The role of gene diversity in the function of denitrifying assemblages in freshwater wetlands

Background/Question/Methods

A central question in microbial ecology is whether the composition of microbial assemblages influences ecological function. Understanding the relationship between structure and function is particularly important for denitrification because of its ecological impact and the wide variety of bacteria that perform this function. Additionally, there is variation among denitrifier assemblages in the release of N₂O, a potent greenhouse gas that is unintentionally increased by wetland restoration. In this study, we investigated the relationship between diversity of denitrifying genes and bacterial taxa that harbor them, as well as the effect of these two factors on denitrification rate in freshwater wetlands. We hypothesized that variation in functional gene sequence and abundance underlies differences in denitrification rates and end products (N₂ and N₂O). Experimentally, two approaches were taken: cultivation-independent analysis of environmental samples and cultivation of denitrifiers for use in microcosm manipulation experiments. Sediment samples were collected from wetlands of the Herrick Aquatic Environmental Research Facility at Kent State University. DNA was extracted from samples for PCR of the nitrous oxide reductase gene (nosZ). Cultivation experiments followed two approaches: anaerobic enrichment of sediments followed by spread plate dilution series and direct spread plate inoculation.

Results/Conclusions

PCR analysis revealed that the nosZ gene was present in 50% of the wetland sediment samples examined to date. Subsequent analysis of this gene via quantitative PCR suggests that denitrifying bacteria were at low abundance or absent from some samples. A total of 223 isolates were obtained from enrichments and direct plating, and were screened for denitrification activity. Of these isolates, 2% produced gas under anaerobic, nitrate-rich conditions, and 80% reduced nitrate to nitrite, but the nosZ gene was not detectable by PCR from any of the isolates. Our findings demonstrate that the occurrence of the nosZ gene is heterogeneous at the wetland study site. Thus, in this environment bacteria appear to be exposed to a variety of environmental conditions that influence the abundance and function of denitrifiers.