『Abstract
　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.

Keywords: Bioremediation; Autotrophic denitrification; Pyrite; DGGE; qPCR; Nitrate isotopes』

1. Introduction
2. Experimental methodology
　2.1. Study site
　2.2. Material and characterization
　2.3. Experimental set-up
　　2.3.1. Batch experiments
　　2.3.2. Flow-through experiments
　2.4. Chemical and isotopic analyses
　2.5. Microbial community analysis
　　2.5.1. DNA extraction
　　2.5.2. PCR and Denaturing Gradient Gel Electrophoresis (DGGE)
　　2.5.3. Quantitative PCR assays
3. Results
　3.1. Batch experiments
　3.2. Flow-through experiments
　3.3. Isotopic fractionation
　3.4. PCR-DGGE analysis of the microbial community
　3.5. Quantification of 16S rRNA and nosZ genes
4. Discussion
　4.1. Enhancement of denitrification by pyrite addition and response of the microbial community
　4.2. Long-term performance
　4.3. Recalculation of the extent of natural attenuation in the Osona aquifer
5. Conclusions
Acknowledgments
Appendix A. Supplementary data
References