One mechanism of silver resistance in microorganisms is accumulation of the metal ions in the cell. Here, we report on the phenomenon of biosynthesis of silver-based single crystals with well-defined compositions and shapes, such as equilateral triangles and hexagons, in Pseudomonas stutzeri AG259. The crystals were up to 200 nm in size and were often located at the cell poles. Transmission electron microscopy, quantitative energy-dispersive x-ray analysis, and electron diffraction established that the crystals comprise at least three different types, found both in whole cells and thin sections. These Ag-containing crystals are embedded in the organic matrix of the bacteria. Their possible potential as organic-metal composites in thin film and surface coating technology is discussed.T here is much current interest in metal-microbial interactions, especially concerning biochemical, toxicological, environmental, and industrial aspects (1). Microbial resistance against heavy metal ions such as Fe, Co, Ni, Cu, Zn, As, Cd, Hg, Pb, or U has been explored for bioleaching processes of ores (2-4) and for biological metal recovery systems (5-9). Much less is known about resistance against the noble metals (10, 11), although it has been stated that gold can serve as a slow acting drug in rheumatology (10). Silver is highly toxic to most microbial cells and can be used as a biocide or antimicrobial agent (12). Nevertheless, it has been reported that several bacterial strains are silver-resistant (13,14) and may even accumulate silver at the cell wall to as much as 25% of the dry weight biomass, thus suggesting their use for industrial recovery of silver from ore materials (13).Here, we describe the biological synthesis of silver-based crystals with sizes up to 200 nm in the periplasmic space of Pseudomonas stutzeri AG259, a bacterial strain that was originally isolated from a silver mine (15).
Materials and MethodsBacterial Strain and Growth Conditions. Pseudomonas stutzeri AG259 was kindly obtained from J. T. Trevors (University of Guelph, ON, Canada). The silver-resistant P. stutzeri AG259 strain was maintained in Lennox L (LB) broth. Bacteria were grown on Lennox L (LB) agar substrate, containing 50 mM AgNO 3 , at 30°C for 48 hr in the dark.
Transmission Electron Microscopy (TEM), Energy Dispersive X-RayAnalysis (EDX), and Electron Diffraction. Growing cells were harvested and fixed for 2 hr in 2.5% glutaraldehyde in distilled water. After fixation at room temperature, the cells were sedimented (1,500 ϫ g, 10 min) and washed three times in distilled water. The pellet was not postfixed. For the preparation of thin sections, the pellet was dehydrated with 30, 50, 70, and 95% (vol͞vol, aq.) ethanol for 15 min each, followed by absolute ethanol. Embedding was in absolute ethanol͞Agar 100 epoxy resin (Agar Scientific) at a ratio of 1:1 overnight at room temperature. Polymerization at 60°C was carried out in 100% Agar 100 epoxy resin for 2 days. Ultrathin sections were cut on an ultramicrotome, and some of the sections were sl...