## 1 Introduction  
Soil is a very complex environment with a high diversity of bacteria [1]. After contamination of soil with crude oil and/or oilfield brine (e.g., water containing salts in solution, produced along with oil), certain groups of bacteria come to dominate the contaminated sites, particularly \(\alpha\)-, \(\beta\)-, \(\gamma\)-, and \(\delta\) Proteobacteria in the crude oil contaminated sites [2, 3, 4] and Firmicutes [5] and some _Pseudomonas_ species [6] in brine contaminated sites. The alteration in bacterial species composition and numbers might be expected to affect biogeochemical cycling in soils. In tallgrass prairie soils, nitrogen is a limiting nutrient for the growth of plants and soil decomposition rates [7]. Therefore, the effect of oil/brine contamination on nitrogen-cycling bacteria is of particular concern in tallgrass prairie soil. In fact, nitrate-reducing (NR) and denitrifying (DN) bacteria are found in oil/brine contaminated soils [8, 9, 10, 11], including Tallgrass Prairie Preserve (TPP) soil contaminated by oil/brine [12, 13], but a detailed examination of their species composition and abundance in oil/brine contaminated TPP soil is unknown. In particular, the effects of long-term residual oil and brine contamination on NR and DN species composition are not known. Nitrate-reducing microorganisms represent a widespread group with members among \(\alpha\)-, \(\beta\)-, and \(\gamma\)-Proteobacteria, some members of Firmicutes, and even _Archaea_. Thesemicroorganisms use nitrate as an alternative electron acceptor that makes them able to obtain energy from dissimilatory reduction of nitrate into nitrite by nitrate reductase enzymes [14]. Nitrite can be reduced into gaseous nitrogen compounds by denitrification or by dissimilatory nitrate reduction into ammonia (DNRA) [15; 16]. Nitrite reduction to nitrite is catalyzed by two different types of nitrate reductases, either membrane-bound encoded by the _narGHJ_I operon or periplasmic encoded by the _napABC_ operon [17]. Some NR bacteria can harbor either _narG_, _napA_, or both [18; 19]. The denitrification process consists of four reaction steps in which nitrate is reduced, and nitrogen is lost in the form of nitrogenous gasses [20]. Because nitrite reductase produces NO, the first gaseous product, nitrite reductase is a significant enzyme in the denitrification pathway [21; 22] and signals the potential for loss of nitrogen from the soil ecosystem. Nitrite reductase is found in two different forms. The first form contains copper and is encoded by _nirK_, while the second contains cytochromes (hemes) \(c\) and \(d_{1}\) and is encoded by _nirS_[14; 22; 23; 24; 25]. The reduction of nitric oxide is catalyzed by nitric oxide reductase, whose small and large subunits are encoded by _norC_ and _norB_, respectively [26]. Based on the European Molecular Biology Laboratory (EMBL) sequence database, norB sequences are grouped in two very distinct branches where the first class encodes the cytochrome bc-type complex (cNorB) while the second class encodes the quinol-oxidizing single-subunit class (qNorB) [26]. The last step in the denitrification pathway is the reduction of nitrous oxide to N\({}_{2}\), which is catalyzed by nitrous oxide reductase encoded by the _nosZ_ gene. Since denitrification also is common among phylogenetically unrelated groups, it is very unsuitable to investigate communities of DN bacteria by using an approach based on 16S rRNA gene sequences [23; 27]. Therefore, genes coding for such key steps as nitrite and nitrous oxide reduction are often used to detect DN bacteria [23; 28; 29; 30; 31; 32]. Nitrate reducers and denitrifiers are an excellent model system for investigating the response of an important component of the N-cycling soil community to the contaminants of crude oil and brine as they contain members from among many different groups of microorganisms.