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Applied and Environmental Microbiology, May 2001, p. 2095-2106, Vol. 67, No. 5
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.5.2095-2106.2001

Attached and Unattached Bacterial Communities in a 120-Meter Corehole in an Acidic, Crystalline Rock Aquifer

R. Michael Lehman,^1,* Francisco F. Roberto,¹ Drummond Earley,^2, Debby F. Bruhn,¹ Susan E. Brink,² Sean P. O'Connell,¹ Mark E. Delwiche,¹ and Frederick S. Colwell¹

Idaho National Engineering and Environmental Laboratory, Biotechnologies Department, Idaho Falls, Idaho 83415-2203,¹ and Twin Cities Research Center, U.S. Bureau of Mines, Minneapolis, Minnesota 55417²

Received 17 October 2000/Accepted 5 March 2001

The bacteria colonizing geologic core sections (attached) were contrasted with those found suspended in the groundwater (unattached) by examining the microbiology of 16 depth-paired core and groundwater samples using a suite of culture-independent and culture-dependent analyses. One hundred twenty-two meters was continuously cored from a buried chalcopyrite ore hosted in a biotite-quartz-monzonite porphyry at the Mineral Park Mine near Kingman, Ariz. Every fourth 1.5-m core was acquired using microbiologically defensible methods, and these core sections were aseptically processed for characterization of the attached bacteria. Groundwater samples containing unattached bacteria were collected from the uncased corehole at depth intervals corresponding to the individual cores using an inflatable straddle packer sampler. The groundwater was acidic (pH 2.8 to 5.0), with low levels of dissolved oxygen and high concentrations of sulfate and metals, including ferrous iron. Total numbers of attached cells were less than 10⁵ cells g of core material¹ while unattached cells numbered about 10⁵ cells ml of groundwater¹. Attached and unattached acidophilic heterotrophs were observed throughout the depth profile. In contrast, acidophilic chemolithotrophs were not found attached to the rock but were commonly observed in the groundwater. Attached communities were composed of low numbers (<40 CFU g¹) of neutrophilic heterotrophs that exhibited a high degree of morphologic diversity, while unattached communities contained higher numbers (ca. 10³ CFU ml¹) of neutrophilic heterotrophs of limited diversity. Sulfate-reducing bacteria were restricted to the deepest samples of both core and groundwater. 16S ribosomal DNA sequence analysis of attached, acidophilic isolates indicated that organisms closely related to heterotrophic, acidophilic mesophiles such as Acidiphilium organovorum and, surprisingly, to the moderately thermophilic Alicyclobacillus acidocaldarius were present. The results indicate that viable (but possibly inactive) microorganisms were present in the buried ore and that there was substantial distinction in biomass and physiological capabilities between attached and unattached populations.

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^* Corresponding author. Mailing address: Idaho National Engineering and Environmental Laboratory, Biotechnologies Department, P.O. Box 1625, Idaho Falls, ID 83415-2203. Phone: (208) 526-3917. Fax: (208) 526-0828. E-mail: mik4{at}inel.gov.

Present address: Daniel B. Stephens & Associates, Albuquerque, NM 87109.

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Applied and Environmental Microbiology, May 2001, p. 2095-2106, Vol. 67, No. 5
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.5.2095-2106.2001

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