The isotope pairing technique (IPT) has been used as the standard measure of anammox activity, most commonly using homogenized sediments (Thamdrup and Dalsgaard 2002). Concentrations of NH4+, NO3-, and NO2- are first determined, the sediments are placed in airtight containers with septums, such as Exetainer tubes, and the headspace is flushed with He for a minimum of 5 minutes to replace ambient O2. Concentrations of residual NOx species are monitored over time until all available NOx is removed from the incubations. Three parallel incubations are then performed: (1) 15NH4+ alone, (2) the combination of 15NH4+ and 14NO2-, and (3) 15NO2- alone. Reactions are stopped by the addition of ZnCl2.The first incubation is used as a control to detect any oxidation of ammonium without the addition of nitrite. The lack of 29N2/30N2 is indicative of the lack of oxidants at the end of the pre-incubations. The second treatment is used to determine if anammox activity is possible. The production of 29N2 indicates anammox activity through the oxidation of ammonium with nitrite. The combination of the first two incubations is used to establish anammox activity. Finally, the third incubation is used to estimate anammox and denitrification rates (Fig. 10.2). Anammox produces 29N2 through the oxidation of the resident NH4+ pool with the added 15NO2-, while denitrification is measured by the production of 30N2. However, evidence that anammox can also
Fig. 10.2 Typical porewater nitrogen profile from a deep ocean sediment, indicating a possible zone of anaerobic ammonium oxidation
Fig. 10.2 Typical porewater nitrogen profile from a deep ocean sediment, indicating a possible zone of anaerobic ammonium oxidation reduce 15NO3- to 15NO2- to 15NH4+ (Kartal et al. 2007a) results in the possibility that the anammox reaction can pair 15NO2- with 15NH4+, and thus some proportion of measured denitrification may be partitioned to the anammox reaction. Several modifications to this protocol are promising, notably the addition of N2O measures to more accurately quantify N2 production, and the use of intact sediment cores (Trimmer et al. 2006).
Molecular methods have been extensively utilized to identify the presence of anammox bacteria in environmental and wastewater samples. Fluorescence in situ hybridization (FISH) targeting the 16S rRNA gene has been used extensively, and is described in detail by Schmid et al. (2005). Anammox bacteria have also been identified using PCR, using a variety of primers, often based on FISH probes, targeting the group as a whole or specific members (Schmid et al. 2005; Penton and Tiedje 2006). The unique ladderane lipids that constitute the anammoxosome have also been used as biomarkers for relative quantification (Kuypers et al. 2003), while distinctive hopanoid lipids may be useful in assessing relative anammox abundance in the sedimentary record (Sinninghe Damste et al. 2004). Quantitative PCR (q-PCR) has been used for direct quantification of all known anammox-like bacteria in water columns (Hamersley et al. 2007), in wastewater enrichment cultures (Tsushima et al. 2007), and for the specific enumeration of Candidatus Scalindua "marine" anammox in sediments.
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