Biosurfactant Complexation of Metals and Applications for Remediation - Biosurfactants: Research Trends and Applications - page 285

hydrogen atoms (Campbell et al., 1994, 1995; Green et al., 1995). It has been
shown that, although HDX occurs in the gas phase, the exchange rates are
directly related to the solution conformation and structure of a molecule. Thus,
HDX was performed for the free monorhamnolipid and the monorhamnolipid-
Pb 2+ complex. Fully protonated free monorhamnolipid contains four exchange-
able hydrogen atoms on the hydroxyls on carbons 2, 3, and 4 of the sugar and
the oxygen of the carboxylic acid involving carbon 7′ in addition to the ionizing
proton which gives the ion its positive charge (Figure 11.3a). Exchange of four of
these hydrogen atoms can be experimentally monitored (exchange of the carbox-
ylic acid hydrogen is too rapid to be measurable). For the free monorhamnolipid,
H/D exchange is complete for all four hydrogen atoms within 1 s. In contrast,
for the complexed monorhamnolipid, which contains only three exchangeable
hydrogen atoms, one of these hydrogen atoms, of the hydroxyl group on the
rhamnose sugar C4 atom, exchanges at the same rate in the complex as in the
free monorhamnolipid, suggesting that this hydroxyl is not involved in the metal
binding. However, the hydrogen atoms of the hydroxyls on the rhamnose C3 and
C2 atoms exchange more slowly by factors of 10 and 100, respectively. These
results clearly indicate that these hydroxyl groups are highly inaccessible in the
complex relative to the free monorhamnolipid, supporting the involvement of the
sugar in a metal cation binding pocket. A model for what this metal binding in a
pocket might look like is shown in Figure 11.3b.
Effects of Metals on Rhamnolipid Production
The strong monorhamnolipid-metal stability constants suggested that there may
be a physiological reason for the interaction of this biosurfactant with metals. In
fact, it has been shown that when P. aeruginosa IGB83 (which produces a mixture
of mono- and dirhamnolipid) was exposed to subtoxic levels of Cd 2+ , expression
of one of the rhamnolipid genes, rhlB , was enhanced in mid-stationary phase and
sustained through late-stationary phase (Neilson et al., 2010). The RhlB enzyme
is responsible for catalyzing the addition of a second rhamnose sugar onto monor-
hamnolipid to form dirhamnolipid. As a result of this increased expression, there
was an increase in the ratio of dirhamnolipid to monorhamnolipid produced. This
is significant because the complexation constant for dirhamnolipid is several orders
of magnitude higher than monorhamnolipid. Thus, it appears that the presence
of Cd 2+ during growth may increase production of dirhamnolipid as a detoxifica-
tion mechanism. This is supported by an earlier study showing that the addition of
monorhamnolipid to soils cocontaminated with phenanthrene and Cd 2+ (levels high
enough to exert toxicity) resulted in enhanced degradation of phenanthrene (Maslin
and Maier, 2000).
Research has also linked rhamnolipid production to iron and magnesium status in
the environment. Multiple studies have reported that iron limitation increases rham-
nolipid production, while sufficient growth levels of iron suppress rhamnolipid pro-
duction (Deziel et al., 2003; Glick et al., 2010; Guerrasantos et al., 1984; Ramana and
Karanth, 1989). In contrast, magnesium limitation reduces rhamnolipid production,
while sufficient levels of magnesium enhance production (Guerrasantos et al., 1986;
Ramana and Karanth, 1989).