Research Article Received: 28 January 2014
Revised: 27 June 2014
Accepted: 27 June 2014
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2014, 28, 1893–1903 (wileyonlinelibrary.com) DOI: 10.1002/rcm.6975
Dual isotope and isotopomer signatures of nitrous oxide from fungal denitrification – a pure culture study Lena Rohe1,2*, Traute-Heidi Anderson1, Gesche Braker3,4, Heinz Flessa1, Anette Giesemann1, Dominika Lewicka-Szczebak1, Nicole Wrage-Mönnig2,5 and Reinhard Well1 1
Thünen Institute of Climate-Smart Agriculture, Bundesallee 50, D-38116 Braunschweig, Germany University of Göttingen, Department of Crop Sciences, Institute of Grassland Science, von-Siebold-Straße 8, D-37075 Göttingen, Germany 3 University of Kiel, Cluster of Excellence ‘The Future Ocean’, Christian-Albrechts-Platz 4, D-24118 Kiel, Germany 4 Max Plank Institute for Terrestrial Microbiology, Karl-von-Frisch-Straße 10, D-35043 Marburg, Germany 5 Rhine-Waal University of Applied Sciences, Faculty of Life Sciences, Marie-Curie-Straße 1, D-47533 Kleve, Germany 2
RATIONALE: The contribution of fungal denitrification to the emission of the greenhouse gas nitrous oxide (N2O) from soil
has not yet been sufficiently investigated. The intramolecular 15N site preference (SP) of N2O could provide a tool to distinguish between N2O produced by bacteria or fungi, since in previous studies fungi exhibited much higher SP values than bacteria. METHODS: To further constrain isotopic evidence of fungal denitrification, we incubated six soil fungal strains under 18 denitrifying conditions, with either NO 3 or NO2 as the electron acceptor, and measured the isotopic signature (δ O, 15 δ Nbulk and SP values) of the N2O produced. The nitrogen isotopic fractionation was calculated and the oxygen isotope exchange associated with particular fungal enzymes was estimated. RESULTS: Five fungi of the order Hypocreales produced N2O with a SP of 35.1 ± 1.7 ‰ after 7 days of anaerobic incubation independent of the electron acceptor, whereas one Sordariales species produced N2O from NO 2 only, with a SP value of 21.9 ± 1.4 ‰. Smaller isotope effects of 15Nbulk were associated with larger N2O production. The δ18O values were influenced by oxygen exchange between water and denitrification intermediates, which occurred primarily at the nitrite reduction step. CONCLUSIONS: Our results confirm that SP of N2O is a promising tool to differentiate between fungal and bacterial N2O from denitrification. Modelling of oxygen isotope fractionation processes indicated that the contribution of the NO 2 and NO reduction steps to the total oxygen exchange differed among the various fungal species studied. However, more information is needed about different biological orders of fungi as they may differ in denitrification enzymes and consequently in the SP and δ18O values of the N2O produced. Copyright © 2014 John Wiley & Sons, Ltd.
Agricultural soils are the most important anthropogenic source of the greenhouse gas nitrous oxide (N2O) and contribute to the rising N2O concentration in the atmosphere.[1] Finding mitigation strategies for N2O emissions is important, and for this purpose better knowledge of the controls of N2O fluxes is urgently needed. One of the main pathways producing N2O is denitrification, the reduction of nitrate (NO 3 ) to nitrite (NO2 ) and to gaseous dinitrogen (N2) via the gaseous intermediates nitric oxide (NO) and N2O under anaerobic conditions.[2] In the 1970s, it was shown that, in addition to bacteria, fungi are capable of denitrification and N2O production.[3] Since then a number of pure culture studies on fungal denitrification have been published.[3–6] Of particular importance was the finding of Shoun et al.[5] that most fungi lack the enzyme N2O reductase (Nos); hence, most fungi produce N2O as the major end
Rapid Commun. Mass Spectrom. 2014, 28, 1893–1903
Copyright © 2014 John Wiley & Sons, Ltd.
1893
* Correspondence to: L. Rohe, Thünen Institute of Climate-Smart Agriculture, Bundesallee 50, D-38116 Braunschweig, Germany. E-mail: [email protected]
product of fungal denitrification. Referring to this, Sutka et al. hypothesized that in soils the lack of Nos potentially enables fungi to produce more N2O than bacteria, assuming that the two groups have identical or similar denitrification rates,[6] and given that, relative to bacteria, fungal biomass is much more abundant in most soils (up to 96 % of biomass).[7,8] Several laboratory studies with soils have indicated that fungi rather than bacteria dominated N2O production by denitrification.[9–13] Commonly, antibiotic inhibitors of either fungal or bacterial growth are used to differentiate between the N2O produced by the two microbial groups.[9–13] However, there are organisms not targeted by the inhibitors. Furthermore, inhibitors do not affect active microorganisms that are not in the growth stage. Thus, the N2O produced by this uninhibited group may distort the results of inhibition experiments. The inhibition of growth may even affect processes other than just the target processes. A direct method to differentiate between the production of N2O by fungal and bacterial denitrification is not yet available. Measuring the dual isotopic composition of N2O (δ15NbulkN2O and δ18O-N2O value, respectively) has become a useful tool for advancing our knowledge of N2O production
L. Rohe et al. pathways and sources.[14] Isotopic fractionation affects the δ15Nbulk and δ18O values of N2O. Hence, during denitrification, isotope fractionation results in the production of N2O with decreased δ15Nbulk values and usually increased δ18O values compared with the precursors, while concurrent N2O reduction results in residual N2O with increased δ15Nbulk and δ18O values compared with the N2O unaffected by reduction.[14,15] Oxygen (O) isotopes are additionally affected by O exchange between water (H2O) and denitrification intermediates.[16–21] Consequently, isotopic fractionation and O exchange during denitrification affect the isotopic composition of N2O and thus have to be taken into account when interpreting isotopic data of emitted N2O. Studies analyzing the enzyme types involved in denitrification found the NO reductase (Nor) of fungi (P450nor) to be distinct from its bacterial counterpart, while all other enzymes involved in fungal denitrification were similar to the bacterial ones,[22] making enzymatic methods inadequate for distinguishing between these processes. These different enzyme types of bacteria and fungi may produce a different intramolecular 15 N site preference (SP) of N2O, which is the difference between the δ15N values of the central and terminal N-positions of the asymmetric N2O molecule.[15] The SP of N2O produced by several bacterial strains ranged from 11 to 0 ‰,[23,24] whereas the two fungal strains analyzed so far (Fusarium oxysporum and Cylindrocarpon tonkinense) produced N2O with a SP of 37 ‰.[6] For the fungal P450nor, a sequential binding of two NO molecules during N2O formation is suggested, whereas a parallel binding mechanism is assumed in bacterial Nors.[25] So far the SP of N2O produced by only two fungi has been determined[6] and consequently it is not clear if there is a species effect additional to the enzyme type effect. Assuming that the current pure culture data are representative for most fungal and bacterial species under natural soil conditions, differences in SP values might be used to quantify the contribution to N2O emission from denitrification of microbial groups in a mixed soil community. So far, information on isotopic fractionation during N2O production by fungal denitrifiers and the resulting SP of N2O is scarce and more data is necessary to improve methods for a reliable quantification of bacterial and fungal contribution to N2O emissions from the soil community. Therefore, we incubated six fungal species under denitrifying conditions, with either NO 3 or NO2 as the electron acceptor, and measured the isotopologue values of the N2O produced (δ18O, δ15Nbulk and SP). The aim of the present study was (1) to test if the SP of the N2O produced by fungi during denitrification is characterized by higher values than that of the N2O produced by bacteria, (2) to determine if the SP of the N2O produced by fungi is species-dependent, and (3) to explore the variation in the δ18O and δ15Nbulk values of the N2O produced by fungal denitrification.
EXPERIMENTAL Microorganisms
1894
For isotopologue analysis of N2O from fungal denitrification, we used six fungal species that are found in soils, described in Domsch et al. (Table 1).[26] In experiments by Sutka et al., the SP of the N2O produced by Cy. lichenicola (synonymous name C. tonkinense, Nite Biological Research Center, Chiba, Japan) and F. oxysporum had already been measured, but the authors
wileyonlinelibrary.com/journal/rcm
Table 1. Fungal species in incubation experiments to determine isotopic composition of N2O from denitrification
Fungal species Chaetomium funicola Fusarium oxysporum Trichoderma hamatum Cylindrocarpon lichenicola Fusarium solani fsp. pisi Fusarium decemcellulare
Catalogue numbera
Biological order
JCM 22733 JCM 11502 JCM 1875 NBRC 30561
Sordariales Hypocreales Hypocreales Hypocreales
NBRC 9425 NBRC 31657
Hypocreales Hypocreales
a
JCM = Japan Collection of Microorganisms, Saitama, Japan and NBRC = Nite Biological Research Center, Chiba, Japan.
used different strains (ATCC 42055 and ATCC 12581) thus enabling us to evaluate whether different strains from the same species produce N2O with identical SP values.[6] Procedure The sterile fungal medium for the preparatory culture (100 mL) contained 1 % glucose, 0.2 % peptone, 0.02 % MgSO4 · 7 H2O, 2 ppm CaCl2 · 6 H2O, 2 ppm FeSO4 · 7 H2O and 0.01 mol potassium phosphate (pH 7.4), in line with the protocols of previous studies.[5,21] We used two setups differing in the volumes used with three fungal species each. Initially, three fungi (Setup A) were incubated in a relatively large amount of medium (1 L flasks) to ensure sufficient N2O production for gas analysis. Because all three fungi produced far more N2O than needed, further incubations were conducted with smaller volumes (Setup B) to save preparation time and resources. We assumed that this modification would have no impact on isotope results because the incubation time and medium concentrations were not changed. In Setup A, the stem cultures of Cy. lichenicola, F. solani fsp. pisi and F. decemcellulare were transferred to 500 mL of medium and incubated aerobically at 22 °C in the dark on a rotary shaker (100 rpm) in a 1 L Erlenmeyer flask with cotton wool plugs for 3 to 7 days (depending on the growth rates of the cells). Of this pre-culture, 60 mL were transferred to a 1 L flask with screw caps and a butyl stopper containing fresh medium (240 mL each). In Setup B, the stem cultures of C. funicola, F. oxysporum and T. hamatum were transferred to 100 mL of medium and incubated aerobically at 22 °C in the dark on a rotary shaker (100 rpm) in a 250 mL Erlenmeyer flask with cotton wool plugs for 3 to 7 days (depending on the growth rates of the cells). Of this pre-culture, 10 mL aliquots were transferred into a 120 mL crimp seal flask with a butyl stopper containing fresh medium (40 mL each). Two treatments were established with either 10 mmol KNO3 or 5 mmol NaNO2 as electron acceptors. All treatments were conducted in triplicate. The headspace of the flasks was purged with filter-sterilized N2 for 10 min while the flasks were stirred manually to expel dissolved O to establish anaerobic conditions. The cultures were incubated in the dark (22 °C) on a rotary shaker (120 rpm). Accumulation of N2O during incubations is assumed to represent N2O production because N2O reduction has been found to be negligible for fungi, with no detectable N2 production by F. oxysporum
Copyright © 2014 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2014, 28, 1893–1903
Isotopomer signatures of N2O from fungal denitrification and F. decemcellulare, while the other fungi produced only a small N2 amount compared with bacteria capable of N2O reduction.[5] Although Shoun et al. found indications by 15 N tracer application that fungal codenitrification might have occurred, the contribution of this process to N2O production was minimal and thus we assume that codenitrification in our incubation played a negligible role.[5] Growth under aerobic conditions and N2O production under anaerobic conditions indicated that all six fungi are facultative anaerobes. Gas samples were collected (2 × 14 mL) after 7 days of incubation and transferred with a syringe to evacuated sampling vials (12 mL Exetainer®, Labco Ltd, Lampeter, UK). Due to the absence of N2O reductase in most fungi we expect constant SP values with ongoing incubation time.[5] The supply of electron acceptors NO 3 and NO2 was much higher than their expected consumption and thus we assume no influence of substrate limitation or isotopic enrichment of substrates on the isotopic signatures during the incubation. To test for the chemical production of N2O, triplicate samples of 50 mL of fresh medium containing either 10 mmol KNO 3 or 5 mmol NaNO2 were incubated for 7 days, without inoculating with pure fungal cultures, using the same protocol as above. No bacterial or fungal growth could be observed by visual inspection after 7 days of incubation, and the controls showed N2O production only in the presence 1 of NO 2 as the electron acceptor (
Revised: 27 June 2014
Accepted: 27 June 2014
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2014, 28, 1893–1903 (wileyonlinelibrary.com) DOI: 10.1002/rcm.6975
Dual isotope and isotopomer signatures of nitrous oxide from fungal denitrification – a pure culture study Lena Rohe1,2*, Traute-Heidi Anderson1, Gesche Braker3,4, Heinz Flessa1, Anette Giesemann1, Dominika Lewicka-Szczebak1, Nicole Wrage-Mönnig2,5 and Reinhard Well1 1
Thünen Institute of Climate-Smart Agriculture, Bundesallee 50, D-38116 Braunschweig, Germany University of Göttingen, Department of Crop Sciences, Institute of Grassland Science, von-Siebold-Straße 8, D-37075 Göttingen, Germany 3 University of Kiel, Cluster of Excellence ‘The Future Ocean’, Christian-Albrechts-Platz 4, D-24118 Kiel, Germany 4 Max Plank Institute for Terrestrial Microbiology, Karl-von-Frisch-Straße 10, D-35043 Marburg, Germany 5 Rhine-Waal University of Applied Sciences, Faculty of Life Sciences, Marie-Curie-Straße 1, D-47533 Kleve, Germany 2
RATIONALE: The contribution of fungal denitrification to the emission of the greenhouse gas nitrous oxide (N2O) from soil
has not yet been sufficiently investigated. The intramolecular 15N site preference (SP) of N2O could provide a tool to distinguish between N2O produced by bacteria or fungi, since in previous studies fungi exhibited much higher SP values than bacteria. METHODS: To further constrain isotopic evidence of fungal denitrification, we incubated six soil fungal strains under 18 denitrifying conditions, with either NO 3 or NO2 as the electron acceptor, and measured the isotopic signature (δ O, 15 δ Nbulk and SP values) of the N2O produced. The nitrogen isotopic fractionation was calculated and the oxygen isotope exchange associated with particular fungal enzymes was estimated. RESULTS: Five fungi of the order Hypocreales produced N2O with a SP of 35.1 ± 1.7 ‰ after 7 days of anaerobic incubation independent of the electron acceptor, whereas one Sordariales species produced N2O from NO 2 only, with a SP value of 21.9 ± 1.4 ‰. Smaller isotope effects of 15Nbulk were associated with larger N2O production. The δ18O values were influenced by oxygen exchange between water and denitrification intermediates, which occurred primarily at the nitrite reduction step. CONCLUSIONS: Our results confirm that SP of N2O is a promising tool to differentiate between fungal and bacterial N2O from denitrification. Modelling of oxygen isotope fractionation processes indicated that the contribution of the NO 2 and NO reduction steps to the total oxygen exchange differed among the various fungal species studied. However, more information is needed about different biological orders of fungi as they may differ in denitrification enzymes and consequently in the SP and δ18O values of the N2O produced. Copyright © 2014 John Wiley & Sons, Ltd.
Agricultural soils are the most important anthropogenic source of the greenhouse gas nitrous oxide (N2O) and contribute to the rising N2O concentration in the atmosphere.[1] Finding mitigation strategies for N2O emissions is important, and for this purpose better knowledge of the controls of N2O fluxes is urgently needed. One of the main pathways producing N2O is denitrification, the reduction of nitrate (NO 3 ) to nitrite (NO2 ) and to gaseous dinitrogen (N2) via the gaseous intermediates nitric oxide (NO) and N2O under anaerobic conditions.[2] In the 1970s, it was shown that, in addition to bacteria, fungi are capable of denitrification and N2O production.[3] Since then a number of pure culture studies on fungal denitrification have been published.[3–6] Of particular importance was the finding of Shoun et al.[5] that most fungi lack the enzyme N2O reductase (Nos); hence, most fungi produce N2O as the major end
Rapid Commun. Mass Spectrom. 2014, 28, 1893–1903
Copyright © 2014 John Wiley & Sons, Ltd.
1893
* Correspondence to: L. Rohe, Thünen Institute of Climate-Smart Agriculture, Bundesallee 50, D-38116 Braunschweig, Germany. E-mail: [email protected]
product of fungal denitrification. Referring to this, Sutka et al. hypothesized that in soils the lack of Nos potentially enables fungi to produce more N2O than bacteria, assuming that the two groups have identical or similar denitrification rates,[6] and given that, relative to bacteria, fungal biomass is much more abundant in most soils (up to 96 % of biomass).[7,8] Several laboratory studies with soils have indicated that fungi rather than bacteria dominated N2O production by denitrification.[9–13] Commonly, antibiotic inhibitors of either fungal or bacterial growth are used to differentiate between the N2O produced by the two microbial groups.[9–13] However, there are organisms not targeted by the inhibitors. Furthermore, inhibitors do not affect active microorganisms that are not in the growth stage. Thus, the N2O produced by this uninhibited group may distort the results of inhibition experiments. The inhibition of growth may even affect processes other than just the target processes. A direct method to differentiate between the production of N2O by fungal and bacterial denitrification is not yet available. Measuring the dual isotopic composition of N2O (δ15NbulkN2O and δ18O-N2O value, respectively) has become a useful tool for advancing our knowledge of N2O production
L. Rohe et al. pathways and sources.[14] Isotopic fractionation affects the δ15Nbulk and δ18O values of N2O. Hence, during denitrification, isotope fractionation results in the production of N2O with decreased δ15Nbulk values and usually increased δ18O values compared with the precursors, while concurrent N2O reduction results in residual N2O with increased δ15Nbulk and δ18O values compared with the N2O unaffected by reduction.[14,15] Oxygen (O) isotopes are additionally affected by O exchange between water (H2O) and denitrification intermediates.[16–21] Consequently, isotopic fractionation and O exchange during denitrification affect the isotopic composition of N2O and thus have to be taken into account when interpreting isotopic data of emitted N2O. Studies analyzing the enzyme types involved in denitrification found the NO reductase (Nor) of fungi (P450nor) to be distinct from its bacterial counterpart, while all other enzymes involved in fungal denitrification were similar to the bacterial ones,[22] making enzymatic methods inadequate for distinguishing between these processes. These different enzyme types of bacteria and fungi may produce a different intramolecular 15 N site preference (SP) of N2O, which is the difference between the δ15N values of the central and terminal N-positions of the asymmetric N2O molecule.[15] The SP of N2O produced by several bacterial strains ranged from 11 to 0 ‰,[23,24] whereas the two fungal strains analyzed so far (Fusarium oxysporum and Cylindrocarpon tonkinense) produced N2O with a SP of 37 ‰.[6] For the fungal P450nor, a sequential binding of two NO molecules during N2O formation is suggested, whereas a parallel binding mechanism is assumed in bacterial Nors.[25] So far the SP of N2O produced by only two fungi has been determined[6] and consequently it is not clear if there is a species effect additional to the enzyme type effect. Assuming that the current pure culture data are representative for most fungal and bacterial species under natural soil conditions, differences in SP values might be used to quantify the contribution to N2O emission from denitrification of microbial groups in a mixed soil community. So far, information on isotopic fractionation during N2O production by fungal denitrifiers and the resulting SP of N2O is scarce and more data is necessary to improve methods for a reliable quantification of bacterial and fungal contribution to N2O emissions from the soil community. Therefore, we incubated six fungal species under denitrifying conditions, with either NO 3 or NO2 as the electron acceptor, and measured the isotopologue values of the N2O produced (δ18O, δ15Nbulk and SP). The aim of the present study was (1) to test if the SP of the N2O produced by fungi during denitrification is characterized by higher values than that of the N2O produced by bacteria, (2) to determine if the SP of the N2O produced by fungi is species-dependent, and (3) to explore the variation in the δ18O and δ15Nbulk values of the N2O produced by fungal denitrification.
EXPERIMENTAL Microorganisms
1894
For isotopologue analysis of N2O from fungal denitrification, we used six fungal species that are found in soils, described in Domsch et al. (Table 1).[26] In experiments by Sutka et al., the SP of the N2O produced by Cy. lichenicola (synonymous name C. tonkinense, Nite Biological Research Center, Chiba, Japan) and F. oxysporum had already been measured, but the authors
wileyonlinelibrary.com/journal/rcm
Table 1. Fungal species in incubation experiments to determine isotopic composition of N2O from denitrification
Fungal species Chaetomium funicola Fusarium oxysporum Trichoderma hamatum Cylindrocarpon lichenicola Fusarium solani fsp. pisi Fusarium decemcellulare
Catalogue numbera
Biological order
JCM 22733 JCM 11502 JCM 1875 NBRC 30561
Sordariales Hypocreales Hypocreales Hypocreales
NBRC 9425 NBRC 31657
Hypocreales Hypocreales
a
JCM = Japan Collection of Microorganisms, Saitama, Japan and NBRC = Nite Biological Research Center, Chiba, Japan.
used different strains (ATCC 42055 and ATCC 12581) thus enabling us to evaluate whether different strains from the same species produce N2O with identical SP values.[6] Procedure The sterile fungal medium for the preparatory culture (100 mL) contained 1 % glucose, 0.2 % peptone, 0.02 % MgSO4 · 7 H2O, 2 ppm CaCl2 · 6 H2O, 2 ppm FeSO4 · 7 H2O and 0.01 mol potassium phosphate (pH 7.4), in line with the protocols of previous studies.[5,21] We used two setups differing in the volumes used with three fungal species each. Initially, three fungi (Setup A) were incubated in a relatively large amount of medium (1 L flasks) to ensure sufficient N2O production for gas analysis. Because all three fungi produced far more N2O than needed, further incubations were conducted with smaller volumes (Setup B) to save preparation time and resources. We assumed that this modification would have no impact on isotope results because the incubation time and medium concentrations were not changed. In Setup A, the stem cultures of Cy. lichenicola, F. solani fsp. pisi and F. decemcellulare were transferred to 500 mL of medium and incubated aerobically at 22 °C in the dark on a rotary shaker (100 rpm) in a 1 L Erlenmeyer flask with cotton wool plugs for 3 to 7 days (depending on the growth rates of the cells). Of this pre-culture, 60 mL were transferred to a 1 L flask with screw caps and a butyl stopper containing fresh medium (240 mL each). In Setup B, the stem cultures of C. funicola, F. oxysporum and T. hamatum were transferred to 100 mL of medium and incubated aerobically at 22 °C in the dark on a rotary shaker (100 rpm) in a 250 mL Erlenmeyer flask with cotton wool plugs for 3 to 7 days (depending on the growth rates of the cells). Of this pre-culture, 10 mL aliquots were transferred into a 120 mL crimp seal flask with a butyl stopper containing fresh medium (40 mL each). Two treatments were established with either 10 mmol KNO3 or 5 mmol NaNO2 as electron acceptors. All treatments were conducted in triplicate. The headspace of the flasks was purged with filter-sterilized N2 for 10 min while the flasks were stirred manually to expel dissolved O to establish anaerobic conditions. The cultures were incubated in the dark (22 °C) on a rotary shaker (120 rpm). Accumulation of N2O during incubations is assumed to represent N2O production because N2O reduction has been found to be negligible for fungi, with no detectable N2 production by F. oxysporum
Copyright © 2014 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2014, 28, 1893–1903
Isotopomer signatures of N2O from fungal denitrification and F. decemcellulare, while the other fungi produced only a small N2 amount compared with bacteria capable of N2O reduction.[5] Although Shoun et al. found indications by 15 N tracer application that fungal codenitrification might have occurred, the contribution of this process to N2O production was minimal and thus we assume that codenitrification in our incubation played a negligible role.[5] Growth under aerobic conditions and N2O production under anaerobic conditions indicated that all six fungi are facultative anaerobes. Gas samples were collected (2 × 14 mL) after 7 days of incubation and transferred with a syringe to evacuated sampling vials (12 mL Exetainer®, Labco Ltd, Lampeter, UK). Due to the absence of N2O reductase in most fungi we expect constant SP values with ongoing incubation time.[5] The supply of electron acceptors NO 3 and NO2 was much higher than their expected consumption and thus we assume no influence of substrate limitation or isotopic enrichment of substrates on the isotopic signatures during the incubation. To test for the chemical production of N2O, triplicate samples of 50 mL of fresh medium containing either 10 mmol KNO 3 or 5 mmol NaNO2 were incubated for 7 days, without inoculating with pure fungal cultures, using the same protocol as above. No bacterial or fungal growth could be observed by visual inspection after 7 days of incubation, and the controls showed N2O production only in the presence 1 of NO 2 as the electron acceptor (
