Article pubs.acs.org/est
Isotope Signatures of N2O in a Mixed Microbial Population System: Constraints on N2O Producing Pathways in Wastewater Treatment Pascal Wunderlin,*,† Moritz F. Lehmann,‡ Hansruedi Siegrist,† Béla Tuzson,§ Adriano Joss,† Lukas Emmenegger,§ and Joachim Mohn*,§ †
Eawag, Swiss Federal Institute of Aquatic Science and Technology, Ueberlandstrasse 133, P.O. Box 611, 8600 Duebendorf, Switzerland ‡ Department of Environmental Science, Bernoullistrasse 30, University of Basel, 4054 Basel, Switzerland § Empa, Swiss Federal Laboratories for Materials Science and Technology, Laboratory for Air Pollution & Environmental Technology, Ueberlandstrasse 129, 8600 Duebendorf, Switzerland S Supporting Information *
ABSTRACT: We present measurements of site preference (SP) and bulk 15N/14N ratios (δ15NbulkN2O) of nitrous oxide (N2O) by quantum cascade laser absorption spectroscopy (QCLAS) as a powerful tool to investigate N2O production pathways in biological wastewater treatment. QCLAS enables high-precision N2O isotopomer analysis in real time. This allowed us to trace short-term fluctuations in SP and δ15NbulkN2O and, hence, microbial transformation pathways during individual batch experiments with activated sludge from a pilot-scale facility treating municipal wastewater. On the basis of previous work with microbial pure cultures, we demonstrate that N2O emitted during ammonia (NH4+) oxidation with a SP of −5.8 to 5.6 ‰ derives mostly from nitrite (NO2−) reduction (e.g., nitrifier denitrification), with a minor contribution from hydroxylamine (NH2OH) oxidation at the beginning of the experiments. SP of N2O produced under anoxic conditions was always positive (1.2 to 26.1 ‰), and SP values at the high end of this spectrum (24.9 to 26.1 ‰) are indicative of N2O reductase activity. The measured δ15NbulkN2O at the initiation of the NH4+ oxidation experiments ranged between −42.3 and −57.6 ‰ (corresponding to a nitrogen isotope effect Δδ15N = δ15Nsubstrate − δ15NbulkN2O of 43.5 to 58.8 ‰), which is considerably higher than under denitrifying conditions (δ15NbulkN2O 2.4 to −17 ‰; Δδ15N = 0.1 to 19.5 ‰). During the course of all NH4+ oxidation and nitrate (NO3−) reduction experiments, δ15NbulkN2O increased significantly, indicating net 15N enrichment in the dissolved inorganic nitrogen substrates (NH4+, NO3−) and transfer into the N2O pool. The decrease in δ15NbulkN2O during NO2− and NH2OH oxidation experiments is best explained by inverse fractionation during the oxidation of NO2− to NO3−.
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of NO2− by ammonia-oxidizing bacteria (AOB), known as nitrifier denitrification,7 and (iii) the production of N2O by heterotrophic denitrifiers (HET), resulting from an unbalanced activity (e.g., due to inhibition) of nitrogen-reducing enzymes (or in some cases from a lack of N2O reductase).8 The reduction of N2O to N2 by HET is currently considered to be the dominant microbial sink for N2O.9 A promising approach to trace N2O source and sink processes is to analyze the nitrogen (and oxygen) isotope composition and intramolecular distribution of 15N on the
INTRODUCTION
Nitrous oxide (N2O) is an important greenhouse gas and a major sink for stratospheric ozone.1,2 It is estimated that about two-thirds of anthropogenic N2O emissions can be attributed to microbial processes occurring mainly in agricultural soils or managed lawns but also in biological wastewater treatment.3,4 Microbial nitrogen (N) transforming processes such as autotrophic nitrification and heterotrophic denitrification have been identified as major N 2 O sources. However, the partitioning between these processes with respect to global N2O emissions and the respective mechanisms are still unclear. There are three main microbial pathways involved in N2O formation (Figure 1):5 (i) N2O production as a side product during hydroxylamine (NH2OH) oxidation to nitrite (NO2−), probably related to high metabolic activity,6 (ii) the reduction © 2012 American Chemical Society
Received: Revised: Accepted: Published: 1339
August 4, 2012 December 14, 2012 December 18, 2012 December 18, 2012 dx.doi.org/10.1021/es303174x | Environ. Sci. Technol. 2013, 47, 1339−1348
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Figure 1. Scheme of relevant N2O production pathways in biological wastewater treatment, adapted from Wunderlin et al.,28 including SP and Δδ15N (= δ15Nsubstrate − δ15NbulkN2O) values reported from cultivation studies.11,17,20−26,29 Additional information is given in the SI, Table S1.
The goal of the present study is to identify N2O production pathways of an activated sludge system in a lab-scale batch reactor operated under specific process conditions. We applied quantum cascade laser absorption spectroscopy (QCLAS) as a novel technique to analyze the site-specific isotope composition of N2O in real time. The net N2O nitrogen isotopic signatures are compared to published pure-culture investigations where the active pathways are known. A mixed population system was chosen to more adequately mimic the situation in a full-scale plant, as compared to pure culture investigations. Our lab-scale experiments were performed to test the following hypotheses: (i) nitrifier denitrification is the dominant N2O production pathway during nitrification (NH4+ oxidation), (ii) NH2OH addition is conducive to N2O production by NH2OH oxidation, while (iii) the addition of NO2− to activated sludge fosters N2O production by nitrifier denitrification, and (iv) HET is assumed to be the main pathway under anoxic conditions with N2O reductase activity being regulated by the dissolved oxygen concentration (Table 1).
central (α) and terminal (β) positions of the asymmetric N2O molecules.10−12 Bulk nitrogen isotope ratios of N2O are reported in the conventional δ-notation, in per-mil (‰), δ15N = [(RN2O/Rreference) − 1] × 1000, where R is the nitrogen isotope ratio (15N/14N) and atmospheric N2 (AIR) serves as the reference.13,14 Analogously, δ15Nα and δ15Nβ denote the relative enrichment of 15N in the central (Nα) position (14N15N16O) or in the terminal (Nβ) position (15N14N16O) with respect to the reference. The site preference (SP) is defined as SP = δ15Nα − δ15Nβ.12,15 Most of the studies that have used N2O isotopic measurements so far were field studies, in which the responsible N2O source processes, for instance, were not well constrained.16−20 In this regard, it is helpful that microbial N transformation processes are typically associated with kinetic isotope fractionation, e.g. the discrimination of the 15N-containing molecules during most enzymatic reactions. During nitrification, N2O is a side-product. Therefore, the nitrogen isotope fractionation is generally higher than during heterotrophic denitrification, where N2O is assumed to be an obligate intermediate and the isotope fractionation is the net result of production and consumption processes (Figure 1; SI, Table S1).17,20 In methodological investigations with pure cultures designed to identify the isotopic signature produced by specific enzymatic pathways, the following SP values were reported: (i) −10.7 ± 2.9 to 0.1 ± 1.7 ‰ for nitrifier denitrification by AOB,11,21−23 (ii) 30.8 ± 5.9 to 36.3 ± 2.4 ‰ for NH2OH oxidation,11,21−24 and (iii) −5 to 0 ‰ for N2O production by HET only (e.g., at inhibited N2O reductase; Figure 1; SI, Table S1 for a detailed overview).11,25 A strongly positive SP might also be the result of the subsequent heterotrophic N2O reduction to N2, as the 14N−O bond is preferentially broken during this enzymatic transformation step, leading to an enrichment in 14N−15N−O in the residual N2O (Figure 1).20,26 For N2O production by the nitrification process, a SP of about 31 ‰ was assumed, mainly on the basis of the findings of a study with N. europaea in a pure culture11 (suggesting the importance of NH2OH oxidation in overall N2O production by this nitrifier). In contrast, Toyoda et al.27 estimated a SP of 4.5 ‰ for N2O production in an aerated (oxic) tank of a biological wastewater treatment plant and suggested that the contribution of NH2OH oxidation (with a presumably much higher SP) during the nitrification pathway is less important in mixed cultures.
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MATERIALS AND METHODS Experimental Setup for Lab-scale Batch Experiments. In the present investigation, batch experiments were carried out with activated sludge sampled from the aerobic (nitrifying) reactor of a pilot scale municipal wastewater treatment plant adapted to NH4+ nitrification before the daily NH4+ peak load (before 8 a.m.; for more details see SI, Text S1). A laboratory batch-scale reactor with a working volume of 6.9 L and a headspace of 1.2 L was used (for more details see ref 28). The wastewater temperature was held at 20 ± 1.2 °C. Continuous gas flow was maintained at 1 standard liter per minute using a mass flow controller (Red-y Smart series, Vögtlin Instruments, Switzerland) during both nitrification and denitrification experiments in order to strip N2O (and NO) from the liquid phase for subsequent analysis. The dissolved oxygen concentration was adjusted by automated oxygen-controlled (by Oxymax H, Endress + Hauser) purging either with high purity nitrogen gas or synthetic air (20.5% O2 in N2). Under anoxic conditions, nitrogen gas was continuously purged through the reactor. The pH was measured with a pH electrode (Orbisint CPS11, Endress + Hauser, calibration at pH 7 and 9), and was held constant at 7.1 ± 0.2 using a pH controller via the addition of CO2 (>99.9% CO2, Carbagas). Nitrification experiments were carried out at oxygen concentrations typical for full-scale plants: 0.5, 1, 2, or 3 mgO2/L. The experiments were started by adding either NH4+, 1340
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Table 1. Summary of Batch Experiments: Added Substrates (Initial Concentrations and Their Isotopic Composition) to Activated Sludge Taken from the Nitrification Reactor of Our Pilot Plant, and Dissolved Oxygen Concentrations; SP, δ15Nbulk of N2O (Both at the Beginning and End of the Experiments), Δδ15N of Produced N2O and the Quantitative Interpretation of SP Dataf δ N 15
experiment
added substrate
NO2− oxidation (δ15N = −30.5‰) hypothesis: nitrifier NO2− denitrification (ND) 15 mgN /L 15 mgN /L 15 mgN /L 15 mgN /L 15 mgN /L NH2OH oxidation (δ15N = −71.3‰) hypothesis: NH2OH NH2OH oxidation (NN) and ND 2 mgN/L 5 mgN/L
NH4+ peak addition (δ15N = 1.15‰) hypothesis: ND and NN
continuous NH4+ addition (δ15N = 1.15‰) hypothesis: ND and NN
NO3− reduction (δ15N = 2.5‰) hypothesis: heterotrophic denitrification (HET) and N2O reductase
O2 [mg/L]
start [‰]
0.6
−64.4
0.6
−56.3
1.1
bulk
site preference (SP)a
a N2O
end [‰]
net N isotope effect (Δδ15N)b [‰]
start [‰]
end [‰]
interpretation of microbial processes based on SPc start
end
-
0.2
−72.9
25.8
−1.1
−2.1
100%ND
100%ND
−54.9
−67.1
24.4
−0.2
−1.1
100%ND
100%ND
2.1
−59.7
−72.4
29.2
−3.0
−3.4
100%ND
100%ND
3.1
−57.5
−69.2
27.0
−2.1
−3.9
100%ND
100%ND
2.1 2.1
−62.3 −76.0
−86.3
4.7
25.2 26.4
19.9
1.1
−63.9
−50.7
−7.4
30.7
3.7
2.2
−68.2
−83.1
−3.1
29.0
20.7
2.3
−70.1
−79.2
−1.2
27.5
25.8
2.1
−65.7
−68.4
−5.6
30.5
30.2
0.6
−57.6
−34.7
58.8
0.9
−2.0
1.0
−55.5
−25.8
56.7
3.0
−1.0
1.9
−54.7
−29.0
55.9
5.6
0.4
2.0
−55.8
−25.3
57.0
2.5
−2.3
3.1
−42.3
−39.6
43.5
1.9
−1.8
1.1
−54.8
−35.7
56.0
−0.5
−4.5
9 mgN/L
2.1
−50.2
−31.7
51.4
−1.2
−5.8
9 mgN/L
3.1
−48
−33.6
49.2
−1.2
−4.9
15 mgN /L NO3− 20 mgN /L 20 mgN /L 10 mgN /L 10 mgN /L 20 mgN /L
2.9
−51.4
−30.9
52.6
1.1
−2.3
0
2.4
50.1
0.1
24.9
25.4
0
−5.3
13.2
7.8
24.9
26.1
4 mgN/L). Under anoxic conditions (0 mgO2/L), initial δ15NbulkN2O values were in the range from −17 to 2.4 ‰ and increased systematically to values between 9.3 and 50.1 ‰ at the end of the experiments (Figure 6; Table 1). That is, the N2O generated had a significantly higher δ15Nbulk than was observed during NH4+ oxidation conditions (−42.3 to −57.6 ‰), albeit similar δ15N substrates (δ15N of NaNO3 = 2.5 ‰; δ15N of NH4HCO3 = 1.15 ‰). The isotopic signature of dissolved NO2− and NO3− (SI, Text S2 and S5, Figure S4 B) indicates that during heterotrophic denitrification the change of the δ15NbulkN2O (−17 to 50.1 ‰) paralleled that of the precursor NO2− (−23.5 to 43 ‰) within the respective experiments, with a consistent δ15N offset between the two N pools due to the N isotope fractionation during NO3− reduction to N2O. The Δδ15N (= δ15NNO3‑ − δ15NbulkN2O) of N2O produced during heterotrophic denitrification ranged between 0.1 and 19.5 ‰, a range that includes values reported from pure culture investigations (∼13 ‰ for NO2− reduction11). However, our values are at the lower end of data reported for heterotrophic denitrification in the natural environment (0 to 39 ‰,17 Figure 1). This might partly be due to an underestimation of our Δδ15N values, since a significant fraction of the initial NO3− was already reduced before the first N2O sample was collected.
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ASSOCIATED CONTENT
S Supporting Information *
Additional information about the analysis of the nitrogen isotopic composition of dissolved nitrogen species, the experimental setup for the analysis of N 2 O isotopic composition, and the time series of NO2− oxidation and NH2OH oxidation experiments. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +41 58 765 5037. Fax: +41 58 765 5389. E-mail:
[email protected] (P.W.). Phone: +41 58 765 4687. Fax: +41 58 765 2211. E-mail:
[email protected] (J.M.). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This study was financially supported by the Swiss Federal Office for the Environment (FOEN), the Swiss National Foundation for Scientific Research, the canton of Bern (AWA), the canton of Basel- Landschaft (AIB), the canton of St. Gallen (AFU), the canton of Thurgau (AfU), the canton of Zurich (AWEL), TBF + Partner AG Consulting Engineers, WWTP region Bern, WWTP ProRheno, WWTP REAL Luzern, WWTP Zurich-Werdhölzli, WWTP Worblental, Abwasserverband Altenrhein, Abwasserverband Morgental, Entsorgung St. Gallen, Zentralschweizer Umweltdirektorenkonferenz 1346
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(ZUDK), Empa, and Eawag. The authors thank Naohiro Yoshida and Sakae Toyoda from the Tokyo Institute of Technology for analysis of the site-specific isotopic composition of our primary N2O calibration gases. Special thanks go also to Carsten Schubert and Gijs Nobbe (Eawag) for the isotopic analysis of the applied substrates and George F. Wells (Eawag) for helpful discussions during the preparation of the manuscript.
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NOMENCLATURE Isotopomer Molecules containing the same isotopes but with differing isotope positions; e.g. N2O with 15N in the central (14N−15N−O) or the end (15N−14N−O) position45 R Nitrogen isotope ratio, 15N/14N 15 δ Ncompound (Rcompound − Rref)/Rref × 1000, with atmospheric nitrogen as the reference material (ref) δ15Nα and δ15Nβ Relative differences of isotope ratios for the inner (α) and the outer (β) nitrogen atom in the asymmetric N2O molecule SP Site preference, the difference between the δ15Nα and δ15Nβ δ15NbulkN2O The average between δ15Nα and δ15Nβ in N2O Δδ15N: The net nitrogen (N) isotope effect, which approximates the apparent fractionation of a multiple step reaction, expressed as the difference between δ 1 5 N s u b s t r a t e and δ15NbulkN2O; according to Koba et al.17 and Sutka et al.11
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