Use of Proteomics in Bioremediation
Rolf U. Halden, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD

Monitoring Bioremediation by Analyzing In Situ Gene Expression
Derek Lovley, University of Massachusetts, Amherst, MA

Barriers to the Adoption of “Cutting Edge” Research to Solve “Real World” Environmental Problems
Robert J. Steffan, Shaw Environmental, Inc., Lawrenceville, NJ

The Use of Stable Isotopes in Nucleic Acid and Lipid Analysis for Assessing Bioremediation Potentials
Aaron Peacock, The University of Tennessee, Knoxville, TN

Investments in Environmental Genomics: Relevance to U.S. DOE Missions
Robert T. Anderson, U.S. Department of Energy, Washington DC

Visualization and Enumeration of Microorganisms Growing Under Denitrifying Conditions with Multi-Color FISH

Eric C. Hince, P.G., Geovation Consultants, Inc., 468 Route 17A, Florida, NY, 10921, Tel: 845-651-4141, Fax: 845-651-0040; e-mail: echince@geovation.com

Molecular, culture-independent methods have revolutionized our current understanding of the microbial ecology of many natural and anthropogenic environments. Fluorescence in-situ hybridization (“FISH”) is emerging as a powerful and quantitative genomic-based method for investigating the relative abundance of microorganisms in environmental samples.  Multi-color FISH (“mFISH”) involves the simultaneous hybridization of a sample with multiple oligonucleotide probes conjugated to different color fluorochromes so as to provide quantitative information on different sub-populations in the same sample.  mFISH also enables the simultaneous visualization of spatial arrangements of microbes in mixed consortia to investigate the potential for mutualistic or syntrophic interactions among different organisms.

mFISH data and images will be presented from an ongoing denitrification-based bioremediation (“DBB”) project at a DoD-owned hydrocarbon contamination site.  Initial DGGE and PCR screening of ground-water samples were used to identify bacteria, archaea and fungi present within the DBB treatment zone and to help design mFISH assays targeting microorganisms suspected to be numerically important members of the DBB consortia.  Multiple probes targeting the 16S or 23S rRNA of various taxonomic classes of microorganisms, from Domain- to Genus-specific probes, are being used to enumerate members of the DBB consortia.  Probes being used include Domain-level probes EUB338 (eubacteria), ARC915 (archaea), and PF2 (yeast) and division- or phyla-specific probes BETA42a (β-proteobacteria), GAM42a (γ‑proteobacteria), ALF968 (α-proteobacteria), DELTA495 (δ‑proteobacteria) and CFB560 (Cytophaga-Flexibacter-Bacteroides).  Group- and genus-specific probes are being used to target sub-populations of bacteria shown by DGGE to be among those consistently found at DBB treatment sites including members of the Comamonadaceae (BONE23a), Burkholderiales (SUBU1237) and Pseudomonas spp. (PS56a).  Probes were labeled with Oregon Green, Cy3, or Cy5 for CCD imaging with a Nikon 90i epi-fluorescent microscope and narrow-band filter sets.

Initial mFISH results have revealed an abundance of β-proteobacteria and dimorphic, Yarrowia-like fungi in dense DBB consortia comprised of ≥10⁷ total cells/mL.  A dual-probe mFISH assay for bacteria (Cy3-labeled EUB338) and yeast (Cy5-labeled PF2) revealed clusters of mixed bacteria and small yeast in contact with one another, raising the possibility of some type of mutualism or opportunistic association.

Preliminary results indicate promising potential for mFISH assays using both the BETA42a (Oregon Green) and GAM42a (Cy5) probes.  BETA42a and GAM42a differ by only one base, so they are normally used with a competitor probe, i.e., the labeled probe to be imaged (e.g., BETA42a) together with the unlabelled “competitor” probe (e.g., GAM42a) to minimize erroneous hybridizations.  Preliminary results suggest both probes can be imaged simultaneously with different color fluorochromes while minimizing errant hybridizations.  mFISH assays that combine multiple probes may enable more rapid and cost effective enumeration and examination of important groups of microorganisms for monitoring in-situ bioremediation programs.

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