Isolation and functional analysis of denitrifiers in an aquifer with high potential for denitrification

Aquifers are among the main freshwater sources. The Raigón aquifer is susceptible to contamination, mainly by nitrate and pesticides, such as atrazine, due to increasing agricultural activities in the area. The capacity of indigenous bacteria to attenuate nitrate contamination in different wells of this aquifer was assessed by measuring denitrification rates with either acetate plus succinate or nitrate amendments. Denitrification activity in nitrate-amended assays was significantly higher than in unamended assays, particularly in groundwater from wells where nitrate concentration was 33.5 mg L⁻¹ or lower. Furthermore, groundwater denitrifiers capable of using acetate or succinate as electron donors were isolated, identified by 16S rRNA gene sequencing and evaluated for functional denitrification genes (nirS, nirK and nosZ). Phylogenetic affiliation of 54 isolates showed that all members belonged to nine different genera within the Proteobacteria (Bosea, Ochrobactrum, Azospira, Zoogloea, Acidovorax, Achromobacter, Vogesella, Stenotrophomonas and Pseudomonas). In addition, isolate AR28 that clustered separately from validly described species could potentially belong to a new genus. The majority of the isolates were related to species belonging to previously reported denitrifying genera. However, the phylogeny of the nirS and nosZ genes revealed new sequences of these functional genes. To our knowledge, this is the first isolation and sequencing of the nirS gene from the genus Vogesella, as well as the nosZ gene from the genera Acidovorax and Zoogloea. The results indicated that indigenous bacteria in the Raigón aquifer had the capacity to overcome high nitrate contamination and exhibited functional gene diversity.