scholarly journals Comparing modified substrate-induced respiration with selective inhibition (SIRIN) and N<sub>2</sub>O isotope approaches to estimate fungal contribution to denitrification in three arable soils under anoxic conditions

2021 ◽  
Vol 18 (15) ◽  
pp. 4629-4650
Author(s):  
Lena Rohe ◽  
Traute-Heidi Anderson ◽  
Heinz Flessa ◽  
Anette Goeske ◽  
Dominika Lewicka-Szczebak ◽  
...  
Keyword(s):  
Fungal Growth ◽  
Selective Inhibition ◽  
15N Tracer ◽  
Site Preference ◽  
Small Contribution ◽  
Anaerobic Conditions ◽  
Arable Soils ◽  
N2o Production ◽  
Substrate Induced Respiration ◽  
N2o Fluxes

Abstract. The coexistence of many N2O production pathways in soil hampers differentiation of microbial pathways. The question of whether fungi are significant contributors to soil emissions of the greenhouse gas nitrous oxide (N2O) from denitrification has not yet been resolved. Here, three approaches to independently investigate the fungal fraction contributing to N2O from denitrification were used simultaneously for, as far as we know, the first time (modified substrate-induced respiration with selective inhibition (SIRIN) approach and two isotopic approaches, i.e. end-member mixing approach (IEM) using the 15N site preference of N2O produced (SPN2O) and the SP/δ18O mapping approach (SP/δ18O Map)). This enabled a comparison of methods and a quantification of the importance of fungal denitrification in soil. Three soils were incubated in four treatments of the SIRIN approach under anaerobic conditions to promote denitrification. While one treatment without microbial inhibition served as a control, the other three treatments were amended with inhibitors to selectively inhibit bacterial, fungal, or bacterial and fungal growth. These treatments were performed in three variants. In one variant, the 15N tracer technique was used to estimate the effect of N2O reduction on the N2O produced, while two other variants were performed under natural isotopic conditions with and without acetylene. All three approaches revealed a small contribution of fungal denitrification to N2O fluxes (fFD) under anaerobic conditions in the soils tested. Quantifying the fungal fraction with modified SIRIN was not successful due to large amounts of uninhibited N2O production. In only one soil could fFD be estimated using modified SIRIN, and this resulted in 28 ± 9 %, which was possibly an overestimation, since results obtained by IEM and SP/δ18O Map for this soil resulted in fFD of below 15 % and 20 %, respectively. As a consequence of the unsuccessful SIRIN approach, estimation of fungal SPN2O values was impossible. While all successful methods consistently suggested a small or missing fungal contribution, further studies with stimulated fungal N2O fluxes by adding fungal C substrates and an improved modified SIRIN approach, including alternative inhibitors, are needed to better cross-validate the methods.

scholarly journals Comparing modified substrate induced respiration with selective inhibition (SIRIN) and N<sub>2</sub>O isotope approaches to estimate fungal contribution to denitrification in three arable soils under anoxic conditions

2020 ◽  
Author(s):  
Lena Rohe ◽  
Traute-Heidi Anderson ◽  
Heinz Flessa ◽  
Anette Giesemann ◽  
Dominika Lewicka-Szczebak ◽  
...  
Keyword(s):  
Fungal Community ◽  
Selective Inhibition ◽  
15N Tracer ◽  
Site Preference ◽  
Anaerobic Conditions ◽  
N2o Reduction ◽  
N2o Production ◽  
Simultaneous Application ◽  
Substrate Induced Respiration ◽  
N2o Fluxes

Abstract. Pure culture studies provide evidence of the ability of soil fungi to produce nitrous oxide (N2O) during denitrification. Soil studies with selective inhibition indicated a possible dominance of fungal compared to bacterial N2O production in soil, which drew more attention to fungal denitrification. Analyzing the isotopic composition of N2O, especially the 15N site preference of N2O produced (SPN2O), showed that N2O of pure bacterial or fungal cultures differed in SPN2O values, which might enable the quantification of fungal N2O based on the isotopic endmember signatures of N2O produced by fungi and bacteria. This study aimed to identify the fungal contribution to N2O emissions under anaerobic conditions in incubated repacked soil samples by using different approaches to disentangle sources of N2O. Three soils were incubated under anaerobic conditions to promote denitrification with four treatments of a modified substrate induced respiration with selective inhibition (SIRIN) approach. While one treatment without microbial inhibition served as a control the other three treatments were amended with inhibitors to selectively inhibit bacterial, fungal or bacterial and fungal growth. These treatments were performed in three varieties. In one variety the 15N tracer technique was used to estimate the effect of N2O reduction on N2O produced, while two other varieties were performed under natural isotopic conditions but with and without acetylene. Three approaches were established to estimate the N2O production by a fungal community in soil: i) A modification of the SIRIN approach was used to calculate N2O evolved from selected organism groups, and ii) SPN2O values from the acetylated treatment were used in the isotope endmember mixing approach (IEM), and iii) the SP/δ18O mapping approach (SP/δ18O Map) was used to estimate the fungal contribution to N2O production and N2O reduction under anaerobic conditions from the non-acetylated treatment. The three approaches tested revealed a small fungal contribution to N2O fluxes under anaerobic conditions in the soils tested. Quantifying the fungal fraction with modified SIRIN was only possible in one soil and totaled 0.28 ± 0.09. This was higher than the results obtained by IEM and SP/δ18O Map, which accounted zero to 0.20 of N2O produced to the fungal community. To our knowledge, this study was the first attempt to quantify the fungal contribution to anaerobic N2O production by simultaneous application of three approaches, i.e. modified SIRIN, IEM and SP/δ18O Map. While all methods coincided by suggesting a small or missing fungal contribution, further studies under conditions ensuring larger fungal N2O fluxes and including alternative inhibitors are needed to better cross-validate the methods.

scholarly journals Trace gas fluxes of CO<sub>2</sub>, CH<sub>4</sub> and N<sub>2</sub>O in a permanent grassland soil exposed to elevated CO<sub>2</sub> in the Giessen FACE study

2011 ◽  
Vol 11 (17) ◽  
pp. 9333-9342 ◽  
Author(s):  
M. Kaleem Abbasi ◽  
C. Müller
Keyword(s):  
Elevated Co2 ◽  
15N Tracer ◽  
Organic N ◽  
N2o Emissions ◽  
Ch4 Oxidation ◽  
N2o Production ◽  
N Application ◽  
Oxidation Rates ◽  
Controlled Conditions ◽  
N2o Fluxes

Abstract. Long-term field observations showed that N2O fluxes observed shortly after N application were not significantly affected by elevated CO2 in the Giessen Free Air Carbon dioxide Enrichment (FACE) study. To further investigate this unexpected result a 15N tracer study was carried out under controlled conditions where in parallel treatments either the NH4+ pool (15NH4NO3) or the NO3− pool (NH415NO3) was enriched with 15N. Fluxes of CO2, CH4, and N2O as well as the 15N enrichment of the N2O were measured. Denitrifying Enzyme Activity (DEA), total denitrification (N2 + N2O) and N2-to-N2O ratios were quantified in separate experiments. Over the 57 day incubation, N2O fluxes averaged 0.090 ng N2O-N g−1 h−1 under ambient and 0.083 ng N2O-N g−1 h−1 under elevated CO2 (not significantly different). The N2O production processes were identified by a two-source model. Results showed that N2O must have also been produced by a third source – possibly related to organic N transformation – which was stimulated by elevated CO2. Soil CO2 fluxes were approximately 20 % higher under elevated CO2 than soil from ambient but the differences were not significant. CH4 oxidation rates were on average −1.75 ng CH4-C g−1 h−1 in the elevated and −1.17 ng CH4-C g−1 h−1 in the ambient indicating that elevated CO2 increased the CH4 oxidation by 49 % compared to ambient CO2 under controlled conditions. N fertilization increased CH4 oxidation by 3-fold in both CO2 treatments. CO2 did not have any significant effect on DEA while total denitrification and N2-to-N2O ratios increased by 36 and 33 %, respectively. The results indicate that shortly after N application elevated CO2 must have stimulated both the N2O production and reduction to N2 to explain the increased N2-to-N2O ratio and at the same time explain the non-responsiveness of the N2O emissions. Thus, the observed variation of the CO2 effect on N2O emissions throughout the year is possibly governed by the dynamics of the N2O reductase activity.

scholarly journals Diversity and N2O Production Potential of Fungi in an Oceanic Oxygen Minimum Zone

2021 ◽  
Vol 7 (3) ◽  
pp. 218
Author(s):  
Xuefeng Peng ◽  
David L. Valentine
Keyword(s):  
Fungal Diversity ◽  
Active Role ◽  
Selective Inhibition ◽  
Oxygen Minimum Zone ◽  
15N Tracer ◽  
Potential Contribution ◽  
Internal Transcribed Spacer Region ◽  
N2o Production ◽  
Minimum Zone ◽  
Oxygen Minimum

Fungi in terrestrial environments are known to play a key role in carbon and nitrogen biogeochemistry and exhibit high diversity. In contrast, the diversity and function of fungi in the ocean has remained underexplored and largely neglected. In the eastern tropical North Pacific oxygen minimum zone, we examined the fungal diversity by sequencing the internal transcribed spacer region 2 (ITS2) and mining a metagenome dataset collected from the same region. Additionally, we coupled 15N-tracer experiments with a selective inhibition method to determine the potential contribution of marine fungi to nitrous oxide (N2O) production. Fungal communities evaluated by ITS2 sequencing were dominated by the phyla Basidiomycota and Ascomycota at most depths. However, the metagenome dataset showed that about one third of the fungal community belong to early-diverging phyla. Fungal N2O production rates peaked at the oxic–anoxic interface of the water column, and when integrated from the oxycline to the top of the anoxic depths, fungi accounted for 18–22% of total N2O production. Our findings highlight the limitation of ITS-based methods typically used to investigate terrestrial fungal diversity and indicate that fungi may play an active role in marine nitrogen cycling.

scholarly journals Isotopomeric characterization of nitrous oxide produced by reaction of enzymes extracted from nitrifying and denitrifying bacteria

2014 ◽  
Vol 11 (10) ◽  
pp. 2679-2689 ◽  
Author(s):  
T. Yamazaki ◽  
T. Hozuki ◽  
K. Arai ◽  
S. Toyoda ◽  
K. Koba ◽  
...  
Keyword(s):  
Nitrous Oxide ◽  
Pure Culture ◽  
Paracoccus Denitrificans ◽  
In Vivo Studies ◽  
Site Preference ◽  
Ammonia Oxidizing Bacteria ◽  
Small Contribution ◽  
N2o Production ◽  
No2 Reduction

Abstract. Nitrous oxide (N2O) is a potent greenhouse gas and produced in denitrification and nitrification by various microorganisms. Site preference (SP) of 15N in N2O, which is defined as the difference in the natural abundance of isotopomers 14N15NO and 15N14NO relative to 14N14NO, has been reported to be a useful tool to quantitatively distinguish N2O production pathways. To determine representative SP values for each microbial process, we firstly measured SP of N2O produced in the enzyme reaction of hydroxylamine oxidoreductase (HAO) purified from two species of ammonia oxidizing bacteria (AOB), Nitrosomonas europaea and Nitrosococcus oceani, and that of nitric oxide reductase (NOR) from Paracoccus denitrificans. The SP value for NOR reaction (−5.9 ± 2.1‰) showed nearly the same value as that reported for N2O produced by P. denitrificans in pure culture. In contrast, SP value for HAO reaction (36.3 ± 2.3‰) was a little higher than the values reported for N2O produced by AOB in aerobic pure culture. Using the SP values obtained by HAO and NOR reactions, we calculated relative contribution of the nitrite (NO2–) reduction (which is followed by NO reduction) to N2O production by N. oceani incubated under different O2 availability. Our calculations revealed that previous in vivo studies might have underestimated the SP value for the NH2OH oxidation pathway possibly due to a small contribution of NO2– reduction pathway. Further evaluation of isotopomer signatures of N2O using common enzymes of other processes related to N2O would improve the isotopomer analysis of N2O in various environments.

scholarly journals Isotopomeric characterization of nitrous oxide produced by reaction of enzymes extracted from nitrifying and denitrifying bacteria

2013 ◽  
Vol 10 (10) ◽  
pp. 16615-16643 ◽  
Author(s):  
T. Yamazaki ◽  
T. Hozuki ◽  
K. Arai ◽  
S. Toyoda ◽  
K. Koba ◽  
...  
Keyword(s):  
Nitrous Oxide ◽  
Pure Culture ◽  
Paracoccus Denitrificans ◽  
In Vivo Studies ◽  
Site Preference ◽  
Ammonia Oxidizing Bacteria ◽  
Small Contribution ◽  
N2o Production ◽  
No2 Reduction

Abstract. Nitrous oxide (N2O) is a potent greenhouse gas and produced in denitrification and nitrification in environmental nitrogen cycle by various microorganism. Site preference (SP) of 15N in N2O, which is defined as the difference in the natural abundance of isotopomers 14N15NO and 15N14NO relative to 14N14NO, has been reported to be a useful tool to quantitatively distinguish N2O production pathway. To determine representative SP value for each microbial process, we firstly measured SP of N2O produced in the enzyme reaction of hydroxylamine oxidoreductase (HAO) purified from two species of ammonia oxidizing bacteria (AOB), Nitrosomonas europaea and Nitrosococcus oceani, and that of nitric oxide reductase (NOR) from Paracoccus denitrificans, respectively. The SP value for NOR reaction (−5.9 ± 2.1‰) showed nearly the same value as that reported for N2O produced by P. denitrificans in pure culture. In contrast, SP value for HAO reaction (36.3 ± 2.3‰) was a little higher than the values reported for N2O produced by AOB in aerobic pure culture. Using the SP values obtained by HAO and NOR reactions, we calculated relative contribution of the nitrite (NO2–) reduction (which is followed by NO reduction) to N2O production by N. oceani incubated under different O2 availability. Our calculations revealed that previous in vivo studies might have underestimated the SP value for NH2OH oxidation pathway possibly due to a small contribution of NO2– reduction pathway. Further evaluation of isotopomer signatures of N2O using common enzymes of other processes related to N2O would improve the isotopomer analysis of N2O in various environments.

scholarly journals Trace gas fluxes of CO<sub>2</sub>, CH<sub>4</sub> and N<sub>2</sub>O in a permanent grassland soil exposed to elevated CO<sub>2</sub> in the Giessen FACE study

2011 ◽  
Vol 11 (2) ◽  
pp. 4199-4227 ◽  
Author(s):  
M. K. Abbasi ◽  
C. Müller
Keyword(s):  
Elevated Co2 ◽  
15N Tracer ◽  
Organic N ◽  
N2o Emissions ◽  
Unexpected Result ◽  
Ch4 Oxidation ◽  
N2o Production ◽  
N Application ◽  
Oxidation Rates ◽  
N2o Fluxes

Abstract. Long-term field observations showed that N2O fluxes were not significantly affected by elevated CO2 shortly after N application in the Giessen Free Air Carbon dioxide Enrichment (FACE) study. To further investigate this unexpected result a 15N tracer study was carried out under controlled conditions where in parallel treatments either the NH4+ pool (15NH4NO3) or the NO3− pool (NH415NO3) was enriched with 15N. Fluxes of CO2, CH4, and N2O as well as the 15N enrichment of the N2O were measured. Denitrifying Enzyme Activity (DEA), total denitrification (N2 + N2O) and N2−to−N2O ratios were quantified in separate experiments. Over the 57 day incubation, N2O fluxes averaged 0.090 ng N2O–N g−1 h−1 under ambient and 0.083 ng N2O–N g−1 h−1 under elevated CO2 (not significantly different). Based on the 15N enrichments of the N2O the N2O production processes were identified by a two-source model. Results showed that N2O must have also been produced by a third source – possibly related to organic N transformation – which was stimulated by elevated CO2. Soil CO2 fluxes were approximately 20% higher under elevated CO2 than soil from ambient but the differences were not significant. CH4 oxidation rates were on average −1.75 ng CH4−C g−1 h−1 in the elevated and −1.17 ng CH4−C g−1 h−1 in the ambient indicating that elevated CO2 increased the CH4 oxidation by 49% compared to ambient CO2. N fertilization increased CH4 oxidation by 3-fold in both CO2 treatments CO2 did not have any significant effect on DEA while total denitrification and N2–to–N2O ratios increased by 36 and 33%, respectively. The results indicate that shortly after N application elevated CO2 must have stimulated both the N2O production and reduction to N2 to explain the increased N2–to–N2O ratio and at the same time explain the non-responsiveness of the N2O emissions. Thus, the observed variation of the CO2 effect on N2O emissions throughout the year is possibly related to the dynamics of the N2O reductase activity.

scholarly journals Spatial and temporal variability of nitrous oxide emissions in a mixed farming landscape of Denmark

2012 ◽  
Vol 9 (8) ◽  
pp. 2989-3002 ◽  
Author(s):  
K. Schelde ◽  
P. Cellier ◽  
T. Bertolini ◽  
T. Dalgaard ◽  
T. Weidinger ◽  
...  
Keyword(s):  
Land Use ◽  
Nitrous Oxide ◽  
Agricultural Land ◽  
Nitrous Oxide Emissions ◽  
Soil Conditions ◽  
Spatial And Temporal Variability ◽  
N2o Emissions ◽  
N2o Production ◽  
Mixed Farming ◽  
N2o Fluxes

Abstract. Nitrous oxide (N2O) emissions from agricultural land are variable at the landscape scale due to variability in land use, management, soil type, and topography. A field experiment was carried out in a typical mixed farming landscape in Denmark, to investigate the main drivers of variations in N2O emissions, measured using static chambers. Measurements were made over a period of 20 months, and sampling was intensified during two weeks in spring 2009 when chambers were installed at ten locations or fields to cover different crops and topography and slurry was applied to three of the fields. N2O emissions during spring 2009 were relatively low, with maximum values below 20 ng N m−2 s−1. This applied to all land use types including winter grain crops, grasslands, meadows, and wetlands. Slurry application to wheat fields resulted in short-lived two-fold increases in emissions. The moderate N2O fluxes and their moderate response to slurry application were attributed to dry soil conditions due to the absence of rain during the four previous weeks. Cumulative annual emissions from two arable fields that were both fertilized with mineral fertilizer and manure were large (17 kg N2O-N ha−1 yr−1 and 5.5 kg N2O-N ha−1 yr−1) during the previous year when soil water conditions were favourable for N2O production during the first month following fertilizer application. Our findings confirm the importance of weather conditions as well as nitrogen management on N2O fluxes.

scholarly journals Vertical segregation among pathways mediating nitrogen loss (N<sub>2</sub> and N<sub>2</sub>O production) across the oxygen gradient in a coastal upwelling ecosystem

2017 ◽  
Vol 14 (20) ◽  
pp. 4795-4813 ◽  
Author(s):  
Alexander Galán ◽  
Bo Thamdrup ◽  
Gonzalo S. Saldías ◽  
Laura Farías
Keyword(s):  
Coastal Upwelling ◽  
Nitrite Reduction ◽  
15N Tracer ◽  
Ammonium Oxidation ◽  
N Loss ◽  
N2o Production ◽  
Coastal System ◽  
Central Chile ◽  
N Removal ◽  
Bottom Waters

Abstract. The upwelling system off central Chile (36.5° S) is seasonally subjected to oxygen (O2)-deficient waters, with a strong vertical gradient in O2 (from oxic to anoxic conditions) that spans a few metres (30–50 m interval) over the shelf. This condition inhibits and/or stimulates processes involved in nitrogen (N) removal (e.g. anammox, denitrification, and nitrification). During austral spring (September 2013) and summer (January 2014), the main pathways involved in N loss and its speciation, in the form of N2 and/or N2O, were studied using 15N-tracer incubations, inhibitor assays, and the natural abundance of nitrate isotopes along with hydrographic information. Incubations were developed using water retrieved from the oxycline (25 m depth) and bottom waters (85 m depth) over the continental shelf off Concepción, Chile. Results of 15N-labelled incubations revealed higher N removal activity during the austral summer, with denitrification as the dominant N2-producing pathway, which occurred together with anammox at all times. Interestingly, in both spring and summer maximum potential N removal rates were observed in the oxycline, where a greater availability of oxygen was observed (maximum O2 fluctuation between 270 and 40 µmol L−1) relative to the hypoxic bottom waters ( <  20 µmol O2 L−1). Different pathways were responsible for N2O produced in the oxycline and bottom waters, with ammonium oxidation and dissimilatory nitrite reduction, respectively, as the main source processes. Ammonium produced by dissimilatory nitrite reduction to ammonium (DNiRA) could sustain both anammox and nitrification rates, including the ammonium utilized for N2O production. The temporal and vertical variability of δ15N-NO3− confirms that multiple N-cycling processes are modulating the isotopic nitrate composition over the shelf off central Chile during spring and summer. N removal processes in this coastal system appear to be related to the availability and distribution of oxygen and particles, which are a source of organic matter and the fuel for the production of other electron donors (i.e. ammonium) and acceptors (i.e. nitrate and nitrite) after its remineralization. These results highlight the links between several pathways involved in N loss. They also establish that different mechanisms supported by alternative N substrates are responsible for substantial accumulation of N2O, which are frequently observed as hotspots in the oxycline and bottom waters. Considering the extreme variation in oxygen observed in several coastal upwelling systems, these findings could help to understand the ecological and biogeochemical implications due to global warming where intensification and/or expansion of the oceanic OMZs is projected.

scholarly journals A new look at an old concept: using <sup>15</sup>N<sub>2</sub>O isotopomers to understand the relationship between soil moisture and N<sub>2</sub>O production pathways

SOIL ◽  
2019 ◽  
Vol 5 (2) ◽  
pp. 265-274 ◽  
Author(s):  
Katelyn A. Congreves ◽  
Trang Phan ◽  
Richard E. Farrell
Keyword(s):  
Soil Moisture ◽  
Mitigation Strategies ◽  
Soil Conditions ◽  
Site Preference ◽  
N2o Emissions ◽  
Spectroscopic Techniques ◽  
N2o Production ◽  
Nitrification And Denitrification ◽  
The Relationship ◽  
Source Partitioning

Abstract. Understanding the production pathways of potent greenhouse gases, such as nitrous oxide (N2O), is essential for accurate flux prediction and for developing effective adaptation and mitigation strategies in response to climate change. Yet there remain surprising gaps in our understanding and precise quantification of the underlying production pathways – such as the relationship between soil moisture and N2O production pathways. A powerful, but arguably underutilized, approach for quantifying the relative contribution of nitrification and denitrification to N2O production involves determining 15N2O isotopomers and 15N site preference (SP) via spectroscopic techniques. Using one such technique, we conducted a short-term incubation where N2O production and 15N2O isotopomers were measured 24 h after soil moisture treatments of 40 % to 105 % water-filled pore space (WFPS) were established for each of three soils that differed in nutrient levels, organic matter, and texture. Relatively low N2O fluxes and high SP values indicted nitrification during dry soil conditions, whereas at higher soil moisture, peak N2O emissions coincided with a sharp decline in SP, indicating denitrification. This pattern supports the classic N2O production curves from nitrification and denitrification as inferred by earlier research; however, our isotopomer data enabled the quantification of source partitioning for either pathway. At soil moisture levels < 53 % WFPS, the fraction of N2O attributed to nitrification (FN) predominated but thereafter decreased rapidly with increasing soil moisture (x), according to FN=3.19-0.041x, until a WFPS of 78 % was reached. Simultaneously, from WFPS of 53 % to 78 %, the fraction of N2O that was attributed to denitrification (FD) was modelled as FD=-2.19+0.041x; at moisture levels of > 78 %, denitrification completely dominated. Clearly, the soil moisture level during transition is a key regulator of N2O production pathways. The presented equations may be helpful for other researchers in estimating N2O source partitioning when soil moisture falls within the transition from nitrification to denitrification.

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