1. Biological Considerations for Successful Vadose Zone Remediation Fred Brockman Pacific Northwest Natl. Lab. Introduction to Microbes Vadose Zone microbiology Bioremediation Research Opportunities

2. What Microbes Can Do Degrade organic compounds Mobilize or immobilize inorganic compounds Affect water & gas geochemistry & quality Increase and reduce porosity Alter unsaturated & saturated flow Cause/enhance corrosion Move to more favorable conditions fortuitous physical transport directed biological (behavioral) “ Microbes are the things that screw up our chemistry and physics” - PNNL non-biologist

3. Morphology Gram-negative - Membrane weaker but protected w/coatings - Does not form spores - “Hibernates” during lean times - Dominant in disturbed environments Gram-positive - Tougher membrane - Many form spores - “Suspended animation” - Dominant in very low nutrient flux environments Generally 0.5 – 2.0 micrometers in width/length Generally spherical or rod-shaped.

4. Physiology A.Source of carbon 1.Organic compounds (heterotroph) 2.CO 2 (autotroph) B.Source of energy (electron donor) 1.Oxidize organic compounds (chemotroph) 2.Oxidize inorganics (lithotroph) 3.Oxidize H 2 (hydrogenstroph) 4.Oxidize CH 4 (methanotroph) 5.Oxidize NH 4 6.Sunlight (phototroph)

5. Physiology C.Fate of electrons (electron acceptor) 1.O 2 (aerobe) 2.NO 3, NO 2 (denitrifier) 3.Split organic (fermentor) 4.Mn (IV) (Mn reducer) 5.Fe (III) (Fe reducer) 6.SO 4 (SO 4 reducer) 7.CO 2 (Methanogen) 8.Contaminants Cr Ur, Tc, Pu TCE, PCE, CT Chlorinated biphenyls, phenols, benzoates } facultative anaerobes } strict anaerobes *Specific physiologies, and specific organisms, degrade specific contaminants.

6. Vadose Zone Microbiology ≈ 10 6 cells/g below the B horizon Live cells Physiological status Spatial distribution All 0.1g samples active No activity in 0.1 and 1g samples Live organisms cluster in isolated pockets Some 0.1 and 1g samples active * Colonization critical to remediation in arid climate V.Z.

7. Bioremediation requires: Order of magnitude increases in contaminant – degrading activity of individual cells Order of magnitude increases in populations of contaminant – degrading organism(s) same fraction of total population increased fraction of total population

8. Bioremediation Natural attenuation – degradation via natural processes Microbes often play a large role in physical/chemical remediation processes soil vapor extraction air sparging b-phase heating chemical oxidation Engineered or accelerated bioremediation – enhance rate of natural remediation or supplement other treatments by optimizing biology. bioventing (air) biosparging (air) nutrient injection – e - donor, donor + acceptor, other nutrients

9. Research Opportunities in Multidisciplinary V.Z. Science Unsaturated zone conditions/experimental systems Fracture flow hydrology-microbiology studies Colloidal transport of inorganic contaminants (microbes as colloids) What physiologies can be amplified in which V.Z. geohydrologic/geochemical environments Functional and genetic stability of communities in the face of natural and engineered perturbations Colonization of locations containing contaminants.

10. Where/How Can Bioremediation Fail? 1.Absence of contaminant – degrader population 2.Microbes, contaminants, nutrients not co-located 3.Other organisms out compete the contaminant-degraders Activity or numbers can not be adequately increased to achieve desired removal rate. 4.Sick contaminant – degrader population substrate: alternative substrate ratio too high or too low mutation & selection favor poorly performing strains crossed signals (microbes communicate w/chemical signals) byproducts toxic/inhibitory (transfer Rates too high) non-target organisms inhibitory * Functional stability is not guaranteed even if contaminant-degrader population is large