruthi and Cameotra (1997) found a direct correlation between hydrophobicity and biosurfactant production. However, it is not clear which method for measuring cell surface hydrophobicity is appropriate for general screening vander Mei et al., 1987, Mozes and Rouxhet, 1987, Neu and Poralla, 1990, Pruthi and Cameotra, 1997, Dillon et al., 1986. Siegmund and Wagner (1991) developed a colorimetric assay, based on the formation of insoluble ion pair between anionic surfactants, cationic cetyl trimethyl ammonium bromide (CTAB), and methylene blue. Since this approach is specific for anionic surfactants, it cannot be used as a general method of screening for biosurfactant producers.
There are a number of approaches that measure directly the surface activity of biosurfactants. These include surface and/or interfacial tension measurement McInerney et al., 1990, Haba et al., 2000, Mercade et al., 1996, axisymmetric drop shape analysis profile (ADSA-P) van der Vegt et al., 1991, Noordmans and Busscher, 1991, glass-slide test (Persson and Molin, 1987), drop collapse method Jain et al., 1991, Bodour and Miller-Maier, 1998, and the oil spreading technique (Morikawa et al., 2000). The measurement of surface tension has traditionally been used to detect biosurfactant production and most of the other methods that measure the surface properties of biosurfactant use surface tension reduction as the standard Noordmans and Busscher, 1991, Persson and Molin, 1987, Willumsen and Karlson, 1997, Makkar and Cameotra, 1997, Makkar and Cameotra, 1998, Neu and Poralla, 1990, Bosch et al., 1988. However, the measurement of surface tension is time-consuming, which makes it inconvenient to use for screening of a large number of isolates. The drop collapse technique depends on the principle that a drop of a liquid containing a biosurfactant will collapse and spread completely over the surface of oil Jain et al., 1991, Bodour and Miller-Maier, 1998. The method is easy and can be used to screen large number of samples (Bodour et al., 2003), but it has not been correlated to surface tension reduction to confirm its reliability. The oil spreading technique measures the diameter of clear zones caused when a drop of a biosurfactant-containing solution is placed on an oil–water surface (Morikawa et al., 2000). Morikawa et al. used this method to compare the activity of both cyclic and linear forms of surfactin and arthrofactin. However, its ability to detect biosurfactant production in diverse microorganisms has not been tested.
The hemolytic activity of biosurfactants was first discovered when Bernheimer and Avigad (1970) reported that the biosurfactant produced by Bacillus subtilis, surfactin, lysed red blood cells. Blood agar lysis has been used to quantify surfactin (Moran et al., 2002) and rhamnolipids (Johnson and Boese-Marrazzo, 1980) and has been used to screen for biosurfactant production by new isolates Carrillo et al., 1996, Banat, 1993, Yonebayashi et al., 2000, Mulligan et al., 1984. Carrillo et al. (1996) found an association between hemolytic activity and surfactant production, and they recommended the use of blood agar lysis as a primary method to screen for biosurfactant activity. However, only 13.5% of the hemolytic strains lowered the surface tension below 40 mN/m. In addition, other microbial products such as virulence factors lyse blood agar and biosurfactants that are poorly diffusible may not lyse blood cells. Thus, it is not clear whether blood agar lysis should be used to screen for biosurfactant production.
In this study, we tested the hemolytic activity of 205 environmental isolates of different phylogenetic affiliations and measured the surface activity of these isolates by using both the drop collapse and the oil spreading techniques. Surface tension was measured for cultures that gave conflicting results between these three methods. We found that the oil spreading and drop collapse methods were correlated with the ability of the cultures to reduce surface tension. However, blood agar gave a large number of false positives and negatives.
Section snippets
Media
All cultures were grown aerobically in liquid medium (medium E) (pH 6.9) that contained (g/l): KH2PO4, 2.7; K2HPO4, 13.9; sucrose, 10; NaCl, 50; yeast extract, 0.5; and NaNO3, 1. After autoclaving, 10 ml each of solutions A, B, and C were added to 1 l of the above medium. Solution A contained 25 g/l of MgSO4; solution B contained 100 g/l of (NH4)2SO4; and solution C contained (g/l): EDTA, 0.5; MnSO4·H2O, 3; NaCl, 1; CaCl2·2H2O, 0.1; ZnSO4·7H2O, 0.1; FeSO4·7H2O, 0.1; CuSO4·5H2O, 0.01; AlK(SO4)2,
Oil spreading technique
Morikawa et al. (2000) showed that the area of displacement by a surfactant-containing solution is directly proportional to the concentration of the two biosurfactants tested. We tested whether the oil spreading technique could be used to detect biosurfactant production by diverse microorganisms. The diameter of the clear zone linearly increased with the concentration of surfactin over a concentration range of 50–400 mg/l (Fig. 1). The coefficient of variation of the diameter of clear zones
Discussion
In this study, we tested the applicability of using the oil spreading technique to detect biosurfactant production in diverse microorganisms. The diameter of clear zones was linearly related to surfactin concentrations (Fig. 1), and replicate analyses had low coefficients of variation (<8.3%). Cultures that gave large diameter of clearing also had low surface tension (Fig. 2). These analyses indicate that the oil spreading technique is reliable in detecting biosurfactant production as
Acknowledgments
We would like to thank Valerie Moody and David Faulkner for their technical assistance. This work was supported by the U.S. Department of Energy (DE-FC26-02NT15321).
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