Measurement of Denitrification in Sediments with the¹⁵N Isotope Pairing Technique

Abstract

Due to increasing nitrogen concentrations in surface waters (6) and eutrophication of coastal waters (14,26,51), the quantification of denitrification rates in sediments of lakes, rivers, and estuaries has gained importance. Different methods for measuring denitrification have been developed: the acetylene inhibition technique (41), measurement of nitrate disappearance (2), calculation of nitrate fluxes into the sediment from pore water profiles (20), the 15 N nitrate dilution method (16), direct measurement of N 2 production (40), the nitrogen massbalance approach (1), and the 15 N isotope pairing technique (IPT) (23). All of these methods have advantages but also potential problems and limitations (38,39).The acetylene inhibition technique is a very simple method. It is widely used, and its sensitivity has been improved by N 2 measurements based on N 2 /Ar ratios. However, many different studies have documented that the acetylene method systematically underestimates denitrification (20). Several causes may be responsible for artifacts: (i) inhibition of coupled nitrification-denitrification by acetylene (19), (ii) incomplete blockage of N 2 O reductase at low nitrate concentrations (3), (iii) incomplete blockage of N 2 O reductase when sulfide is present (3), (iv) diffusion of N 2 O toward deeper sediment layers and reduction to N 2 (39), and (v) catalytic oxidation of NO into NO 2 (4). The disappearance of nitrate can overestimate denitrification because nitrate may not only be denitrified but also reduced to ammonia (16,20) or assimilated (36). On the other hand, it may also underestimate denitrification because it does not consider coupled nitrification-denitrification. The calculation of nitrate fluxes into the sediment from pore water profiles has the same problems as described for the nitrate disappearance method. In addition, the resulting fluxes across the sediment-water interface and the diffusive boundary layer often underestimate denitrification, because of insufficient vertical resolution of the profiles and because only diffusive and not turbulent transport is considered when the fluxes are calculated (11, 21). The 15 N nitrate dilution method with subsequent measurement of 15 N nitrate disappearance and 15 N ammonia production quantifies denitrification and nitrification but still neglects coupled nitrification-denitrification and assimilation. Direct measurement of N 2 production has the advantage to include coupled nitrification-denitrification but requires a very sensitive analysis because of the small N 2 production compared to the N 2 background. The mass-balance approach on larger systems such as whole lakes may lead to considerable errors due to a combination of the errors of each term in the mass-balance.The 15 N IPT has the advantage that denitrification of both NO 3 Ϫ diffusing from the overlaying water and NO 3 Ϫ from nitrification within the sediment can be quantified.The purpose of this study is to briefly describe the principles of the 15 N IPT, to review the different applications ...

Seasonal oxygen, nitrogen and phosphorus benthic cycling along an impacted Baltic Sea estuary: regulation and spatial patterns

“…Our rates are at least twice as high as rates previously measured in sediment from the open Baltic Proper (Tuominen et al 1998;Deutsch et al 2010), generally comparable to those measured in the Gulf of Finland (Hietanen and Kuparinen 2008;Jäntti et al 2011), but 1.5 to twofold lower than those in muddy sediments of the southern Baltic Sea (Deutsch et al 2010). Compared to measurements in other coastal settings worldwide, our rates and those reported in previous studies from the Baltic are generally low (Seitzinger 1988;Steingruber et al 2001). Denitrification rates did not correlate with organic carbon content, contrasting with the observations from a previous work on sedimentary denitrification in the Baltic (Deutsch et al 2010).…”

“…It is conspicuous that the threefold drop in denitrification agrees so well with the decrease in DIN discharge from the STP. The drop in denitrification rates could be even more significant when considering that the acetylene inhibition method used in the Elmgren and Larsson (1997) study tends to underestimate rates of denitrification because of potential coinhibition of nitrifying bacteria (Steingruber et al 2001). Furthermore, in the 1980s and early 1990s the sediment hosted different communities of bioturbating fauna dominated by the amphipods Monoporeia affinis and Pontoporeia femorata (Elmgren and Larsson 1997), whereas today the widespread polychaete Marenzelleria spp.…”

Archaeal ammonia oxidizers and nirS-type denitrifiers dominate sediment nitrifying and denitrifying populations in a subtropical macrotidal estuary

“…Â 25 cm) taken from the original sediment cores used for the flux measurements. The isotope pairing method involved the spiking sediment cores with 15 NO À 3 and measuring the rate of conversion to 15 Nlabelled N 2 gas by isotope ratio mass spectrometry (Steingruber et al, 2001).…”

New Method of Denitrification Analysis of Bradyrhizobium Field Isolates by Gas Chromatographic Determination of 15 N-Labeled N 2

“…For this reason, the activities and kinetic parameters of N 2 O reductase have been studied directly by measurement of the N 2 O decrease (4) or by spectrophotometric assay of reduced benzyl viologen for nitrous oxide reductase activity in vitro (12,23). Moreover, the underestimation of denitrification capability may occur because of incomplete blockage of N 2 O reductase at a low nitrate concentration or in the presence of sulfide (24). In practice, two determinations of N 2 O concentration per sample-in the presence and absence of acetylene-are required for estimation of the amount of N 2 evolved by N 2 O reductase.…”

“…Most denitrifying bacteria reduce the electron acceptors sequentially as NO 3 Ϫ 3 NO 2 Ϫ 3 NO 3 N 2 O 3 N 2 , and each step of these reactions is catalyzed by specific respective reductases (31). To evaluate denitrification capability, the acetylene inhibition assay (30) has been widely used in pure cultures (13) as well as for environmental samples such as soils (8,14,27) and water (24) because of sensitivity and atmospheric N 2 contamination (13). In this assay, the excess of N 2 O emission in the presence of acetylene over the level of N 2 O emission in the absence of acetylene is regarded as the N 2 evolution (because acetylene inhibits N 2 O reductase).…”