Abstract: Denitrifier method is a novel method for nitrogen and oxygen isotopic analysis of nitrate, which has many advantages, e.g., low nitrate water sample, simple pretreatment, no crosscontamination, and small volume water, and obtained rapid popularization. In this study, the method was first developed in China for nitrate isotopic measurement in our laboratory. The denitrifier (Pseudomonas aureofaciens) was grown in an amended medium of Tryptic Soy Broth. After 5-10 days culture, the cells were harvested and concentrated 5 folds, and split into 3 mL aliquots in 20 mL headspace vials. The vials were sealed and then purged for at least 3 h with highpurity N₂ to ensure anaerobic condition. A sample amount corresponding to 50 nmol of nitrate was added to the sample vials, and cultured overnight to allow the complete conversion of NO₃^- to N₂O. On the following day, 0.1-0.2 mL of 10 mol·L^-1 NaOH was injected into the vials to lyse the bacterium and to scrub any CO₂ gas. Simultaneous δ¹⁵N and δ¹⁸O analysis of the produced N₂O was carried out by using a Trace Gas Preconcentrator unit coupled to an IRMS instrument. The measured data showed good reproducibility and precision, in close with the similar international studies. The establishment of this method would promote our studies in nitrogen biogeochemical cycle in rivers and lakes (reservoirs) and in precipitation.

Key words: anaerobic dechlorination, dechlorination enrichment culture, electron donor, DGGE, microbial community structure.