Novel laser spectroscopic technique for continuous analysis of N2O isotopomers – application and intercomparison with isotope ratio mass spectrometry

RATIONALE
Nitrous oxide (N(2)O), a highly climate-relevant trace gas, is mainly derived from microbial denitrification and nitrification processes in soils. Apportioning N(2)O to these source processes is a challenging task, but better understanding of the processes is required to improve mitigation strategies. The N(2)O site-specific (15)N signatures from denitrification and nitrification have been shown to be clearly different, making this signature a potential tool for N(2)O source identification. We have applied for the first time quantum cascade laser absorption spectroscopy (QCLAS) for the continuous analysis of the intramolecular (15)N distribution of soil-derived N(2)O and compared this with state-of-the-art isotope ratio mass spectrometry (IRMS).


METHODS
Soil was amended with nitrate and sucrose and incubated in a laboratory setup. The N(2)O release was quantified by FTIR spectroscopy, while the N(2)O intramolecular (15)N distribution was continuously analyzed by online QCLAS at 1 Hz resolution. The QCLAS results on time-integrating flask samples were compared with those from the IRMS analysis.


RESULTS
The analytical precision (2σ) of QCLAS was around 0.3‰ for the δ(15)N(bulk) and the (15)N site preference (SP) for 1-min average values. Comparing the two techniques on flask samples, excellent agreement (R(2)= 0.99; offset of 1.2‰) was observed for the δ(15)N(bulk) values while for the SP values the correlation was less good (R(2 )= 0.76; offset of 0.9‰), presumably due to the lower precision of the IRMS SP measurements.


CONCLUSIONS
These findings validate QCLAS as a viable alternative technique with even higher precision than state-of-the-art IRMS. Thus, laser spectroscopy has the potential to contribute significantly to a better understanding of N turnover in soils, which is crucial for advancing strategies to mitigate emissions of this efficient greenhouse gas.

enzymatic pathways associated with N 2 O production from nitrification and denitrification induce 15 N depletion in the emitted N 2 O which is considerably higher for nitrifying bacteria than for denitrifying bacteria. [3,4] Therefore, measurement of the 15 N content in N 2 O (d 15 N bulk value) is an excellent tool to study these processes, although it has to be considered that its d 15 N bulk value also depends on the precursor signature, fractionation during N 2 O to N 2 reduction, [5] and transport limitations as well as physiological controls. [6,7] In addition to the bulk 15 N isotopic composition of N 2 O, the site preference (SP = d 15 N ad 15 N b ), which specifies the intramolecular 15 N distribution on the central (a) and the end (b) positions of the linear asymmetric N 2 O molecule, has been shown to differ significantly between different microbial N 2 O-releasing processes in soil. SP values for nitrification (i.e. NH 3 oxidation via hydroxylamine) were found to be between 31 and 37 %, and in the range of -10 to 0 % for denitrification (heterotrophic as well as nitrifier denitrification). [8][9][10][11] Therefore, analysis of the N 2 O site-specific isotopic composition to allocate N 2 O production processes in soil studies is of increasing interest. [12][13][14][15][16] However, N 2 O isotopic source signatures for distinct microbial processes are still based on a limited number of pure culture studies. Furthermore, a simple two source mixing model might not always be adequate as, for example, N 2 O production by fungal denitrification (ca. 37 %) [7] and N 2 O to N 2 reduction by heterotrophic denitrifiers (eSP = 2.9 -6.8 %) [12,17] significantly increase the N 2 O site preference and might result in an overestimation of nitrification-derived N 2 O.
Most reported studies analyzing N 2 O isotopomers are based on mass spectrometric determination of molecular (N 2 O + ) and fragment (NO + ) ions of N 2 O, allowing the calculation of d 15 N bulk and SP values. [18,19] In contrast, novel spectroscopic techniques such as Fourier transform infrared (FTIR) spectroscopy, [20] or quantum cascade laser absorption spectroscopy (QCLAS), [21][22][23] enable the direct quantification of N 2 O isotopomers based on their characteristic rotationalvibrational absorption spectra, and hold advantages over isotope ratio mass spectrometry (IRMS) in terms of field applicability.
The aim of the present study was to demonstrate the feasibility of continuous N 2 O isotopomer analysis by laser spectroscopy for source identification of soil-derived N 2 O and its validation by intercomparison with IRMS as standard technique.

EXPERIMENTAL Setup
An arable soil, which had been used in previous studies, [24,25] taken from the top horizon of a Luvisol at the Hohenschulen experimental farm of Kiel University, Germany, was sieved and ca. 3 dm 3 soil was repacked into 4.25 L glass jars to a bulk density of 1.4 g cm -1 . Potassium nitrate and sucrose solution were applied on top of the soil at rates equivalent to 0.21 g sucrose and 0.025 g nitrate-N kg -1 soil dry matter (DM) (equivalent to 1200 kg sucrose ha -1 and 60 kg nitrate-N ha -1 , respectively) to foster N 2 O production by heterotrophic denitrification. The soil moisture was adjusted to 80 % waterfilled pore space. A control treatment was amended with nitrate only. Both treatments were set up in triplicate.
Pressurized air (Messer Schweiz AG, Lenzburg, Switzerland) was passed through the headspace of each incubation vessel at a flow rate of 20 mL min -1 (Fig. 1). To assess the variability between different soil cores and to perform an offline intercomparison between QCLAS and IRMS on N 2 O isotopomer concentrations, the outlet air of individual soil cores was sampled in Tedlar W bags deploying a peristaltic pump (Ecoline VC-MS/CA 8-6 with Tygon LFL tubing i.d. 0.63 or 0.89 mm; Ismatec, IDEX Health & Science SA, Glattbrugg, Switzerland) at 3.5 mL min -1 (nitrate sucrose treatment) and 6 mL min -1 (control treatment). The remaining outflow gas from the replicates of each treatment was combined and directed to a FTIR spectrometer for trace gas analysis (N 2 O, CO 2 ). For the nitrate-sucrose treatment a FTIR spectrometer (Avatar 370, Thermo Fisher Scientific, Waltham, MA, USA) with a low-volume (50 mL) flow-through gas cell with a 1 m optical path length (model LFT-210; Axiom Analytical Inc., Tustin, CA, USA) and InSb detector was applied. [26] For the control cores, a FTIR spectrometer (CX4000; Gasmet Technologies Oy, Helsinki, Finland), with a 9.8 m optical path cell and MCT detector was deployed. Continuous trace gas analysis was initiated 8 h prior to fertilizer addition and continued until the N 2 O mixing ratios decreased to background concentrations.
Prior to online N 2 O isotopomer analysis by QCLAS, H 2 O and CO 2 were quantitatively removed from the gas flow of the nitrate sucrose-treated soil cores, by means of a permeation drier (MD-070-24S; Perma Pure Inc., Toms River, NY, USA) and a chemical trap filled with Ascarite (20 g, 10-35 mesh; Sigma Aldrich, Buchs, Switzerland) bracketed by Mg(ClO 4 ) 2 (2 Â 8 g; Sigma Aldrich). For N 2 O concentrations above 100 ppm, the dried and CO 2 -scrubbed sample gas was dynamically diluted with synthetic air (Messer Schweiz AG) to a constant N 2 O mixing ratio (100 ppm) using a LabVIEW ™ controlled mass flow controller (MFC, Red-y Smart series; Vögtlin Instruments AG, Aesch, Switzerland), based on the N 2 O concentrations determined by FTIR spectroscopy. This experimental setup greatly reduced the need for non-linearity corrections of the QCLAS results and allowed optimal accuracy.

Laser spectroscopy
The laser spectrometer consisted of a single-mode, pulsed QCL (Alpes Lasers SA, Neuchâtel, Switzerland) emitting at 2188 cm -1 , a multipass absorption cell (AMAC-56; optical path length 56 m, volume 500 mL; Aerodyne Research Inc., Billerica, MA, USA) and a detection scheme with pulse normalization. [22] Laser control, data acquisition and simultaneous quantification of the three main N 2 O isotopic species ( 14 [23] Primary laboratory standards were analyzed for their d 15 N a , d 15 N b and d 15 N bulk values by IRMS at the Tokyo Institute of Technology. [19] Secondary working standards applied in the presented project were measured against primary standards by QCLAS: standard 1: at À150 C. Desorption is accomplished by resistive heating of the trap to +10 C and purging the released N 2 O with 10 mL min -1 of synthetic air into the evacuated multipass cell of the laser spectrometer. [21,22] To confirm the accuracy of our measurements, N 2 O isotopomer concentrations in the pres-  [27] with minor contributions of a 15 N-depleted N 2 O emission source.

Mass spectrometry
The gas samples collected in the Tedlar W bags were analyzed for their d 15 N a , d 15 N b , and d 18 O value by IRMS as a direct intercomparison between the two techniques at the von Thuenen Institute in Braunschweig, Germany. Isotopologue signatures of N 2 O were determined by analyzing m/z 44, 45, and 46 of intact N 2 O + molecular ions as well as m/z 30, 31 of NO + fragment ions. [19] A modified preconcentration unit consisting of a set of automated cryotraps (PreCon; ThermoFinnigan, Bremen, Germany) equipped with an autosampler (Combi-PAL; CTC-Analytics, Zwingen, Switzerland) was coupled to a gas chromatograph (Trace GC Ultra; Thermo Fisher Scientific, Bremen, Germany) which was connected via a Conflo IV interface to a Delta V isotope ratio mass spectrometer (Thermo Fisher Scientific). Simultaneous detection of m/z 30, 31, 44, 45, and 46 was hence possible. N-exchange between N 2 O + and NO + in the ion source of the mass spectrometer, the so-called scrambling factor, was determined by analyzing defined mixtures of non-labeled N 2 O with a N 2 O standard labeled at the b-N position (98 atom %; CK Gas Products Ltd., Hook, UK) as described by Röckmann et al., [28] giving a scrambling factor of 0.08 (a scrambling factor of 0.5 would mask the site preference entirely). The isotopologue ratios of 15 R bulk , 18 R and 15 R a were determined, and 15 R b was obtained by the relationship of 15  Linde, Munich, Germany) was used as reference gas which was analyzed for isotopologue signatures in the laboratory of the Tokyo Institute of Technology using the calibration procedures developed earlier. [19] This reference signature was used to correct the raw d 15 N a value determined by our IRMS instrumentation. The linear regression between the d 15 N a value and m/z 30 peak areas, as determined by analysis of reference gas standards with concentrations between 200 and 10000 ppb, was used to correct for non-linearity of the NO + isotope ratios. The m/z 30 and m/z 44 peak areas were used to determine N 2 O concentrations. The correction for 17 O for the d 15 N-N 2 O value was made according to the method described by Brand. [29] RESULTS AND DISCUSSION Continuous analysis of trace gas concentrations and N 2 O isotope ratios by infrared spectroscopy  values were around À35 %, but they then increased by more than 50 % in an almost linear way, reaching +16 % after 3 days (Fig. 3(a)). Similar results were reported by Meijide et al. [30] who observed an increase in d 15 [4] Although the emphasis of this study is on the implementation of a novel analytical technique and intercomparison measurements and the detailed discussion of the involved microbial source processes is beyond its scope, it should be pointed out that d 15 N bulk value observed in this study is in agreement with typical values reported for microbial N 2 O production processes. The 15 N site preference (SP, Fig. 3(b)) of the N 2 O released from the nitrate sucrose treatment was À1 % at the beginning of the incubation experiment and declined to around À2 to À3 % within the first day after onset. Two short-term shifts in SP and N 2 O mixing ratios within this period (around 20 and 55 h after onset) are due to pressure fluctuations in the headspace caused by replacement of the Ascarite/Mg(ClO 4 ) 2 trap. The SP reached a maximum value of +5 % around 40 h after fertilizer addition, which coincided with the highest N 2 O emissions (Fig. 2). Subsequently, the SP decreased to around +3 % before it leveled out at +5 %. The observed range of SP values is consistent with the dominance of heterotrophic denitrification as the main N 2 O source process for the nitrate sucrose-amended soil cores. The predominance of    [16,31] While SP values around 0 % or slightly negative have been reported for N 2 O production by denitrification (heterotrophic as well as nitrifier denitrification), [8][9][10][11] it has been shown that fractionation during partial N 2 O reduction favors 15 N 14 N 16 O reduction relative to 14 N 15 N 16 O reduction, resulting in increasing SP. [12,17,31] The increase in SP in the nitrate sucrose-addition treatment, therefore, could be explained by an increasing importance of N 2 O reduction with rising N 2 O emissions. However, as nitrification and fungal denitrification have been reported to produce N 2 O with SP values of 31 to 37 % or 37 %, respectively, we cannot exclude a contribution of these processes to the observed SP shift. [7,9] For the control treatment, no continuous N 2 O isotopic analysis was conducted, but Tedlar W bag gas samples were analyzed by IRMS and QCLAS. The d 15 N bulk values of the emitted N 2 O displayed only a minor, but still significant increase from À38.7 to À34.2 % (QCLAS) from day 1 to day 3 (data not shown), while the N 2 O SP increased from 4.3 to 7.7 % (QCLAS). These results are included in the following section on the method intercomparison without detailed discussion of the underlying microbial production processes.

Intercomparison of QCLAS and IRMS
In addition to real-time d 15 N bulk and SP analysis by QCLAS performed on N 2 O from the nitrate sucrose-treated soil cores, N 2 O isotopomers were determined in time-integrating bag samples by laser spectroscopy and IRMS. Figures 3(a)-3(d) indicate a considerable agreement between online N 2 O SP isotopic composition and offline analysis of Tedlar W bag gas samples by laser spectroscopy and IRMS. The results of both techniques follow a similar trend and exhibit an excellent correlation, with R 2 = 0.99 and p <0.0001 (Fig. 4(a)). However, the d 15 N bulk values determined by QCLAS show a systematic offset of 1.2 AE 0.1 % (p <0.0001) compared with those for the Tedlar W bag samples analyzed by IRMS. The source of this disagreement has not yet been identified, and it might be due to any one (or both) of the involved methods. As similar d 15 N bulk values were obtained with both techniques for N 2 O calibration gases, the discrepancy might be due to differences in the gas matrix (e.g. CO 2 ), transportation, or gas conditioning prior to analysis, and this will be the subject of an upcoming research project. For SP the level of agreement is clearly lower (Fig. 4(b), R 2 = 0.76; p <0.0001). However, the SP values from the two techniques were not significantly different. Both may be explained to some extent by the considerably higher uncertainty of IRMS for SP (1 %, 2s) than for d 15 N bulk (0.4 %, 2s) as SP includes the uncertainties of the d 15 N a and d 15 N bulk values. [32] In contrast, the analytical precision (2s) of the laser spectrometer at current elevated N 2 O mixing ratios (100 ppm) is higher, around 0.3 % for both d 15 N bulk and SP, for 1-min average values.

CONCLUSIONS
This study demonstrates the performance of QCLAS in terms of precision and temporal resolution when measuring N 2 O isotopomers. Laser spectroscopy was applied for the first time for the continuous analysis of the site-specific 15 N isotopic composition of soil-derived N 2 O at high temporal resolution. In our intercomparison study using time-integrating bag samples, excellent agreement was observed for the N 2 O d 15 N bulk value between the QCLAS results and the IRMS analysis. For the 15 N site preference, the correlation suffered from the lower precision of IRMS for SP. These results confirm that laser spectroscopy is a feasible alternative technique to IRMS that will facilitate a large range of new process studies based on its capability for realtime N 2 O isotopic analysis. Moreover, the higher precision of QCLAS than of IRMS will enable more accurate analysis of isotope ratios of soil-derived N 2 O which will improve the investigation of N 2 O processes using the isotopomer approach. Currently, the amount of sample needed for QCLAS is significantly larger than for IRMS. However, this will soon be significantly improved as more sensitive laser spectrometers become available. In addition, we expect that laser spectrometers will be capable of providing data on N 2 O d 18 O values in addition to d 15 N a and d 15 N b values in the near future. This may allow the investigation of further processes, such as N 2 O reduction, based on additional isotopic discrimination patterns. Finally, robust field instruments will enable extended field studies with the additional advantage of immediate data availability.