Rhamnolipids Characteristics, Production, Applications, and Analysis - Biosurfactants: Research Trends and Applications - page 68

and emulsification activities. And between those 12 strains, two of them showed the
highest rate of biosurfactant production by growing on paraffin and glycerol as the
sole carbon source. In this section, a few common methods for isolating rhamno-
lipid-producing bacteria are discussed. The advantage of these methods are that they
are easy to apply and don't need complicated instruments. For more precise results,
one could use more advanced methods, such as real-time PCR and DNA microarray-
based assays.

CTAB Agar Test
Pinzon and Lu-Kwang (2009) described a method for the detection of rhamnolipids
by culturing the microorganisms on the agar plates containing methylene blue and
cetyl trimethylammonium bromide (CTAB). Also according to Abdel-Mawgoud
et al. (2010), CTAB agar can be used for identifying rhamnolipid-producing strains.
Rhamnolipid produced by microorganisms is able to react with bromide salt, so the
agar around the colonies of rhamnolipid-producing microorganisms becomes dark
blue. In this test, the microorganisms are cultured on a CTAB methylene blue agar
plate. After 24 h if the strain was able to produce anionic biosurfactants (e.g., rham-
nolipids), there will be a purple-blue haze with a sharp defined edge around the
culture (Jadhav et al., 2011) (Figure 3.2).

Hemolytic Test
The hemolytic property of the rhamnolipids is another method for identifying and
isolating the rhamnolipid-producing strains. McClure and Schiller (1992) indicated
that the presence of rhamnolipids is responsible for the heat-stable hemolytic activ-
ity of the released fluids of P. aeruginosa . In the hemolytic method, blood agar
is being used for culturing the strains, and after incubating the colonies, a halo
is observed around the colonies due to lysis of the cells potentially by biosurfac-
tant production. Further investigations are performed to determine if the clearing
halos are created by rhamnolipids and not by other hemolytic components (Abdel-
Mawgoud et al., 2010; Anandaraj and Thivakaran, 2010; Mulligan et al., 1989c;
Siegmund and Wagner 1991). The other approach is to separate the supernatant
and then inject into the holes made in the blood agar Petri dishes that are then left
for 24-48 h for the observation and measurement of the hemolysis zones (Rahman
et al., 2010) (Figure 3.3).

24 h

FIGURE 3.2 CTAB assay to identify the rhamnolipid-producing strains.