## 4 Discussion
### Long-Term Impact of Brine/Oil Contamination on Nitrogen Cycling in Tallgrass Prairie Soil  
Our results support the hypothesis that the species composition of NR/DN bacteria in brine and oil contamination continued to be affected years after the contamination event. In particular, strains of some groups (e.g., Bacilli) that are known to be salt tolerant [5; 78] were found in brine/oil-contaminated soils but not in crude oil-contaminated soils, while most NR/DN strains isolated from the oil-contaminated sites were Gammaproteobacteria bacteria. Although it has been reported that many hydrocarbonelastic bacteria responsible for the first steps of hydrocarbon degradation are classified as \(\gamma\)-Proteobacteria [79], it is not known whether hydrocarbons represent a readily usable carbon source for our NR and DN \(\gamma\)-Proteobacteria strains. Our previous studies of bioremediation at these sites did document the substantial loss of hydrocarbons in association with higher numbers of hydrocarbon-degrading bacteria [34] and of \(\gamma\)-Proteobacteria [37]; however, the hydrocarbon-degrading bacteria were not identified in those studies.  
Other long-term effects from previous research indicated that sites contaminated 5-10 years prior had at least ten times the level of nitrate as nearby uncontaminated prairie sampled in parallel [13] despite our findings that NR and DN bacteria were not eliminated by brine/oil contamination. Although certain groups were relatively more or less abundant in oil or brine sites than in the parallel prairie sites, it is not known to what extent this shift in species composition directly affected the rate of nitrogen transformations in tallgrass prairie soil. There were a number of differences between contaminated and uncontaminated sites in terms of environmental parameters, such as soil moisture potentially impacting the activity of nitrate-reducing and denitrifying bacteria. In addition, plant community composition, nematode community structure, and the overall microbial community composition were affected as well [12; 13; 34; 37]. Dominant groups of bacteria in undisturbed TPP included Actinobacteria and Acidobacteria as determined by 16S rRNA sequence analysis but were relatively low in Gammaproteobacteria in contrast to soil contaminated with crude oil [37], which contained a higher proportion of Gammaproteobacteria and lower proportions of Actinobacteria and Acidobacteria. For primarily brine-impacted sites, there was initially about half the PLGA concentration (phospholipid fatty acids, an indication of live cell biomass, [37]) and differences in PFLA structural groups (which roughly correspond to different higher-level taxonomic groups) compared to samples from native prairie. These differences had decreased but not vanished after four years [36; 37]. As with oil-contaminated soil, 16S rRNA sequence analysis of brine-contaminated soil had a lower proportion of Acidobacteria and Actinobacteria but higher proportions of Firmicutes, Gammaproteobacteria, and Bacteroidetes [37]. It is noteworthy that at the time of the current study, as much as a decade post-contamination, native prairie had approximately 1/10 the concentration of nitrate as did adjacent contaminated sites, which had never been remediated [13]. Future research should investigate if an alteration in the rate of nitrogen transformations via nitrate reduction and denitrification has a long-term impact on contamination by crude oil and brine.