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The diverse properties of biosurfactants do not make it possible to identify the molecules directly. Moreover, the biological roles of surfactant producers and the conditions of culture have a major impact on the production of biosurfactants. Instead, the physical shift in liquid media is the only general indicator for biosurfactant production. A number of screening techniques for biosurfactants have been developed and every technique has its own advantages and limitations.

For example, in the drop collapse method an aliquot of bacterial culture or culture supernatant is placed over the surface of the oil. The drops that collapse are considered positive for biosurfactants, while the drops that remain beaded are considered negative. Improved methods, such as the atomized spray method, have several advantages over the commonly used drop collapse method. The method helps to identify strains that have bypassed other detection methods and also helps to detect surfactants that are present in very low concentrations [61]. Another common method used for biosurfactant screening is the hemolytic method. In the hemolytic method, biosurfactant producing bacterial strains are tested on the basis of hemolytic activity. However, there are some bacterial strains that are capable of producing biosurfactant‐like substances but are non‐hemolytic. For example, glucose lipids produced by Alcaligenes sp. have properties similar to biosurfactants but are non‐hemolytic [62]. Although it is a rapid test method, the breakdown and destruction of blood cells in the blood agar plate may be mediated by factors other than biosurfactants, resulting in false positive results. In addition, the quantity of biosurfactants may not be sufficient to lyse the blood cells in a given experimental condition. The other rapid screening method is the emulsification assay. The production of biosurfactants in the emulsification assay is determined by the emulsification index. The higher the emulsification index the more will be the emulsifying capacity of the biosurfactants in emulsifying oil hydrocarbons [63]. Detailed features of each screening method are presented in Table 2.1. A combination of screening methods is warranted for the high‐throughput screening of biosurfactant producers. The combined assay will facilitate the detection of surfactants produced at a much lower concentration. Biosurfactants produced may be subjected to an analysis of Fourier transform infrared spectroscopy (FTIR) for the determination of chemical bonds and functional groups [70]. Compounds present in biosurfactants may be analyzed using gas chromatography–mass spectrometry (GCMS) [71].

Table 2.1 Comparison of screening techniques for determination of biosurfactants production.

Screening techniques	Key features	Limitations	References
CTAB‐methylene blue agar	Allows the identification of biological anionic glycolipid biosurfactants.Biological surfactants form insoluble ion pairs with cationic surfactant cetyl trimethyl ammonium bromide and base dye methylene blue, indicated by the formation of dark blue halo around the culture colonies.Ideal method for detecting extracellular glycolipids (rhamnolipids).	CTAB is toxic to certain bacterial strains, e.g. E. coli.The method is only appropriate for anionic biosurfactants.	[64]
Drop‐collapse test	Suitable for the detection of large‐scale metagenomic clones.Sensitive method for the determination of surfactant activity using a small volume of cell‐free broth culture.It can be used for both qualitative and quantitative tests.Cell‐free broth droplets are transferred to an oil‐coated surface. Surfactant containing droplets collapses, whereas those lacking remain beaded.In quantitative test diameter of droplet is measured. Droplet diameter of test broth larger than control indicates positive for biosurfactants.	Bacterial strains producing low levels of surfactant cannot be detected. May show false positive due to the hydrophobicity of certain bacterial cells acting as biosurfactants themselves.	[61, 65]
Atomized oil assay	The oil droplet/liquid paraffin mist is sprayed over the culture plates. The formation of a bright zone or halo around the bacterial colonies indicates the production of biosurfactants. Facilitates the simultaneous assessment of the number of colonies and is ideal for the library of metagenomic clones. The production of surfactants can be detected even at very low concentrations.	Method uses the detection of surfactant producers on the basis of the formation of bright halos around the bacterial colonies. Some synthetic surfactants may imitate the formation of bright halos. The distinction between “bright” and “dark” halo is arbitrary.The assay is limited to cultivable microbes only.The assay shows positive results on a solid medium.	[61, 66]
Oil‐spreading technique	Crude oil is added to the surface of the distilled water taken on a petri dish. An aliquot of bacterial culture is placed on the surface of the oil. Biosurfactant production is indicated by the formation of a dispersion zone.	The method is suitable for primary screening and qualitative testing.	[37]
Haemolytic method (Blood plate method)	Rapid biosurfactant detection test is indicated by the formation of halo around the spot‐inoculated bacterial colony on the blood agar plate. Ideal method for the detection of rhamnolipids or surfactins.	Extracellular metabolites other than biosurfactants can provide false‐positive results.	[37, 67]
Emulsification assay	The production of biosurfactants is determined by the ability of cell‐free broth to emulsify crude oil in the test solution.The activity of emulsification is calculated on an emulsification index basis.	The activity of emulsification may not be correlated with reduction in surface tension.The method only indicates for biosurfactant presence.	[68]
Bacterial adhesion to hydrocarbons (BATH) assay	Measures the hydrophobicity of bacterial cells to hydrocarbons.An indirect method to screen biosurfactants producing bacteria.Increase in cell adhesion to liquid hydrocarbons indicates the production of biosurfactants.	Affinity for hydrocarbon may vary between different bacterial strains.Cell adherence may also be due to other bacterial cellular components.	[69]