Image and numerical data detailing interactions between metal reducing bacteria and metals (NERC grant NE/J024732/1)

Preparation of thin film experiment bottles A spin coating technique was used to produce surfaces with thin films of Fe(III) mineral. A solution of 0.15 M Fe gel and methanol at a v:v ratio of 3:1 was added to microscope slide cover glasses fixed to the base of a salad spinner. They were spun vigorously for 15 seconds and allowed to air dry at room temperature and were then were cut into 1 cm2 pieces using a diamond-tipped scriber. The final mass of Fe(III) on the slides was 0.08 ± 0.007 µM (n=6) and the coatings were found to be saturating but variable in thickness. Mineral-coated cover slides were added to serum bottles and placed vertically in slide holders prior to inoculation. Anaerobic modified minimal medium with 25 mM acetate as the electron donor and no electron acceptor was added to the bottles to cover the films. Fluorescence microscopy Cover glasses were removed from serum bottles, under a stream of argon gas in a box, and placed mineral side up onto microscope slides inside a window of PAP, which had been drawn on with liquid blocker pen. A solution of 150 µl of anaerobic 30 mM NaHCO3 buffer and fluorescent probes was added to the mineral film, the excess removed, and a cover slide was placed on top and the edges sealed to the slide using nail varnish. The probes were DAPI, SYTO9 and Redox Sensor Green (all from Invitrogen) used at final concentrations of 25 µg l-1, 20 µM and 1 µM in 30 mM NaHCO3, respectively. To visualise Fe(II), we synthesised the highly selective turn-on fluorescent probe RhoNox-1 and used it at a final concentration of 5 µM in 30 mM NaHCO3. The slides were imaged using a Zeiss Axio Scope A1 microscope with CCD monochrome camera and EC PLAN 100 × 1.3 oil lens. Confocal laser scanning microscopy was performed using a Leica SP5 inverted tandem head microscope and HCX PL APO lambda blue 63× 1.40 oil lens. The excitation and emission wavelengths were as follows; DAPI: Ex. 350 nm, Em. 420 – 480 nm; SYTO9: Ex. 488 nm, Em. 495 – 550 nm; Redox Sensor Green: Ex. 488 nm, Em. 500 – 540 nm; RhoNox-1: Ex. 543 nm, Em. 560 – 600 nm. Multiple channels were recorded sequentially with 2 × line averaging. Atomic force microscopy After 12 days of incubation in the experiment bottles, slides were removed from the serum bottles and mounted directly onto the Veeco NanoScope V. Samples were scanned in tapping mode and data was analysed using WSxM 5.0 software. Image analysis Image analysis was performed using the software imageJ [55]. Cells were counted by measuring the area of representative individual G. sulfurreducens cells (n=29) in segmented images. In other images the total area of bacteria fluorescence was divided by the mean area of 1 cell to estimate the total number of cells in an image [56] of a known area. The first step of comparing the location of G. sulfurreducens cells with the intensity RhoNox-1 fluorescence (and therefore Fe(II) concentration) and the location of thick regions of mineral coating was to segment the cells, patches of Fe(II) and thick regions of mineral from the background in respective channels of the same image. These were saved as regions of interest (ROIs) and the RhoNox-1 fluorescence intensity for each ROI was measured and categorised into areas which corresponded with a cell, a thick piece of mineral, with one of these or without either. Statistical analyses were performed using OriginPro 8.5. Extracellular appendages were measured from 10 CLSM images using the straight line tool in image J; pili extending beyond the field of view were not included and a total of 80 features were measured.

INSPIRE Implementing rules laying down technical arrangements for the interoperability and harmonisation of Geology

Commission Regulation (EU) No 1089/2010 of 23 November 2010 implementing Directive 2007/2/EC of the European Parliament and of the Council as regards interoperability of spatial data sets and services