Nitrous oxide from fungal denitrification - Pure culture and soil studies using stable isotope and microbial inhibitor approaches

Abstract

The trace gas nitrous oxide (N₂O) contributes to climate change as well as to the depletion of the ozone layer in the stratosphere. Agricultural soils account for about 70% of the high anthropogenic N₂O emissions. Microbial processes in soil use, for instance fertilizer N to produce N₂O, are an important factor. An understanding of N₂O production pathways is imperative to evaluate reliable mitigation methods for N₂O emissions. The present study focused on denitrification, which, besides nitrification and nitrifier denitrification, is one of the main N₂O production pathways in soils. Denitrification describes the reduction from nitrate (NO₃^-) to N₂, with nitrite (NO₂^-), nitrous monoxide (NO) and N₂O as intermediates. For a long time denitrification was attributed only to heterotrophic bacteria. In 1972, however, pure culture studies showed that fungi are also capable of denitrification, and two decades later most fungi were found to lack the N₂O reductase, resulting in N₂O being the main product of fungal denitrification instead of N₂. This could indicate that fungi might produce more N₂O compared to bacteria, providing that both groups have the same production rates. However, the contribution of different microbial groups to N₂O emissions from soil has not yet been sufficiently investigated. Analysis of the isotopic signature of N₂O found this to be a promising tool to distinguish between N₂O produced by different microbial groups. Especially the site preference of ¹⁵N in N₂O (SP = difference between δ¹⁵N of the outer and central N atoms in N₂O) from denitrification revealed differences between pure bacterial cultures (SP = -11 to 0 ‰) and two studied pure fungal cultures (SP ~ 37 ‰). Although it is known that all enzymes involved in fungal denitrification, with the exception of the N₂O reductase, equals the enzymes of bacteria, most denitrification studies with pure cultures covered the bacterial pathway. The different N₂O reductases might be the reason for different SP of N₂O produced by bacteria or fungi. An O exchange between denitrification intermediates and water between 4 and 100% was found during bacterial denitrification, while there has been no study analyzing the existence of O exchange during fungal denitrification so far. If O exchange were not to occur during fungal denitrification, this could provide an additional ability to differentiate between N₂O produced by fungi or bacteria. The O isotopic signature of N₂O produced by fungi would significantly differ from that produced by bacteria. The present study focused on three subjects. With an isotope tracer experiment with ¹⁸O labeled water, the existence of O exchange between denitrification intermediates and water during denitrification was studied with six fungal species. The fungi showed an O exchange of up to 100% and consequently a differentiation between fungal and bacterial denitrification with an O isotopic signature is impossible. The second subject was verification of the high SP values of N₂O from fungal denitrification in four additionally tested species and consideration of whether it was reproducible for the two tested species known from literature. This study confirmed higher SP values of N₂O (SP = 19.7 to 31.7 ‰) compared to the SP of N₂O known from bacteria. Based on the results of the isotope tracer experiment and the O isotopic signature of N₂O under natural conditions, mechanisms of the O isotope fractionation were analyzed by applying values of fractionation effects known from the literature in an isotope fractionation model to estimate the involved enzymes on O exchange during denitrification. The O exchange of NO₂^- reductase was high compared to O exchange of NO₃^- and NO reductases. The knowledge obtained from pure fungal culture studies was used in Subject Three to test the transferability to microbial communities in soils by using microbial inhibitors for bacteria or fungi in soil incubation experiments. A modification of substrate induced respiration with selective inhibition (SIRIN) was used to determine whether the specific SP values of N₂O known for bacteria and fungi are measurable after selective growth inhibition by specific antibiotic application. The expected effect of growth inhibition on SP of N₂O was not found. In most cases the SP of N₂O was in the range known from pure bacterial cultures and bacterial growth inhibition did not result in the expected shift of SP values. Consequently the SP values of this incubation experiment did not serve to associate the N₂O production in inhibited treatments to different microbial groups. It remained unclear if this was due to the modified SIRIN method or if transferability of differences in SP of N₂O known from fungi and bacteria on a microbial community in soil is possible. Future studies should approach the existing problems regarding the methods to identify fungal denitrification in soil.