Robert R. Sharp

Subsurface Biofilm Barriers for the Containment and Remediation of Contaminated Groundwater

An engineered microbial biofilm barrier capable of reducing aquifer hydraulic conductivity while simultaneously biodegrading nitrate has been developed and tested at a field-relevant scale. The 22-month demonstration project was conducted at the MSE Technology Applications Inc. test facility in Butte, Montana, which consisted of a 130 ft wide, 180 ft long, 21 ft deep, polyvinylchloride (PVC)-lined test cell, with an initial hydraulic conductivity of 4.2 × 10−2 cm/s. A flow field was established across the test cell by injecting water upgradient while simultaneously pumping from an effluent well located approximately 82 ft down gradient. A 30 ft wide biofilm barrier was developed along the centerline of the test cell by injecting a starved bacterial inoculum of Pseudomonas fluorescens strain CPC211a, followed by injection of a growth nutrient mixture composed of molasses, nitrate, and other additives. A 99% reduction of average hydraulic conductivity across the barrier was accomplished after three months of weekly or bi-weekly injections of growth nutrient. Reduced hydraulic conductivity was maintained by additional nutrient injections at intervals ranging from three to ten months. After the barrier was in place, a sustained concentration of 100 mg/l nitrate nitrogen, along with a 100 mg/l concentration of conservative (chloride) tracer, was added to the test cell influent over a six-month period. At the test cell effluent the concentration of chloride increased to about 80 mg/l while the effluent nitrate concentration varied between 0.0 and 6.4 mg/l.

Activity and stability of a recombinant plasmid‐borne TCE degradative pathway in biofilm cultures

The retention and expression of the plasmid-borne, TCE degradative toluene-ortho-monooxygenase (TOM) pathway in suspended continuous cultures of transconjugant Burkholderia cepacia 17616 (TOM31c) were studied. Acetate growth and TCE degradation kinetics for the transconjugant host are described and utilized in a plasmid loss model. Plasmid maintenance did not have a significant effect on the growth rate of the transconjugant. Both plasmid-bearing and plasmid-free strains followed Andrews inhibition growth kinetics when grown on acetate and had maximum growth rates of 0.22 h-1. The transconjugant was capable of degrading TCE at a maximum rate of 9.7 nmol TCE/min. mg protein, which is comparable to the rates found for the original plasmid host, Burkholderia cepacia PR131 (TOM31c). The specific activity of the TOM pathway was found to be a linear function of growth rate. Plasmid maintenance was studied at three different growth rates: 0.17/h, 0.1/h, and 0.065/h. Plasmid maintenance was found to be a function of growth rate, with the probability of loss ranging from 0.027 at a growth rate of 0.065/h to 0.034 at a growth rate 0.17/h.

International Specialized Conference on Sludge Management, Czestochowa, Poland, 26-28 June 1997

Exposure of plasmid recombinant microorganisms to an open environment, either inadvertently or intentionally, requires research into those fundamental processes that govern plasmid retention, transfer and expression. In the open environment, a majority of the microbial activity occurs associated with an interface, within thin biological layers consisting of cells and their insoluble extracellular polymer, layers known as biofilms . Current toxic wastewater or wastegas treatment reactors exploit bacterial biofilm systems for certain system operating advantages. Using recombinant bacteria within a biofilm reactor to degrade xenobiotic wastes requires finding a suitable host to harbor and express the desired plasmid phenotype. Suitable host characteristics include: the ability to produce copious amounts of biofilm, resistance to waste-related injury and toxicity, and the ability to retain and express the desired plasmid during long term operation. This paper reports on a laboratory evaluation of factors governing plasmid retention and the expression of trichloroethene (TCE) degradative capacity in both suspended and biofilm cultures.