Figure 2. Consumption of Ag+ ions and Ag
nanoparticle formation of D.r. bacteria. (a) Anodic stripping
voltammograms of bacterial solutions initially containing 2 mM
AgNO3 as a function of time within 9 hours. (b) UV-vis
spectra of the bacteria with (red) and without (blue)
Ag+ incubation. (c) SEM image and (d) Ag-element
mapping.
The product of the bacterial silver consumption is further investigated
by looking into a single bacterium using scanning electron microscopy.
Figure 2c shows a representative SEM image of a silver
particle-containing bacterium. A total number of 106 bright features
with an average diameter of 95±22 nm within the bacterial cell is
evident (see Figure S1). Furthermore, elemental mapping based on EDS
(Figure 2d) shows the presence of silver and importantly the spatial
distribution of the Ag element is well consistent with that in the
electron microscopic image. These observations strongly confirm that
inside the bacteria silver nanoparticles form as a result of the silver
ion uptake. As such, the characterisations above clearly demonstrate the
ability of individual D.r. bacteria to reduce the silver metal
ions.
On the basis of the strongly scattering properties of silver
nanoparticles, we next turn to optically explore the differences in the
silver ion reducing ability of bacteria within their cultures. To this
end, the high-throughput method of flow cytometry was conducted for
detecting the bacteria after the incubation with Ag+and both the forward scattering and side scattering signals were
collected for individual bacteria. It is recognised that forward scatter
detection is mainly related to the size of a cell and thus for a certain
bacterial strain a larger cell gives higher scattering intensity, whilst
side scatter detection is relatively sensitive to the strongly
scattering particles inside the cell.32, 33 The two
kinds of scatter signals are plotted for each detected bacteria in form
of a two dimensional intensity distribution graph as shown in Figure
3a& 3b. As a result, the scattering signals of bacteria after
incubation with Ag+ exhibit a bimodal distribution
(Figure 3b) where the first contour peak at the SSC intensity ofca. 105 becomes larger than that for the
bacteria before Ag+ incubation (i.e. some
104) as shown in Figure 3a, which is consistent with
the formation of the strongly scattering silver nanoparticles for most
bacteria. Strikingly, a second, though smaller, peak emerges at a much
higher SSC intensity of nearly 106, indicating a
unusually considerable number of silver nanoparticles formed in those
bacteria. In contrast, the FSC intensity of the bacteria before and
after Ag+ incubation stays almost constant.
Nevertheless, the bacteria synthesising large quantities of silver
nanoparticles show a relatively higher FSC intensity than most bacteria
on average, to which the scattering silver particles possibly
contribute. Figure 3c shows the number distributions of bacteria with
different SSC intensity before and after the Ag+incubation. While the total number of bacteria seems unchanged despite
being incubated, the after-incubation bacteria with the SSC values
within the second peak account for a small portion of nearly 10% by
integral. These observations implies the emergence of a minority of
‘strongly reducing’ bacteria that are distinct from the others in silver
metal metabolism when exposed to silver ions. The phenomenon points to
the interesting fact that when facing environmental threats, bacteria
owning the same genome can evolve to a subpopulation of a very different
phenotype – in this case, those that can reduce silver ions much more
greatly than the rest. This natural strategy is consistent with the
extraordinary adapting ability of the polyextremophile.