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Evaluation and Modeling of Subsurface Biobarrier Formation and Persistence

Principal Investigators
A.B. Cunningham, Montana State University; and B.M. Chen, University of Wyoming


Goal: The overall goal of this project is to understand factors which promote or retard biomass accumulation in porous media with an intent to apply such understanding toward prediction and beneficial manipulation of permeability and mass transport properties.

Rationale: A concept which appears promising in the manipulation of biological and chemical processes for remediation of subsurface hazardous waste sites is the creation of biobarriers for containment and remediation of soil and ground water contaminated with organics and heavy metals. Biobarriers are formed by stimulating growth of microbial biomass so as to plug the free pore space flow paths through porous media, thereby reducing permeability and mass transport. Selective plugging of permeable strata is currently being explored as a means of preventing contaminant migration of ground water contaminants from hazardous waste sites. Penetration of bacteria through porous media varies between extensive penetration of ultramicrobacteria and formation of plugging biofilms on the proximal formations by well-fed cells of the same organisms. Investigators will attempt to use simple nutritional differences to deliver bacteria to any location in the subsurface environment to resuscitate and either plug the formation or carry out specific biodegradation.

Approach: Test organisms will include a Klebsiella pneumoniae as well as these same bacteria starved for ultramicrobacteria size. Experimental objectives will be carried out using a series of flowing packed bed reactors including flat plate flow cells and packed columns. Procedures will be developed for applying bacterial inoculum, along with subsequent resuscitation with nutrients, so as to produce controlled reduction of porous media permeability and dissolved oxygen transport. Researchers will quantify and model temporal and spatial variability in the biofilm accumulation (and mass transport) using bioluminescence. Finally, a mathematical model for biofilm accumulation and corresponding permeability and dissolved oxygen gradients in porous media will be developed and evaluated.

Status: Columns have been inoculated with streptomycin-resistant K. pneumoniae. The effluent cell density was not significantly higher than the initial effluent cell density. However, the number of streptomycin-resistant cells in the column effluent increased. Although inoculated cell recovery was less than 10%, the streptomycin-resistant inoculum comprised a significant proportion of the culturable microbial community in the column effluent. Next, the columns were treated with sodium citrate medium (SCM). Analysis of fluid samples taken throughout the column revealed that viable cells and nutrient medium components were uniformly distributed throughout the column. Nutrient resuscitation led to a uniform increase in bacterial numbers throughout the column. Examination of colony morphotypes on HPC enumeration plates suggested that use of SCM for resuscitation led to a selective advantage for colonization success of the inoculated K. pneumoniae population over the culturable indigenous bacterial population. Following 16 days of nutrient resuscitation, nearly 100% of the microbial population in the column effluent were culturable. Nutrient resuscitation of the starved bacterium inoculum resulted in a reduction in hydraulic conductivity throughout the length of the columns. This project is in its first year.

Clients/Users: This project will interest the U.S. Department of Energy, U.S. Department of Defense, environmental contractors, regulators, and those in the petroleum industry.

Key words: biofilms, hydraulic conductivity, ultramicrobacteria, waste containment, barriers.

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