Time-dependent growth of crystalline Au0-nanoparticles in cyanobacteria as self-reproducing bioreactors: 2. Anabaena cylindrica

Microbial biosynthesis of metal nanoparticles as needed in catalysis has shown its theoretical ability as an extremely environmentally friendly production method in the last few years, even though the separation of the nanoparticles is challenging. Biosynthesis, summing up biosorption and bioreduction of diluted metal ions to zero valent metals, is especially ecofriendly, when the bioreactor itself is harmless and needs no further harmful reagents. The cyanobacterium Anabaena cylindrica (SAG 1403.2) is able to form crystalline Au0-nanoparticles from Au3+ ions and does not release toxic anatoxin-a. X-ray powder diffraction (XRD), transmission electron microscopy (TEM) and laser-induced breakdown spectroscopy (LIBS) are applied to monitor the time-dependent development of gold nanoparticles for up to 40 hours. Some vegetative cells (VC) are filled with nanoparticles within minutes, while the extracellular polymeric substances (EPS) of vegetative cells and the heterocyst polysaccharide layer (HEP) are the regions, where the first nanoparticles are detected on most other cells. The uptake of gold starts immediately after incubation and within four hours the average size remains constant around 10 nm. Analyzing the TEM images with an image processing program reveals a wide distribution for the diameter of the nanoparticles at all times and in all regions of the cyanobacteria. Finally, the nanoparticle concentration in vegetative cells of Anabaena cylindrica is about 50% higher than in heterocysts (HC). These nanoparticles are found to be located along the thylakoid membranes.


Microscopy
In the beginning of the experiment fluorescence microscopy (Axioimager M2, Zeiss) and optical microscopy (Axioskop 50, Zeiss) were used. The condition of the cultures has been monitored by red fluorescence.
A fluorescence microscopy image is depicted in Figure S1. An Anabaena cylindrica culture grown for twelve weeks in modified BBM shows a significant red fluorescence in the vegetative cells because of chlorophyll a. The large cell without red fluorescence is a heterocyst, the smaller ones with significant red fluorescence are vegetative cells.

X-ray powder diffraction
X-ray powder diffraction (XRD) is applied to determine the average size of the formed nanoparticles.
Therefore the peaks, showing up in Figure 2 of the main manuscript, are fitted following the LeBail method [S8 = 60; S9 = 61] first and the so determined FWHM is converted using the Scherrerequation into a characteristic size of the crystallites [S10 = 62, S11 = 63].
The normalized fitted data are shown in Figure S2. In the left panel data from the peak (111) are plotted, in the right panel those for (002). The later ones contain less data points, since the angular position is larger and the signal intensity is less. Therefore the fits are as expected not as good as for the peak (111)  The average size of gold nanoparticles found in Anabaena cylindrica samples is given in Figure S3 as a function of time. As seen in the lower panel the diameter calculated from peaks (111) and (002) differs. This shows an imperfect spherical shape of the nanoparticles [S11 = 63]. Assuming an ellipsoid the aspect ratio of the formed gold nanoparticles is with a value of about 1.15 in average, see upper panel in Figure S3. Transmission electron microscopy Figure S4 shows TEM of Anabaena cylindrica. In Figure S4a a single vegetative cell from a chain of cells is shown, in Figure S4b a heterocyst between two vegetative cells in another cell chain is visible.
Thylakoid membranes (limited by light curved lines) and lipid droplets (dark dots) are clearly visible inside the cell as the EPS outside. One prominent feature of the heterocysts is the polar plugs, also called cyanophycin granules (CPG) [S5 = 28]. In the picture shown, only the polar plug at the left side of the heterocyst is visible, because of the sectional plane being not median but somewhat peripheral and slanted. Normally the CPGs are seen as an electron dense dark structure, see Figure S4b, but it is well known that it can be lost during sample preparation, leaving only an electron translucent area behind, as seen here [S5 = 28]. Since XRD detects only the average size of the formed nanoparticles, the size of each individual nanoparticle was determined from TEM images with a magnification of 50k and 85k to reveal the size distribution of the nanoparticles in specific regions of the organism using an image processing tool.
The difference between the two methods is that XRD preferably detects large nanoparticles since S6 crystallinity is needed and image processing of TEM images is very likely to detect more very small ones because of the electron dense and structured background. One pixel in a TEM image is 1.15 nm x 1.15 nm for 85k resp. 1.94 nm x 1.94 nm for 50k. Taking this into account only nanoparticles of more than 3 nm could be detected reliably.

Laser-induced breakdown spectroscopy
A standard experimental LIBS setup as shown in Figure S5 has been used for the experiments presented. The homemade LIBS setup consisted of a pulsed laser source (shown as a blue box in Figure S5), focusing optics (white box), and Czerny-Turner spectrometers (pink box). A micro-plasma (shown as green cloud in Figure S5) is generated by a low-power passively Q-switched Nd:YAG laser (CryLas, model DSS1064-3000, wavelength 1064 nm, pulse energy 2.5 mJ, pulse duration 2 ns (FWHM), repetition rate 80 pulses per second) focused on the sample surface (shown as dark gray tray in Figure S5)) by a quartz glass lens with a focal length of 20 mm.
The plasma emission (shown as yellow arrow and red, green and blue waves in Figure S5) was imaged by a combination of two quartz lenses (focal length 20 and 25 mm) into a glass fiber (core diameter S7 (from 190 to 420 nm, spectral resolution of 0.1 nm) and USB2000 (190 to 860 nm, spectral resolution 0.3 nm)). S8