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.