Structural and chemical characterization of a natural fracture surface from 2.8 kilometers below land surface: Biofilms in the deep subsurface

A freshly intersected water-bearing fracture zone from the Mponeng Au mine located in the Witwatersrand Basin, Republic of South Africa was sampled, providing an opportunity to examine the natural, deep subsurface biosphere. The fracture, intersected by an advancing tunnel 2.8 kilometers below land surface, possessed a millimeter thick layer of chlorite group minerals, i.e., chamosite, at the water-mineral interface.Water flowing out from the fracture zone had a temperature of 52ºC, pH of 9.16 and Eh of −263 mV. Using scanning electron microscopy, the water-mineral interface was generally found to be clean, i.e., it did not possess any secondary mineral or dominant organic coatings. Irregular patches (10’s ofμm2) of organic material, however, resembling bacterial exopolysaccharides, occurred in the presence or absence of bacteria. The surface was colonized by highly dispersed individual bacteria or by microcolonies containing up to 5 cells, with an overall cell density of 5×10⁴ bacteria cm⁻². This biofilm population, although low, was 2 orders of magnitude greater than the bacteria present within the aqueous phase and provides the first direct observation of the sessile population from the terrestrial deep subsurface. Time of Flight-Secondary Ion Mass spectrometry revealed that the fracture surface was actually coated with a thin, i.e., molecular, organic conditioning film over much of its surface that was separate from the exopolysaccharide layers associated with the mineral water interface and with some of the attached cells.