Enhanced denitrification in groundwater and sediments from a nitrate-contaminated aquifer after addition of pyrite

Using chemical, isotopic and microbiologic techniques we tested in laboratory experiments the extent to which the addition of pyrite to groundwater and sediments from a nitrate-contaminated aquifer could stimulate denitrification by indigenous bacteria. In addition to this biostimulated approach, a combined biostimulated and bioaugmented treatment was also evaluated by inoculating the well-known autotrophic denitrifying bacterium Thiobacillus denitrificans. Results showed that the addition of pyrite enhanced nitrate removal and that denitrifying bacteria existing in the aquifer material were able to reduce nitrate using pyrite as the electron donor, obviating the need for the inoculation of T. denitrificans. The results of the 16S rRNA and nosZ gene-based DGGE and the quantitative PCR (qPCR) showed that the addition of pyrite led to an increase in the proportion of denitrifying bacteria and that bacterial populations closely related to the Xanthomonadaceae might probably be the autotrophic denitrifiers that used pyrite as the electron donor. Not only autotrophic but also heterotrophic denitrifying bacteria were stimulated through pyrite addition and both populations probably contributed to nitrate removal. Isotopic analyses (δ15N and δ18ONO3) were used to monitor enhanced denitrification and the N and O isotopic enrichment factors (− 26.3 ± 1.8‰ and − 20.4 ± 1.3‰, respectively) allowed to calculate the degree of natural nitrate attenuation in the aquifer. Furthermore, flow-through experiments amended with pyrite confirmed the long-term efficiency of the process under the study conditions. Further research under field conditions is needed to determine whether stimulation of denitrification by pyrite addition constitutes a feasible bioremediation strategy for nitrate-contaminated aquifers.

Research highlights
► Biostimulation of the aquifer material with pyrite enhances denitrification.
► Addition of pyrite increases the proportion of native denitrifying bacteria.
► Both autotrophic and heterotrophic denitrifiers are stimulated after pyrite addition.
► The durability of the effects of the biostimulation over time is proved.
► The extent of natural denitrification is recalculated using isotopic fractionation.