ISOLATION AND CHARACTERIZATION OF NITRATE REDUCING/DENITRIFYING BACTERIA FROM TALLGRASS PRAIRIE SOILS

This dissertation addresses the long term impact of crude oil/brine (e.g. salt water) spills on biogeochemical cycling of nitrogen in tallgrass prairie soils. The study sites were prairie soils that were accidently contaminated with crude oil and/or brine 5-10 years prior to this study. These sites had been partially bioremediated but still had low levels of oil and brine which could affect the abundance and species composition of denitrifying bacteria. The abundance of culturable nitrate reducing (NR) and denitrifying (DN) bacteria in the soil samples was estimated by 5-tube Most Probable Number (MPN) method using nitrate broth. The strength of the association of the abundance of NR and DN bacteria with various environmental factors including soil moisture, brine and/or crude oil, oxygen, and nitrate was estimated using multivariate statistics. The MPN tubes were the source of NR and DN isolates used to determine if molecular detection methods for NR and DN genes matched the ability of these strains to perform nitrate or nitrite reduction. My results showed that the NR and DN bacteria were as abundant in the long-term contaminated sites as in the uncontaminated sites. Soil moisture had a slight positive effect on the abundance of NR and DN bacteria in both contaminated and uncontaminated sites. In general, nitrate treatment did not produce an increase in numbers of NR and DN bacteria in contaminated sites. The degree of culturable bacterial diversity in the contaminated prairie sites was not lower than that in prairie uncontaminated sites. However, species composition of nitrate-reducing bacteria varied among different sites where brine contaminated soils selected for salt tolerant bacteria like Bacillus while crude oil-contaminated sites selected for ã-Proteobacteria. Nitrate reducing and DN functional genes were detected in roughly half the strains that reduced nitrate or nitrite which suggests that the PCR-molecular detection methods underestimate the number of NR and DN bacteria. However, a high proportion of ã-Proteobacteria was correctly identified by PCR detection methods. These results indicate that both molecular and phenotypic methods are needed to correctly identify NR and DN bacteria. Examining a link to bioremediation through bioavailability of organic contaminants, I showed that naphthalene 1,2-dioxygenase (NDO) can enzymatically alter a variety of humic and fulvic acids and the extent of NDO-specific NADH oxidation paralleled the percent aromaticity of the humic and fulvic acids. Humic substances have not previously been known to be substrates for dioxygenases; even more significant was that dioxygenase enzymes can facilitate condensation between indole-like functional groups well-known to be present in humic and fulvic acids. The NDO enzyme retained activity for two weeks under ambient conditions suggesting prolonged extra cellular activity. These results illustrate how enzymes like NDO might alter the bioavailability of organic contaminants associated with soil when released into the environment upon microbial death