In Situ Analysis of a Silver Nanoparticle-Precipitating Shewanella Biofilm by Surface Enhanced Confocal Raman Microscopy

Abstract

Shewanella oneidensis MR-1 is an electroactive bacterium, capable of reducing extracellular insoluble electron acceptors, making it important for both nutrient cycling in nature and microbial electrochemical technologies, such as microbial fuel cells and microbial electrosynthesis. When allowed to anaerobically colonize an Ag/AgCl solid interface, S. oneidensis has precipitated silver nanoparticles (AgNp), thus providing the means for a surface enhanced confocal Raman microscopy (SECRaM) investigation of its biofilm. The result is the in-situ chemical mapping of the biofilm as it developed over time, where the distribution of cytochromes, reduced and oxidized flavins, polysaccharides and phosphate in the undisturbed biofilm is monitored. Utilizing AgNp bio-produced by the bacteria colonizing the Ag/AgCl interface, we could perform SECRaM while avoiding the use of a patterned or roughened support or the introduction of noble metal salts and reducing agents. This new method will allow a spatially and temporally resolved chemical investigation not only of Shewanella biofilms at an insoluble electron acceptor, but also of other noble metal nanoparticle-precipitating bacteria in laboratory cultures or in complex microbial communities in their natural habitats.

Fig 1. SERS spectra of expected biofilm component proxies.

Each component was dissolved either in water (Sodium Alginate) or in phosphate buffer (hhcytc and riboflavin phosphate) and mixed with colloidal silver for Raman surface enhancement. a) reduced (red) and oxidized (blue) hhcytc; b) reduced (black) and oxidized (olive) riboflavin phosphate; c) two different SERS spectra of sodium alginate; d) spectra of reduced hhcytc, reduced riboflavin phosphate and sodium alginate overlaid, for comparison.

Fig 2. Dark field microscopy (x10) of S. oneidensis MR-1 at the Ag/AgCl solid interface.

Bacteria appear as bright dots, and the Ag/AgCl patch is the dark area at bottom right corner. a) 30 min after sealing the setup, b) 7 days after sealing the setup: a brownish-orange light refracting substance has accumulated at the Ag/AgCl patch. Scale bar 50 μm.

Fig 3. SEM-EDX analysis of a Ag/AgCl patch with (b, c, e, f) and without (a and d) freshly deposited S. oneidensis cells.

Left column: SEM images, right column: overlay SEM-EDX images. Color code: Red: Ag, Cyan: C, Green: O, Magenta: P, Yellow: S, Blue: Si. Yellow bar is 1 μm and applies to all images but d. Images a and b were obtained at 1.5 kV and c to f at 5 kV, compare with Fig 4. kV stands for kilovolts and represents the acceleration voltage of the electron beam.

Fig 4. SEM-EDX images of 7 days (a-e) and 14 days (f-g) old biofilms.

Scale bar 2 μm applicable for panels a, b and c (see bar at a) d, and e (see bar at e). For panels f and g scale bar is 3 μm (see bar at g). Panels a, c and f were acquired at 1.5 kV; d, e and g at 5 kV and b at 18 kV. In EDX images red is silver (Ag) and cyan is carbon (C). Image e is a superposition of the EDX color maps for Ag and C and a contour image produced from panel d using ImageJ plugin FeatureJ Edges. From panels a-e it is evident that AgNp in various shapes and sizes can be found both on the EPS and under the outer membrane of some bacteria (see text for more details). In panels f and g both the EPS and the outer membrane of some bacteria are completely covered with roughly spherical AgNp or agglomerations thereof. For a higher resolution segment of image f, see S4 Fig.

Fig 5. SECRaM time series of the S. oneidensis biofilm at the Ag/AgCl patch (patch seen on the left): 1, 3, 6, 9 and 35 days after sealing the setup.

The Raman signal is summed over 1400–1600 cm^-1. The 20 μm scale bar applies to all images. Intensity scale bar on left. All images normalized to 1000 a.u., without background subtraction. With time, Raman intensity hotspots develop where Raman-active biofilm components directly touch bio-produced AgNp. Raman hotspot intensity and distribution change with biofilm development, for discussion see text and Fig 6.

Fig 6. Time series of the S. oneidensis biofilm at the Ag/AgCl patch—surface enhanced Raman signal overlaid on light microscopy.

6, 9 and 35 days after sealing the setup. Ag/AgCl patch seen as dark area on the left. Grayscale: light microscopy, bright field, 20×. Black features are bacteria or bacterial aggregates, which have already precipitated Ag. Before precipitating Ag the bacteria are not visible in bright field, see also S1–S4 videos. Overlay: SECRaM image summed over 1400–1600 cm^-1, intensity scale bar on left, all images normalized to 1000 a.u., without background subtraction. Yellow scale bar: 20 μm.

Fig 7. Chemical maps of a typical AgNp-precipitating S. oneidensis biofilm, produced by SECRaM.

Top row: 6 days, middle row: 9 days, bottom row: 35 days after sealing the setup. Mapped biofilm components: Red: cytochromes, yellow: oxidized flavins, magenta: reduced flavins (white: reduced and oxidized flavins coinciding), cyan: polysaccharides, green: phosphate. Extreme right column is a superposition of all components. Scale bar: 20 μm.

Fig 8. Component profiles, extracted using ImageJ from the chemical maps in Fig 7 and smoothed using Origin.

Teal: polysaccharides; Maroon: cytochromes; Black: reduced flavins; Olive: oxidized flavins; Green: phosphates; Grey: SERS hotspots, normalized to fit the scale of the other components.