1. Archaea DLO-like N-fixers Metal-reducers Methanotroph NH 3 + oxidizer Acetogens S-oxidizer High Density Microarray analysis of prokaryotes in planktonic and biofilm gold mine communities. Comparison of planktonic and biofilm microbial communities in fracture water from the deep subsurface of the Witwatersrand Basin, South Africa. Terry Hazen 1, Eoin Brodie 1, Fred Brockman 2, Duane Moser 2, Tom Gihring 2, David Culley 2, Li-Hung Lin 3, Thomas Pray 4, Gary Andersen 1, Paul Richardson 1,5, Lisa Pratt 6 and Tullis Onstott 4. 1 Lawrence Berkeley National Lab, 2 Pacific Northwest National Lab, 3 National Taiwan University, 4 Princeton University, 5 Joint Genome Institute, 6 Indiana University Background A deep-branching clade of nearly identical Desulfotomaculum-like, or DLO, 16S rDNA sequences (>99% homology) has been identified as the dominant microorganism in fracture water (14 Kyr to 20 Myr old) from multiple Au mines of the Witwatersrand Basin. The DLO is only the dominant form in the planktonic phase of the deepest (2 – 3 km depth), most saline fracture water and has not been reported as part of the community structure in biofilms that occupy deep, open boreholes with flowing fracture water. Here we compare fracture water planktonic communities from a recently drilled borehole in one mine with biofilm samples collected from an existing borehole at another mine in the Witwatersrand Basin. Methods DNA from fissure water and rock biofilm samples collected from 3m, 6m and 9m into a borehole with fracture water ~ 3 Myr old, was amplified, fragmented and hybridized to our high density 16S microarrays containing 500,000 probes capable of detecting 9,381 prokaryotic OTUs. Clone libraries of 16S rDNA were also generated in order to validate array results. Scanning electron microscopy was used to visualize biofilm architecture, while XRD and EDX-S were used to confirm mineral identities.Results Planktonic community composition Biofilm community composition = detected in clone library and array = detected by array only = present in planktonic also = only present in biofilm FeS 2 framboid BaSO 3 crystals Possibly FeS 2 ZnS mineral coating Biofilm interior with various mineral morphologies Biofilm cross section showing dense exopolysaccharide matrix Conclusions Biofilm microbial communities in open boreholes are considerably more diverse than planktonic communities present in fracture water and appear to contain many of the functional groups expected given the geochemistry of an ecosystem where highly reduced, metal, sulfide, H 2 and CH 4 rich water encounter oxygenated mine air. Acknowledgements PlanktonicBiofilm Temperature ( o C)6031 Eh (mV)-330 to -260-250 pH9.2 to 9.37.4 Depth sampled (km below surface)2.831.47 CH 4 9 to 1729 SO 4 2- /S 2- 0.5 to 1.9/1.1 to 1.40.55/2.7x10 3 Fe< 2 to 7x10 -4 1.3x10 3 Zn< 2x10 -4 2.8x10 3 Ba0.2 to 2.5x10 -3 0.55 δ 13 C-CH 4 (‰VPDB) δ 2 H-CH 4 (‰VSMOW) -31.6 to -33.2 -364 to -390 mM/L -56.9 -175 mM/kg Harmony Gold Mine, Inc. for providing access to the boreholes at Evander Mine. Evander geologists Colin Ralston and Pete Roberts. This research was supported by grant EAR-9978267 from the National Science Foundation LExEn program to T.C. Onstott. Physical and chemical characteristics of planktonic and biofilm samples Contacts: Contacts: tullis@princeton.edu – elbrodie@lbl.gov – tchazen@lbl.gov tullis@princeton.eduelbrodie@lbl.govtchazen@lbl.gov Low intensity High intensity