**4.1 Biosurfactant screening methods**

A variety of methods, both qualitative and quantitative, have been applied to screen microbial cultures and cell-free media for total (intracellular, surface-bound, and freely released) and freely released biosurfactants, respectively. As biosurfactants are structurally diverse, complex molecules, most of these methods are indirect, reliant on physico-chemical properties such as emulsification, surface activity or hydrophobicity. Commonly reported screening methods used to detect biosurfactant production amongst MACA strains are listed in **Table 4**. Besides the bacterial adhesion to hydrocarbons (BATH) assay [37] other tests based on cell surface hydrophobicity include salt aggregation [38] and hydrocarbon overlay [39] assays. The atomized oil assay [40] may be used to directly screen colonies growing on primary isolation plates and is therefore useful as an initial screen for novel-producing strains recovered from the environment. The microplate assay [41] which relies on the wetting properties of biosurfactants and the penetration assay [42], which relies on the reduction of interfacial tension are also considered useful for screening large numbers of strains. Recently, a rapid, high throughput assay that utilises Victoria pure blue BO dye, and is based on surface-active properties, has been developed for quantitative screening, but has not yet been applied to MACA [43].


#### **Table 4.**

*Examples of screening methods used to detect biosurfactant production by MACA.*

*Biosurfactant Production by Mycolic Acid-Containing Actinobacteria DOI: http://dx.doi.org/10.5772/intechopen.104576*

These assays are simpler and more rapid than chemical analytical procedures, and most enable larger-scale screening for biosurfactant production. However, perhaps owing to the general and indirect nature of these assays and various limitations associated with some, test results between assays are not always congruent and no one assay is considered definitive for biosurfactant production. It is thus advisable to use several methods in combination, adopting simple methods to undertake preliminary screening of large strains collections prior to further investigation of those found to be most promising. The development of high-throughput screening, metabolic profiling technologies, and whole-genome analysis promise a more thorough investigation of potential biosurfactant producing strain in the future [28].

#### **4.2 Extraction and structural analysis of biosurfactants**

Crude biosurfactant extracts may be obtained from cell cultures (cell-associated and free surfactants) or cell-free broth (free surfactant only) by acidification and solidification followed by solvent extraction of the precipitate. In the case of MACA commonly used solvents include MTBE, dichloromethane, or varying ratios of chloroform–methanol or MTBE–chloroform [44]. Various analytical techniques are used in combination to detect, quantify, and characterise biosurfactants. Thin layer chromatography (TLC) is a straightforward method to separate biosurfactant fractions present in crude extracts. Samples are spotted at the base of a silica plate before development in a solvent system, then air-dried and sprayed with a particular reagent to detect certain chemical groups based on spot colour and/or *R*<sup>f</sup> values. Orcinol, for example, allows detection and differentiation of glycolipids and can distinguish mono-rhamnolipid (MRL) and DRL congeners [45]. However, TLC provides little further detail on congener structure, and it is not generally considered suitable for quantitative analysis although densiometry has been used for this purpose [46]. Biosurfactants may be further separated by silica gel column chromatography.

High-performance liquid chromatography-mass spectrometry (HPLC-MS) allows more precise and accurate characterisation and quantitation of biosurfactant compounds. Isocratic HPLC-UV has been reported for structural and yield determination of THLs produced by *R. erythropolis* strain MTCC 2794 from semi-purified extractions of whole-cell broth [47]. Nuclear magnetic resonance spectroscopy (NMR) is considered the gold standard method to characterise the chemical structure of novel biosurfactants. This has been used in combination with matrixassisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-ToF/MS) to elucidate the structure of two novel extracellular THLs TL A and TL B from *Tsukamurella* spp. [18].

A combination of Fourier transform infrared spectroscopy (FTIR), NMR, and liquid chromatography-mass spectrometry (LC-MS) enabled structural characterisation of a novel cyclic lipopeptide, Coryxin, produced by *Corynebacterium xerosis* NS5 [48]. Multiple-Stage Linear Ion-Trap Mass Spectrometry with Electrospray Ionization has been used to determine the structure of trehalose monomycolate (TMM) and trehalose dimycolate (TDM) in the cell wall of *Rhodococcus equi* and *R. opacus* [49]. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) has been utilised successfully for the purification and characterisation of sophorolipids and rhamnolipids in *Pseudomonas aeruginosa* [50] and could be applied to similar compounds produced by mycolate species. Gas chromatography-mass spectrometry (GC-MS) is used to characterisation of the fatty acid and mycolic acid components and for the carbohydrate portion of THLs.
