Dissecting the link between community composition and function in the laboratory: Denitrification in pure and mixed cultures

Background/Question/Methods

Denitrification provides an important ecosystem service via removal of excess bioavailable nitrogen.  On the other hand, intermediates released during denitrification—nitrite, nitric oxide, and nitrous oxide—detract from this service.  This has led to efforts to determine conditions that regulate denitrification rates and production of intermediates.  Field studies that have attempted to link denitrifier community structure and production of denitrification end products have yielded mixed results.  This study examined the relationship between specific denitrifiers and denitrification reaction kinetics, which in turn regulates accumulation of end products.  To address interactions among denitrifiers, responses were examined in mono-cultures and compared to mixed cultures.  Mixing cultures in pairs was hypothesized to be non-additive, meaning that responses in mono-culture would not be predictive of responses in co-culture. This is predicted because each isolate will affect the concentrations and stoichiometry of denitrification intermediates, which will in turn influence reaction kinetics.

Batch cultures of single denitrifying isolates (mono-culture) and pairs of isolates (co-culture) were established in nitrate broth, under anaerobic conditions.  Denitrification was approximated as acetylene-inhibited nitrous oxide production (ANOP).  Headspace samples were collected every hour for 5 hours and analyzed for nitrous oxide with gas chromatography.  Nitrous oxide accumulation was measured using the same batch culture technique with no acetylene addition.

Results/Conclusions

 ANOP of both mono-cultures and the mixture were closely fit to exponential functions.  Mean ANOP increased each hour by 204%, 144%, and 197% for isolates J, S2, and the mixture.  Mean accumulation of acetylene-inhibited nitrous oxide (ANO) at the end of 5 hours was 224 mg/L (± 43 mg/L), 47.1 mg/L (± 2.2 mg/L), and 152 mg/L (± 14.7 mg/L) respectively.  Nitrous oxide accumulation was more variable than ANOP and could not be fit to linear or exponential functions.  Mean accumulation of nitrous oxide for J, S2, and the mixture were 5.52 mg/L (± 0.72 mg/L), 2.52 mg/L (± 0.44 mg/L), and 6.05 mg/L (± 0.86 mg/L) respectively.  These results imply that although the 2 isolates displayed different denitrification and nitrous oxide accumulation rates, the mixture seemed to reflect the denitrification rate of the more robust denitrifier isolate.  Therefore, the mixture did not appear to have interaction effects that either negatively or positively influenced denitrification rate.  However, more work needs to be done to quantify other denitrification intermediates (NO₂^- and NO₃^-) and the number of cells of each isolate in culture.  This information will shed light on the influence of isolate interactions on denitrification kinetics.