Molecular Ecology Studies Of The NosZ-encoding Denitrifying Bacteria In High Temperature Oil Reservoirs Of The Shengli Oilfield

Nitrogen is one of the basic elements for essential life activities. The nitrogen cycle is the process of biogeochemical transformation of various forms of nitrogen among molecular nitrogen, organic nitrogen and inorganic nitrogen. Denitrification is a microbial process of nitrate reduction that ultimately produces molecular nitrogen (N2) or other nitrogen oxides as the intermediate gaseous products. Nitrite reductase genes (nirS and nirK) and nitrous oxide reductase gene (nosZ) are widely used in the studies of denitrifying microbial diversity. In the current study, methods of molecular ecology employing the environmental microbial nosZ gene clone library construction and sequence phylogenetic analysis were used to investigate the diversity, community structure and spatial distribution of the denitrifying bacteria in the high temperature oil reservoirs of the Shengli oilfield, in order to decode their basic ecological characteristics and roles in the nitrogen biogeochemical cycling process in this extreme environment.Five denitrifying bacterial nosZ gene clone libraries were constructed from the oil well production fluid samples of the high temperature oil reservoirs. A total of 375 gene clones, from all the samples, were obtained, which were grouped into a total of 90 OTUs (Operational Taxonomic Units) after the nosZ gene sequences were translated into NosZ protein sequences. Some of the NosZ sequences shared quite distant evolutionary relationship with those from known denitrifying bacteria, possibly representing novel unknown denitrifying bacterial species or groups specific to high temperature oil reservoirs. According to the result of molecular phylogenetic analysis, the NosZ protein sequences were assigned into 10 clusters (ClustersⅠtoⅩ). Most of the NosZ sequences were related to Pseudomonadales (ClusterⅧ), Alteromonadales (ClusterⅨ) and Rhodobacteraceae (ClusterⅡ). Some NosZ sequences had their closest matches obtained in coastal and marine environments, including the Jiaozhou Bay, the South Atlantic and the continental shelf sediments. Multivariate statistical approaches were used for the molecular ecology analysis, including the UniFrac environmental clustering, the principal coordinates analyses (PCoA) and the canonical correspondence analysis (CCA). The analytical results indicated that the nosZ-encoding denitrifying bacteria were highly diverse and they formed complicated communities in the high temperature oil reservoirs of the Shengli oilfield. Furthermore, the spatial distribution of the nosZ-encoding bacterial assemblages was apparently correlated with environmental factors, where the oil reservoir depth possibly as the most influential environmental factor, along with other oil reservoir physicochemical properties (such as reservoir fluid conductivity and Eh), may cooperatively influence the structure and spatial variation of the nosZ-encoding denitrifying bacterial assemblages in these oil reservoirs.