CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 39, Issue 8, August 2011 Online English edition of the Chinese language journal

Cite this article as: Chin J Anal Chem, 2011, 39(8), 1141–1146.

RESEARCH PAPER

Isotopic Confirmation of Occurrence of Microbial Denitrification Based on N2 and N2O Production Monitored by Gas Chromatography/Isotope Ratio Mass Spectrometry and Gas Chromatography/Mass Spectrometry
AI Guo-Min*, ZHENG Hai-Yan, ZHANG Min, LIU Zhi-Pei
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China

Abstract:

In this study, a new

15

N-labeled procedure based on isotopic ratio monitoring of N2 by gas chromatography/isolink/

isotope ratio mass spectrometry with great precision and of N2O by gas chromatography/mass spectrometry in SIM mode with high sensitivity was developed and proposed for the identification and confirmation of in vitro microbial denitrification. The mixture of gaseous metabolites produced by Alcaligenes faecalis and atmospheric gases in the confined cultivation tube was analyzed on a GS-CarbonPlot column. A baseline separation of N2/O2, CO2, N2O and water vapour was obtained in a single run, which eliminated CO2 and H2O interference with isotopic analysis of N2 and N2O. In
15 15

N analysis of N2, combustion oven/interface in GC isolink can
15

remove all of the O2 in the sample gases, thereby providing accurate N-natural abundance control and
15

N measurement. The N2O and
15,15

15

N value of N2 in 15N-labled sample,
14,14

N-KNO3 blank control were 2.394‰ ± 0.261‰, 0.022‰ ± 0.044‰ and 0.315‰ ± 0.045‰,
14,15

respectively. Besides, significant increases in isotopic abundance of

N2O (RT = 2.99 min) relative to

N2O were

observed, indicating N2 and N2O production from denitrificaiton by A. faecalis. This procedure provides isotopic evidence of N2 and/or N2O production based on the marked increase in the 15N isotope abundance, and is rapid, sensitive and accurate to indentify and confirm the occurrence of microbial denitrification. We have confirmed the denitrifying activity of several strains of microorganisms screened from the environment using this procedure. This procedure has also been applied to confirm the N2 formation by nitrifier denitrification under defined conditions. Key Words: Gas chromatography/isotope ratio mass spectrometry; Gas chromatography/mass spectrometry; Denitrification; Nitrogen; Nitrous oxide; Alcaligenes faecalis

1

Introduction

Microbial nitrification and denitrification are playing ecologically important roles in global N cycling pathway, acting as a key regulator of water and air quality at regional and global scales[1]. Nitrification is the oxidation process of NH4+ or NH3 to NO3– via NO2–; Denitrification is the stepwise reduction process of NO3– to N2O and/or N2; In the nitrifier denitrification process, the ammonia is firstly oxidized to NO2– then reduced to N2O and/or N2 (Fig.1)[2]. Nowadays, a

lot of direct or indirect methods estimating denitrification rates are available, including measurement of NO 3 disappearance; of N2O accumulation after acetylene inhibition of N2O reduction; and of labeled N2 levels (14N15N, 15N15N) by GC/TCD or by various mass spectrometers [1] . These approaches are focused mainly on N2 quantitation, including direct 15N-N2 quantification after addition of 15N-labeled tracers measured by gas chromatography coupled with mass spectrometry or with isotope ratio mass spectrometry (GC/MS or GC/IRMS), and N2 : Ar ratio quantification measured by

Received 12 January 2011; accepted 26 March 2011 * Corresponding author. Email: agm2006@163.com This work was supported by the Knowledge Innovation Program of the Chinese Academy of Sciences (No. KJCX2-YW-L08). Copyright © 2011, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(10)60460-4

AI Guo-Min et al. / Chinese Journal of Analytical Chemistry, 2011, 39(8): 1141–1146

Fig.1 Microbial transformation pathway of nitrogenous compounds in N cycling by nitrification, denitrification and nitrifier denitrification (modified from Wrage et al, 2001[2])

especially for highly sensitive and accurate measurement of isotope ratio of N2 sample with high background of air. In the present study, we developed a GC/IRMS method measuring the isotope ratio of N2 in gas mixture produced by microbial denitrification cultivation. The effect of the oxygen present in the sample on the isotope ratio analysis of N2, without the removal of N2, O2, CO2 and H2O in the air prior to analysis, was examined at the same time. The established GC/IRMS method, combined with highly sensitive screening for N2O using GC/MS in SIM mode, could work efficiently to confirm the formation of trace N2 and/or N2O, and for the rapid identification of in vitro microbial denitrification.

2
2.1

Experimental
Apparatus and reagents

membrane inlet mass spectrometry (MIMS)[3–5]. For example, the 15N (N isotope ratio, reported in conventional 15N notation and calculated from the relative difference in the isotope ratio 15N/14N between sample and International Standard of atmospheric N2 in units of per mil) of dissolved N2 was measured in aquatic environments[6] by GC/IRMS. During this analytical protocol, N2 was extracted into a headspace to eliminate contamination by N2 in air, and the gas samples were subjected to water remover and to a packed column of molecular sieve (5A) for separation before introduction of N2 into the inlet of the mass spectrometer. It was also reported to measure N2O and N2 mixed with air for characterizing denitrification or isotopic analysis, based on either the pre-concentration of a large volume of gas sample and on the separation with the aid of an in-line N2 liquid trap[7,8]; or the removal of water vapor and carbon dioxide from the sample air using a pre-column filled with sodium hydroxide-coated silica prior to N2O analysis, alternatively the removal of O2 from the sample with reduced copper prior to N2 isotopic analysis[9]. GC/IRMS methods based on the baseline separation of N2, CO2, N2O and water vapor for measuring isotopic composition of N2 in air or in gas mixture from in vitro microbial denitrification have not yet been reported. A lot of assay methods measuring microbial denitrification rate are available. However, if the confirmation or identification of the occurrence of denitrification solely relys on the existing quantitative methods for measuring denitrification rate, the omission of certain metabolic pathways because of the low sensitivity of the methods or a false identification due to the poor capacity of qualitative screening might occur. Previous confirmation of microbial denitrification was based on the formation of N2O and/or N2 produced by microorganisms from 15N-labelled NO3– or NO2– using GC/MS[10,11] which was appropriate to measure the relative high concentration of N2O and/or N2. The methods identifying or confirming denitrification with high sensitivity based on the monitoring of relative isotopic abundances of N in N2O and N2 (enrichment of 15N) have been rarely reported,

The isotope ratio analysis of N2 was performed on Trace GC/IsoLink/Delta V Advantage GC/IRMS (Thermo Fisher Scientific, Bremen, Germany) with a 10- L gas-tight syringe (Agilent Company, USA); The N2O screen in SIM mode was conducted on Agilent 7890A/5975C GC/MS with a 1-mL gas-tight syringe (Agilent Company, USA); 15N-KNO3 was purchased from Spectra Gases Inc. (USA); Other analytical grade reagents were purchased from Beijing Chemical Reagents Company (Beijing, China). 2.2 Media

Denitrification medium (DM, g L–1): KNO3 1.0; sodium succinate 10; MgSO4·7H2O 0.2; CaCl2 0.01; EDTA (0.5 M) 0.5 mL; KH2PO4 0.5; Na2HPO4 0.5; FeSO4 0.01; trace element solution 5 mL; pH 7.0–7.5; Trace element solution (g L–1): EDTA 50.0; ZnSO4 2.2; CaCl2 5.5; MnCl2·4H2O 5.06; FeSO4·7H2O 5.0; (NH4)6Mo7O2·4H2O 1.1; CuSO4·5H2O 1.57; CoCl2·6H2O 1.61; pH 7.0. 2.3 Bacterial strain

Alcaligenes faecalis was provided by China General Microbiological Culture Collection Center (CGMCC). 5 mL of denitrification medium containing 50% 15N-labeled KNO3 was placed in an anaerobic tube (16 mm × 160 mm), and then 5% (V/V) of pre-cultured Alcaligenes faecalis grown in DM to log phase was inoculated. After 7 d of enclosed shaking cultivation at 160 rpm and 30 °C, the resulting headspace gas was subjected to GC/IRMS and GC/MS analysis. Additionally, natural abundance KNO3 control incubations and 15N-labeled KNO3 blank incubations (without Alcaligenes faecalis) were prepared in order to differentiate metabolites (N2 and N2O) originating from denitrification by Alcaligenes faecalis and possible metabolites from chemodenitrification. Each treatment was processed at least in triplicate.

AI Guo-Min et al. / Chinese Journal of Analytical Chemistry, 2011, 39(8): 1141–1146

2.4

15N

analysis of N2 by GC/IRMS

3.1.1

Effect of background of 32O2 in analysis system on 15N analysis of N2

The combustion oven temperature was elevated gradually from 25 ºC to 1030 ºC, and the effect of the background of 32 O2 in the analysis system on the 15N analysis of N2 at different temperatures was examined. An accurate 5- L gas sample was subjected to 15N analysis by GC/IRMS equipped with a 30 m × 0.320 mm i.d., 3.00- m film GS-CarbonPlot column on the GC. The following conditions were applied: split injection(injection temperature 160 ºC, split flow 30 mL min–1); oven temperature program, 35 ºC for 6 min raised from 35 ºC to 140 ºC at 20 ºC min–1, maintained at 140 ºC for 2 min; carrier gas and flow, He 1 mL min–1. IRMS analysis was in N2 mode, monitoring masses at m/z 28 and 29. The relative isotope ratio of 15N/14N is expressed in 15N notation calculated from the following equation:
15

N vs Air-N2 = 1000‰ × [(15N/14N)Sample – (15N/14N)Air-N2]/(15N/14N)Air-N2

2.5

Screen of N2O in gas sample by GC/MS in SIM mode

100 L of gas sample was injected for the confirmation of the N2O production on GC/MS, using a 30 m × 0.320 mm i.d., 3.00- m film GS-CarbonPlot column. GC was used under the following conditions: split injection (injector temperature 160 ºC, split flow 6.5 mL min–1); oven temperature program, 35 ºC for 5 min raised from 35 ºC to 140 ºC at 20 ºC min–1, maintained at 140 ºC for 2 min; carrier gas and flow, He 1.3 mL min–1. Under this condition, N2/O2, CO2, N2O and H2O were eluted at 1.36, 2.34, 2.99 and 4.38 min, respectively. The screen of N2O was performed in SIM mode, monitoring masses at m/z 44, 45 and 46 at about 2.99 min. The solvent delay was set as 2.45 min.

3
3.1

Results and discussion
15N

analysis of N2 by GC/IRMS
15

All organic compounds separated by GC are continuously and quantitatively converted to CO2 by combustion operating at 1030 ºC, and to N2 by combustion and subsequent reduction operating at 1030 ºC ( with the aid of an in-line liquid N2 trap to fix the simultaneously produced CO2). The combined effect of the absorption of O2 from the gas sample by the combustion oven and the release of the fixed O2 in the form of metal oxide in the combustion oven under different temperatures during the 15N analysis of N2 in air was examined. As shown in Table 1, 99.8% of the O2 in the gas sample was removed when the combustion oven temperature was above 400 ºC; The background of 32O2 was minimum when the temperature was 600 ºC to 900 ºC, and then increase when the temperature was 1030 ºC; The in-line liquid N2 trap can subtly remove O2 and reduce the background of 32O2. No significant 15N difference was observed when the combustion oven temperature change from 400 ºC to 1030 ºC; with the combustion oven temperature set at 650 ºC and 1030 ºC, the application of an in-line liquid N2 trap had no effect on the 15N value of N2. All the results showed that the combustion oven could effectively remove O2 from the gas sample with temperature above 400 ºC, and a precise and accurate 15N measurement of 15N of N2 in air could be achieved, on the basis of the baseline separation of N2/O2, CO2, N2O and water vapour on a GS-CarbonPlot column (Fig.2). If O2 and N2 enter simultaneously into isotope ratio mass spectrometers or conventional “organic” mass spectrometers, increased ionization energy will lead to an increase in apparent mass 14 (N+ and N2++) relative to 28N2+, and the formation of NO+ induced by the combination of N and O in the instrument[4,5]. The formation of NO+ will interfere with 30N2 measurement. Oxygen removal from the gas sample is desirable and the introduction of a copper reduction column operated at 600 ºC

Table 1 Effect of background of 32O2 on
Combustion oven temperature (ºC) Injection volume ( L) 5 5 5 5 5 5 5 5 5 5 5 5 5

N value of atmospheric nitrogen at different temperatures of combustion oven
Air Sample Background of O2 (mV) 1033 830 535 546 491 477 468 466 472 479 660 411 462
32

Response of 32O2 (mV) 50000 47164 9456 70 68 67 67 69 69 71 80 51 42

15

Nvs Air-N2 (‰, n = 6) S.D. 0.099 0.061 0.042 0.071 0.040 0.031 0.074 0.039 0.049 0.082 0.049 0.033 0.017

Mean 1.347 1.225 0.658 0.305 0.314 0.284 0.305 0.316 0.257 0.330 0.359 0.323 0.303

25 (off) 200 300 400 500 600 650 700 800 900 1030 650-LN2 1030-LN2 Note LN2: an in-line liquid N2 trap.

AI Guo-Min et al. / Chinese Journal of Analytical Chemistry, 2011, 39(8): 1141–1146

Fig.2 Chromatograms and peak shapes for N2 in a typical air sample by GC/IRMS

before mass spectrometers ion source can effectively remove O2 from the sample, obtaining an accurate N2/Ar ratio analysis[4,5]. 3.1.2 analysis of N2 in gas mixture produced by denitrification cultivation
15N

After 7 d denitrification cultivation by A. faecalis, the 32O2 concentration in the cultivation tube with the combustion oven at 25 ºC and 200 ºC significantly decreased compared with air control; no significant differences in the responses of 32O2 between 650 ºC and 1030 ºC of the combustion oven were observed (Table 1, Table 2). The 15N values of N2 samples with O2 entirely removed (650 and 1030 ºC) were comparable with those obtained when O2 was not entirely removed (25 and 200 ºC), which was consistent with the literature results[4]. It would be very difficult to directly quantify small changes in N2 concentration induced by denitrification, against the 79% N2 in atmospheric background, without addition of 15N tracer or inhibitor such as C2H2[1]. Seitzinger et al[12] made the first extensive direct N2 flux measurement with a pre-incubation period to remove N2 by flushing with N2-free gases. Later, the N2:Ar method was developed without altering the background N2 concentration using MIMS[3,13]. In addition, Meyer et al. presented an improved isotope pairing techniques for the quantification of N2 released from denitrification by replacing the background air with a gas mixture of He and O2[9]. In order to confirm the N2 production,
Table 2
15

a precise GC/IRMS determination of N2 in gas mixtures released from 15N-tracers denitrification cultivation should be performed. By taking advantages of the high precision in GC/IRMS measurement of 13C isotopic abundance at natural abundance and low enrichment level, even small changes in 13 C isotopic abundance of 1‰ can be reliably detected[14]. In this study, because the interference of O2, CO2 and H2O with the 15N analysis of N2 was eliminated, and neither combustion nor reduction process was necessary for N2 analysis, the small changes in 15N isotopic abundance of 1‰ could be reliably detected. The 15N value of N2 from 15 N-labled sample was 2‰ higher (9‰ higher for other batch experiments) than that from 15N-natural abundance control and that from15N-KNO3 blank control (Table 2), suggesting N2 formation from 15N-KNO3 by A. faecalis, that is, providing evidence for the occurrence of denitrification. The stable isotope labeling approach to confirmation of the occurrence of denitrification can avoid the constrains (such as incomplete inhibition of N2O reduction ) and the adverse effects (such as inhibition of nitrification) that may be introduced by inhibitors[2]. Several strains of microorganisms screened from the environment were confirmed as denitrifier producing N2 with a 15N value of 2‰–11‰ using our newly established GC/IRMS method. 3.2 Isotopic screening and identification of N2O produced by microbial denitrification

It is relatively easy to directly quantify small changes in N2O concentration by GC[9, 15] or by GC/MS[16] because of its low atmospheric concentration. The N2O formation by microbial denitrification cultivation can be confirmed on the basis of the relative isotopic abundance of 14,15N2O and 15,15 N2O in gas mixtures produced by denitrification cultivation by GC/MS in SIM mode with 15N-labled substrate. The screening results are reported in Fig.3 and Fig.4. After a 7-day denitrification cultivation by A. faecalis, the N2O signal in gas mixtures increased significantly compared with control, confirming N2O formation by A. faecalis. However, with a

N analysis of nitrogen in gas mixture by A. faecalis
15

N-labled sample Nvs Air-N2 (‰, n = 3) (Mean ± S.D.) 2.628 ± 0.104 2.530 ± 0.109 2.423 ± 0.160 2.601 ± 0.063 2.394 ± 0.261 2.462 ± 0.298
15

Unlabled sample Nvs Air-N2 (‰, n = 3) (Mean ± S.D.)
15

Blank control Nvs Air-N2 (‰, n = 3) (Mean ± S.D.)
15

Combustion oven temperature Injection volume (ºC) ( L)

Background of 32O2 (mV)

Response of 32O2 (mV)

25 (off) 200 650 1030 650-LN2 1030-LN2

5 5 5 5 5 5

1090 798 473 646 428 459

4325 3084 80 91 49 56

0.022 ± 0.044

0.315 ± 0.045

AI Guo-Min et al. / Chinese Journal of Analytical Chemistry, 2011, 39(8): 1141–1146

isotopic abundance of 45N2O and 46N2O enhanced sharply, exceeding that of 44N2O. What limits the use of GC/IRMS analysis of 15N in N2O with a low concentration in gas mixtures is the low sensitivity of the method compared with GC/MS analysis. When the N2O concentration is high enough, 15N analysis of N2 and N2O could be performed by GC/IRMS in a single run.

4

Conclusions

Fig.3 SIM chromatograms of N2O (m/z 44 45 46, RT = 2.99 min) were obtained by analyzing 100 L of gas sample, 15N-labled sample (7 d), 15N-KNO3 blank control, and 15N-labled sample (20 h), from the top, respectively

20-h denitrification cultivation, the N2O response in gas mixtures increased only slightly against control, which is not obvious enough to confirm N2O production by A. faecalis (Fig.3). Figure 4 provided the differences in N2O isotope ratio instead of in total N2O responses, after 20-hour denitrification cultivation, the relative isotopic abundances of 14,15N2O and 15,15 N2O (m/z 45 and 46) increased significantly with 45/44 and 46/44 higher than 35%, while the natural relative abundance of 45N2O and 46N2O is 1.1757% and 0.4033%, respectively, which confirming the formation of 14,15N2O and 15. 15 N2O. After 7 d of denitrification cultivation, the relative

Microbial nitrification, denitrification and nitrifier denitrification are ecologically and environmentally important for their role in the transformation of nitrogenous compounds in an ecosystem to gaseous nitrogen species. In order to analyze microorganisms screened from the environment and subjected to subsequent cultivation and modification, a sensitive method is required for the rapid and accurate identification or confirmation of their functions and the metabolic pathways involved. In this paper, we presented a novel 15N-labled procedure based on isotopic ratio monitoring of N2 by GC/isolink/IRMS with high precision and of N2O by GC/MS in SIM mode with high sensitivity, for the confirmation of trace N2 and/or N2O formation by microbial cultivation. The gas metabolites produced by microorganisms mixed with air were directly subjected to analysis. This procedure could also be applied to study the N2 production by nitrifier denitrification under defined conditions with 15N-labled substrates.

Fig.4 N2O isotopic abundance ratios under different culture media, indicating production of 14,15N2O and 15,15N2O
1. 15N-KNO3 blank control; 2. 15N-natural abundance control; 3. 15N-labled sample (20 h); 4. 15N-labled sample (7 d)

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