Pitfalls in measuring nitrous oxide production by nitrifiers

Nitrous oxide (N ₂ O) is an important greenhouse gas. At present, it causes 6% of global warming. The atmospheric concentration of N ₂ O continues to increase at a rate of 0.8 ppb per year. The main known sink of N ₂ O is its destruction in the stratosphere to nitric oxide (NO). Via that destruction product, N ₂ O contributes to the decomposition of stratospheric ozone.

The most important sources of N ₂ O are the microbial soil processes nitrification and denitrification. Especially after fertilization of the soil, large amounts of N ₂ O can be emitted. Nitrifiers produce N ₂ O by nitrification and by nitrifier denitrification. In nitrification, N ₂ O develops during the oxidation of hydroxylamine (NH ₂ OH). In nitrifier denitrification, nitrifiers reduce nitrite (NO ₂^-) via N ₂ O to N ₂ . Not much is known about nitrifier denitrification yet. The discovery of several intermediates and enzymes is in line with a suspected similarity between nitrifier denitrification and denitrification. Denitrifiers reduce nitrate (NO ₃^-) to N ₂ . N ₂ O is an intermediate in that process. It is important to be able to differentiate between N ₂ O produced by the different processes in soils, since they are influenced by different factors. Only with a profound knowledge of the sources is a mitigation of N ₂ O emission from soils possible.

The objectives of this study were to quantitatively assess N ₂ O production by nitrifier denitrification under a range of conditions and to come up with a best estimate for N ₂ O produced by nitrifier denitrification in The Netherlands. A review of nitrifier denitrification and related processes in soils (Chapter 2) revealed how important it is to get to know more about this poorly studied pathway. Up to 30% of the total N ₂ O production in soils has been attributed to nitrifier denitrification. Especially low oxygen (O ₂ ) conditions coupled with low organic carbon contents might favour this pathway. It was concluded that there was a need to quantify the N ₂ O production by nitrifier denitrification under different conditions. Therefore, a soil study was carried out with different soils in a range of conditions. Rather than leading to new quantitative insights, this study gave rise to questions concerning the prevailing measurement method for nitrifier denitrification (Chapter 3). In this method, the differentiation between nitrification, nitrifier denitrification, denitrification and other soil sources of N ₂ O is based on incubations with combinations of 0.02 kPa acetylene (C ₂ H ₂ ) and 100 kPa O ₂ . C ₂ H ₂ is supposed to inhibit nitrification and nitrifier denitrification without influencing denitrification, and O ₂ is supposed to inhibit nitrifier denitrification and denitrification, without affecting nitrification. However, this method did not seem to be suitable for all soils. In some conditions, the addition of inhibitors seemed to stimulate the production of N ₂ O compared to the controls. Furthermore, negative fluxes were calculated for some sources of N ₂ O, especially for nitrifier denitrification (Chapter 3). Due to these methodological difficulties, the objectives of this study were adapted and became i) to test the prominent methodology for quantifying the N ₂ O production by nitrifier denitrification, and ii) to assess the importance of nitrifier denitrification for N ₂ O production in pure cultures of Nitrosomonas europaea and Nitrosospira briensis . N. europaea is often used as a model organism in laboratory studies. It has frequently been found in environments high in N like water treatment plants. N. briensis is better adapted to environments less abundant in N and is common in a number of fertilized arable soils of neutral pH.

The first objective has been addressed in Chapter 3, 4 and 5. We have seen in Chapter 3 that the prevailing measurement method using the inhibitors C ₂ H ₂ (0.02 kPa) and O ₂ (100 kPa) in different combinations to quantify the N ₂ O production by nitrifier denitrification was not suitable for all soils. Pure culture studies revealed some reasons for the observed problems (Chapter 4 and 5). O ₂ was not suitable as an inhibitor of nitrifier denitrification, since it also had a negative effect on ammonia oxidation, the first step of nitrification (Chapter 4 and 5). C ₂ H ₂ only inhibited the N ₂ O production by N. europaea , but not that by N. briensis (Chapter 4). C ₂ H ₂ did furthermore not inhibit the N ₂ O production by a transformant of N. europaea lacking nitric oxide reductase, an enzyme catalyzing the reduction of nitric oxide to N ₂ O in the nitrifier denitrification pathway (Chapter 5). While it is not clear yet whether the reason for the insensitivity to C ₂ H ₂ was the same in the transformant and in N. briensis , we can conclude that C ₂ H ₂ was not reliable as an inhibitor of N ₂ O production by all nitrifiers.

Due to the consistent results of soil studies and pure culture experiments, we reach the conclusion that the method using C ₂ H ₂ and O ₂ is not suitable for differentiating reliably between sources of N ₂ O in soils. In the past, especially C ₂ H ₂ has been used extensively to differentiate between nitrification and denitrification in soils. If C ₂ H ₂ does not inhibit N ₂ O production by nitrifiers reliably, the share of nitrifiers in N ₂ O production might have been underestimated in these studies.

The importance of nitrifier denitrification for N ₂ O production has been studied in pure culture experiments (Chapter 4 and 5). In Chapter 4, a study of the production of N ₂ O by pure cultures of N. europaea and N. briensis is described. Large concentrations (100 kPa) of O ₂ were used to inhibit nitrifier denitrification. The results suggested that nitrifier denitrification was the most important pathway in this respect, causing about 80% of the N ₂ O production by N. europaea and about 65% of that by N. briensis . However, there were indications that nitrification might have been underestimated due to adverse effects of O ₂ on ammonia oxidation. In Chapter 5, the N ₂ O production was studied in mutants of N. europaea that were deficient in either nitrite reductase (NirK) or nitric oxide reductase (NORB), two enzymes of the nitrifier denitrification pathway. The NirK-deficient cells produced similar amounts of N ₂ O as the wild-type. Since the NirK-deficient cells could not have produced this N ₂ O via the known pathway of nitrifier denitrification, this result suggests that nitrifier denitrification is not so important for N ₂ O production in this mutant. The NORB-deficient cells produced even more N ₂ O, about 60 times as much as the wild-type. At the same time, the NORB-deficient cells consumed NO ₂^-. While side-effects of the mutation on pathways of N ₂ O production cannot be excluded, there are indications for a role of the enzyme NORB in directing ammonia oxidation towards NO ₂^-rather than N ₂ O. Large concentrations of O ₂ inhibited the N ₂ O production and NO ₂^-consumption in this mutant and might therefore be able to fulfil a role similar to NORB in directing the reaction to NO ₂^-. The N ₂ O production of the NORB-deficient cells was not inhibited by C ₂ H ₂ . This could hint at an unknown pathway of N ₂ O production in nitrifiers (Chapter 5).

A sensitivity analysis (Chapter 6) revealed that an inhibition of the N ₂ O reductase of denitrifiers by C ₂ H ₂ most likely caused some of the observed over- and underestimations of sources of N ₂ O in the soil survey. Furthermore, it is likely that C ₂ H ₂ only inhibited part of nitrification and nitrifier denitrification and that O ₂ also partly inhibited nitrification in the soil. This suggests that nitrifiers have probably been underestimated as producers of N ₂ O in studies using C ₂ H ₂ and O ₂ as inhibitors. Future studies should further investigate the pathways of N ₂ O production, including the indicated possible unknown pathway of nitrifiers. A combination of stable isotope studies of N and O and incubation studies with inhibitors might enable the differentiation between sources of N ₂ O in soils. Since this study shows that 0.02 kPa C ₂ H ₂ and 100 kPa O ₂ are not suitable as inhibitors of different N ₂ O producing processes, alternatives need to be found.