08558nam a2200169 a 450000100080000000500110000800800410001910000190006024500880007926000160016730000110018350000650019452080850025965000140834465000170835865300130837512469512004-01-20       2003    bl uuuu  m   00u1 u    #d1 aMARIANO, E. D.  aCitrate exudation by maize rootsba possible mechanisms of resistance to aliminium.  a2003.c2003  a124 p.  aThesis (Doctor) - Wageningen Universiteit, Wageningen, 2003.  aNitrous oxide (N2O) is an important greenhouse gas. At present, it causes 6% of global warming. The atmospheric concentration of N2O continues to increase at a rate of 0.8 ppb per year. The main known sink of N2O is its destruction in the stratosphere to nitric oxide (NO). Via that destruction product, N2O contributes to the decomposition of stratospheric ozone. The most important sources of N2O are the microbial soil processes nitrification and denitrification. Especially after fertilization of the soil, large amounts of N2O can be emitted. Nitrifiers produce N2O by nitrification and by nitrifier denitrification. In nitrification, N2O develops during the oxidation of hydroxylamine (NH2OH). In nitrifier denitrification, nitrifiers reduce nitrite (NO2-) via N2O to N2. 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 (NO3-) to N2. N2O is an intermediate in that process. It is important to be able to differentiate between N2O 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 N2O emission from soils possible.  The objectives of this study were to quantitatively assess N2O production by nitrifier denitrification under a range of conditions and to come up with a best estimate for N2O 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 N2O production in soils has been attributed to nitrifier denitrification. Especially low oxygen (O2) conditions coupled with low organic carbon contents might favour this pathway. It was concluded that there was a need to quantify the N2O 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 N2O is based on incubations with combinations of 0.02 kPa acetylene (C2H2) and 100 kPa O2. C2H2 is supposed to inhibit nitrification and nitrifier denitrification without influencing denitrification, and O2 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 N2O compared to the controls. Furthermore, negative fluxes were calculated for some sources of N2O, 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 N2O production by nitrifier denitrification, and ii) to assess the importance of nitrifier denitrification for N2O 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 C2H2 (0.02 kPa) and O2 (100 kPa) in different combinations to quantify the N2O production by nitrifier denitrification was not suitable for all soils. Pure culture studies revealed some reasons for the observed problems (Chapter 4 and 5). O2 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). C2H2 only inhibited the N2O production by N. europaea, but not that by N. briensis (Chapter 4). C2H2 did furthermore not inhibit the N2O production by a transformant of N. europaea lacking nitric oxide reductase, an enzyme catalyzing the reduction of nitric oxide to N2O in the nitrifier denitrification pathway (Chapter 5). While it is not clear yet whether the reason for the insensitivity to C2H2 was the same in the transformant and in N. briensis, we can conclude that C2H2 was not reliable as an inhibitor of N2O production by all nitrifiers. Due to the consistent results of soil studies and pure culture experiments, we reach the conclusion that the method using C2H2 and O2 is not suitable for differentiating reliably between sources of N2O in soils. In the past, especially C2H2 has been used extensively to differentiate between nitrification and denitrification in soils. If C2H2 does not inhibit N2O production by nitrifiers reliably, the share of nitrifiers in N2O production might have been underestimated in these studies. The importance of nitrifier denitrification for N2O production has been studied in pure culture experiments (Chapter 4 and 5). In Chapter 4, a study of the production of N2O by pure cultures of N. europaea and N. briensis is described. Large concentrations (100 kPa) of O2 were used to inhibit nitrifier denitrification. The results sugested that nitrifier denitrification was the most important pathway in this respect, causing about 80% of the N2O 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 O2 on ammonia oxidation. In Chapter 5, the N2O 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 N2O as the wild-type. Since the NirK-deficient cells could not have produced this N2O via the known pathway of nitrifier denitrification, this result suggests that nitrifier denitrification is not so important for N2O production in this mutant. The NORB-deficient cells produced even more N2O, about 60 times as much as the wild-type. At the same time, the NORB-deficient cells consumed NO2-. While side-effects of the mutation on pathways of N2O production cannot be excluded, there are indications for a role of the enzyme NORB in directing ammonia oxidation towards NO2- rather than N2O. Large concentrations of O2 inhibited the N2O production and NO2- consumption in this mutant and might therefore be able to fulfil a role similar to NORB in directing the reaction to NO2-. The N2O production of the NORB-deficient cells was not inhibited by C2H2. This could hint at an unknown pathway of N2O production in nitrifiers (Chapter 5).  A sensitivity analysis (Chapter 6) revealed that an inhibition of the N2O reductase of denitrifiers by C2H2 most likely caused some of the observed over- and underestimations of sources of N2O in the soil survey. Furthermore, it is likely that C2H2 only inhibited part of nitrification and nitrifier denitrification and that O2 also partly inhibited nitrification in the soil. This suggests that nitrifiers have probably been underestimated as producers of N2O in studies using C2H2 and O2 as inhibitors. Future studies should further investigate the pathways of N2O 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 N2O in soils. Since this study shows that 0.02 kPa C2H2 and 100 kPa O2 are not suitable as inhibitors of different N2O producing processes, alternatives need to be found.  aAlumínio  aResistência  aAliminio