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a U.S. Geological Survey, Box 25046 M.S. 408, Denver Federal Center, Denver, CO 80225 USA
b Dep. of Chemistry, California State University, Bakersfield, CA 93311 USA
kathorn{at}usgs.gov
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Abbreviations: IHSS, International Humic Substances Society • LFA, Laurentian soil fulvic acid • NMR, nuclear magnetic resonance • NOE, nuclear Overhauser enhancement • SRFA, Suwannee River fulvic acid
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMaterials and methodsNOTESResultsDiscussionREFERENCES |
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25, 80, and 40 Tg N yr-1, respectively (15). The concentration of N2O in the atmosphere has increased by 0.25% per year during the last 20 yr (6). Fertilizer usage is considered the major cause of this increase, compared with other sources of N2O such as biomass burning, fossil fuel combustion, deforestation, and increasing amounts of wet and dry NOx deposition and NH4 in rainwater. One report estimated that fertilizer-induced N2O emission was 0.4 to 1.4 Tg N2O–N yr-1 in 1987. The relative contributions of nitrification, biological denitrification, and chemodenitrification processes to the anthropogenic production of N2O are not well understood, although microbial processes are considered most important (17). Ammonia-based fertilizers are the most abundant form of nitrogenous fertilizer applied to agricultural soils (9, 21). Ammonia not taken up by plants, lost through volatilization, or fixed by clay minerals or soil organic matter can undergo nitrification to NO-2 and NO-3 under aerobic conditions. Nitrate and NO-2 in turn may undergo denitrification to NO, N2O, and N2 under anaerobic conditions. The occurrence of denitrification under aerobic conditions has also been documented (22).
One reaction potentially affecting these pathways is that between soil organic matter and NO-2 itself. Under conditions of heavy application of NH3, NO-2 may accumulate due to inhibition of nitrification. This inhibition is presumed to result from NH3 toxicity to nitrobacter (23–27). The ability of soil organic matter to both fix NO-2, and, in one of the processes associated with chemodenitrification, effect its transformation into NO, N2O, and N2, was first documented in the work of Bremner and of Stevenson (23–25, 28–30). Early studies into the phenomenon of chemodenitrification were motivated by a need to understand soil processes affecting the efficiency of N fertilization. Interest in this process has expanded to include environmental impacts and its overall significance in the biogeochemical cycling of N. Conditions favoring chemodenitrification have been outlined by Nelson (20); N2O losses via chemodenitrification are most significant during nitrification in acidic soils. It is also conceivable that humic substances react with NO-2 under natural conditions in soils, sediments, and natural waters. In fact, Azhar et al. (31) concluded that apparently significant nitrosation of organic matter can occur in soil environments upon normal nitrification at neutral to slightly acidic pH values, without the accumulation of large amounts of NO-2. The possibility that nitrosation of organic matter may contribute to the immobilization of N in soils also has been suggested by Smith and Chalck (32–33).
Bremner (23) proposed a scheme for the reaction of NO-2 with soil organic matter wherein phenolic moieties are nitrosated under acidic conditions to form nitrosophenols, which tautomerize to quinone monoximes. A second molecule of NO-2 reacts with the oxime to form N2O and water.
Several related studies have examined reaction conditions leading to the formation of NO, N2O, N2, and CO2 upon nitrosation of humic substances, lignins, and model compounds (28–30, 34). In addition to aromatic C-nitrosation, these reports have also examined demethoxylation (formation of methyl nitrite), nitrosative decarboxylation, and N-nitrosation reactions.
Before a more accurate assessment of the significance of NO-2 fixation and corresponding chemodenitrification reactions in the soil environment can be made, it is desirable to first learn in more detail on a molecular level how NO-2 reacts with humic substances. We previously observed that liquid phase 15N NMR could be used to observe the nitrosophenol–quinone monoxime equilibrium in humic substances reacted with hydroxylamine (35). This prompted us to use 15N NMR to investigate the model for the nitrosation of humic substances proposed by Bremner (23). Nitrogen-15 NMR afforded the possibility of directly observing the incorporation of NO-2–N into humic substances, and also distinguishing among nitrosation of aromatic C, aliphatic C, and N, as well as secondary reactions. To this end, liquid-phase 15N NMR spectra were recorded on soil and aquatic fulvic acids nitrosated with 15N-labeled NaNO2, and of the aquatic fulvic acid sequentially treated with 15N-labeled NH3 and unlabeled NO-2. The effect of pH on nitrosation of the fulvic acids was also examined. Carbon-13 NMR was used to help identify the nature of the aliphatic carbons undergoing nitrosation, and to observe nitrosative demethoxylation in a soil humic acid. Finally, the incorporation of NO-2 into the organic matter of a whole peat, and a comparison of the reaction of NO-2 with the humic acid extracted from the peat, were examined using solid state 15N NMR.
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Materials and methods |
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Reactions
Oximation of SRFA was performed by dissolving 0.2 g 15NH2OH · HCl and 500 mg of the H+-saturated fulvic acid in 100 mL of H2O, titrating to pH 6 with 1.0 M NaOH, and stirring at room temperature for 24 h. The sample was then H+-saturated on a Dowex MSC-1 cation-exchange resin (Dow Corning, Midland, MI), freeze-dried, and a portion dissolved in DMSO-d6 for 15N NMR analysis.
The following nitrosation procedures were varied to accommodate the differing solubilities of the individual samples, and to investigate pH effects and reaction conditions. Workups of the nitrosated samples were manipulated to retain or remove the low molecular weight degradation products of NaNO2 (e.g., NO-3, hydroxylamine, and NH3). For nitrosation of SRFA, 80 mg of Na15NO2 dissolved in 0.5 mL of H2O and 300 mg SRFA (H+-saturated) dissolved in 1.0 mL of H2O were mixed in a 10-mm NMR tube, to which was added 0.5 mL of D2O. An ACOUSTIC 15N NMR spectrum was recorded after cessation of gas evolution. The final pH was in the range 3.2 to 3.6. The Armadale was nitrosated similarly, but with 392 mg of sample (H+-saturated) and 104 mg of Na15NO2 in a volume of 5.5 mL of H2O. After reaction, the sample was freeze-dried and redissolved in 2.5 mL of 50% D2O in H2O for NMR analysis. Nitrosation of SRFA at the higher pH was performed by dissolving 461 mg of the Na+ salt of the fulvic acid and 124 mg of Na15NO2 in 2 mL of H2O and 1 mL of D2O. The initial pH was 6.15. The sample was allowed to react for 3 d before the 15N NMR spectrum was recorded. The pH had risen to 7.05 by the end of the NMR acquisition. Laurentian fulvic acid was nitrosated at initial pH levels of 3 and 6 similar to SRFA. The LFA was also nitrosated under more dilute conditions: 300 mg of LFA and 40 mg of NaNO2 dissolved in 500 mL of H2O, with pH adjusted to 4.0 with 0.1 M NaOH, was allowed to stir for 9 d. The sample was then dialyzed (1000 MW cut off) to remove NO-3 and any residual NO-2, freeze-dried, and redissolved in 2 mL of 50% D2O for NMR. For nitrosation of the IHSS soil humic acid, 120 mg of Na15NO2 was dissolved in 3 to 5 mL of H2O. Approximately 300 mg of H+-saturated humic acid was slowly added and stirred for 1 d. For nitrosation of peat humic acid, 400 mg of the H+-saturated humic acid was added to 15 mL of H2O, titrated to pH 4 with 1 M NaOH, charged with 82 mg Na15NO2 dissolved in 2 mL of H2O, and stirred for 6 d. After reaction, both the soil and peat humic acid solutions were diluted, H+-saturated on the exchange resin, dialyzed, freeze-dried, and redissolved in DMSO-d6 for NMR. Two grams of the Pahokee peat were homogenized with 8 mL of H2O in a 250-mL teflon centrifuge bottle, charged with 400 mg Na15NO2 dissolved in 7 mL of H2O, shaken for 3 d, dialyzed, and then freeze-dried.
Approximately 600 mg of SRFA were reacted for 2 wk with 15NH4OH as previously described (36) and then H+-saturated and freeze-dried. The sample was redissolved in 3 mL of H2O along with 160 mg of unlabeled
. After cessation of gas evolution, the sample was H+-saturated again, freeze-dried, and a portion dissolved in DMSO-d6 for 15N NMR analysis.
NMR Spectrometry
Liquid-phase NMR spectra were recorded on a Varian XL-300 or Varian Gemini 2000 NMR spectrometer (Varian, Sunnyvale, CA) at 13C and 15N resonant frequencies of 75.4 and 30.4 MHz, respectively, using a 10-mm broadband probe. Quantitative liquid-phase 13C NMR spectra of the unreacted fulvic and humic acids were recorded as previously described (35), employing 8- to 10-s pulse delays in conjunction with 45° pulse angles to eliminate differential T1 effects, and inverse gated decoupling to eliminate nuclear Overhauser enhancement (NOE). ACOUSTIC (37) 15N NMR spectra of SRFA, Armadale, and Laurentian fulvic acids were recorded in aqueous solution without paramagnetic relaxation reagent using a 15649.5 Hz (514.8 ppm) spectral window, 0.5-s acquisition time, 90° pulse angle, 2.0-s pulse delay, and 
delay of 0.1 ms. The ACOUSTIC 15N NMR spectrum of the peat humic acid was recorded in DMSO-d6 with the addition of Cr(Acac)3 using the same acquisition parameters, but with a spectral window of 24325.1 Hz (800 ppm). The DEPT (38) 15N NMR spectra of the peat humic acid were recorded using a 15649.5-Hz spectral window, 0.2-s acquisition time, 2.0-s delay for proton relaxation, and 1JNH of 90 Hz. The DEPTGL (39) 13C NMR spectra of the soil humic acid were recorded as previously described (35). Solid-state CP/MAS 15N NMR spectra of peat fulvic acid, peat humic acid, and peat were recorded on a Chemagnetics CMX-200 NMR spectrometer (Chemagnetics, Fort Collins, CO) at a N-resonant frequency of 20.3 MHz, using a 7.5-mm ceramic probe (zirconium pencil rotors). Natural abundance spectra were recorded using a 30000-Hz spectral window, 17.051-ms acquisition time, 2.0-ms contact time, 0.5-s pulse delay, and spinning rate of 5 KHz. Nitrosated samples were acquired similarly, but with a 1.0-s pulse delay and spinning rate of 6 KHz. Chemical shifts were referenced to glycine, taken as 32.6 ppm.
Quantitation in 15N NMR
The NOE is retained in the liquid-phase ACOUSTIC 15N NMR spectra recorded without paramagnetic relaxation reagent, so the spectra of the nitrosated SRFA, Armadale, and Laurentian fulvic acids can only be interpreted semiquantitatively. The ACOUSTIC 15N NMR spectrum of the nitrosated peat humic acid, recorded with the use of relaxation reagent and a 2-s pulse delay, represents the quantitative distribution of nitrogens incorporated into the sample. The DEPT experiments performed on the nitrosated peat humic acid detect only nitrogens directly bonded to protons. In CP–MAS experiments, peak areas can accurately represent the number of nuclei resonating, when the time constant for cross polarization is significantly less than the time constant for proton spin lattice relaxation in the rotating frame, (TNH << T1
H). Since no analyses of the spin dynamics were performed, or a comparison made with direct polarization experiments, the CP/MAS spectra of the unreacted and nitrosated peat samples can only be interpreted semiquantitatively. Further discussion of quantitation in the CP/MAS experiment can be found in the review of Kinchesh et al. (40).
Background
Nitrosation
Several reviews on nitrosation chemistry are available (41–45). The nitrosating species in acidic solutions of nitrous acid may be dinitrogen trioxide or nitrosonium ion, depending on pH and the concentration of the nitrous acid. Nitrosation of aliphatic and aromatic carbons is favored at acidic pH. Activated methyl and methylene carbons are nitrosated to aldoximes and ketoximes, respectively.
With unsymmetrical ketones such as methyl alkyl ketones, the normal product is the ketoxime arising from nitrosation in the methylene rather than the methyl group (44).
Nitrosation of activated carbons adjacent to a carboxylic acid group may result in a decarboxylation reaction (42). Nitrosative decarboxylation reactions can account in part for the CO2 produced upon nitrosation of humic substances (25).
Phenols and naphthols readily undergo nitrosation to nitrosophenols, which in solution exist primarily as their tautomeric quinone monoximes.
Certain aromatic acids can also undergo nitrosative decarboxylation reactions (42).
One feature of aromatic nitrosation reactions is that the initially formed nitroso compound can be further oxidized to the nitro compound when excess nitrous acid is used (44). This was documented in the nitrosation of ferulic acid, in conjunction with decarboxylation reactions, and the formation of 1,2(4H)-benzoxazin-4-one among other compounds in a complex reaction mixture (46).
Secondary aliphatic and secondary aromatic amines (including 2° amino acids and amino sugars) form stable N-nitrosamines upon nitrosation, whereas primary aliphatic and primary aromatic amines usually undergo deamination to a variety of products.
Likewise, nitrosation of secondary amides results in the corresponding nitrosamides, whereas nitrosation of primary amides results in a deamination reaction giving the carboxylic acid and N2 gas as products.
Nitrogen-15 NMR chemical shifts corresponding with the products from these nitrosation and other related reactions are illustrated in Fig. 1 .
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Intact protein or peptide structures are assumed not to co-isolate with the fulvic and humic acid fractions. The shoulders at 169 and 173 ppm in the humic and fulvic acids, respectively, correspond with heterocyclic nitrogens. These nitrogens occur in the approximate range from 135 to 230 ppm, and may include heterocyclic sp3 hybridized nitrogens such as indoles, pyrroles, and the imide or lactam nitrogens of nucleosides. Heterocyclic nitrogens are not clearly resolved in the spectrum of the whole peat. The peaks at 29 to 31 ppm in all three samples correspond with the free amino nitrogens of amino sugars and amino acids. Notably absent in all three spectra are any peaks downfield of 250 ppm. In other words, imine, pyridine, and other sp2 hybridized nitrogens involved in heterocyclic linkages are not observed. The fact that these nitrogens are not observed in the spectra does not necessarily mean they do not occur in the samples. For example, some classes of nitrogens (e.g., porphyrins, phthalocyanins, and related enamino-imino systems) may not be observed in the CP/MAS experiment because of problems of molecular motion or chemical exchange at room temperature (53) and require low temperature experiments for detection. Additionally, nitrogens not directly bonded to protons may have an optimal contact time longer than the 2 ms employed in these acquisitions. Finally, it is possible that nitrogens that have chemical shifts downfield from 250 ppm occur at concentrations below the detection limit of NMR at the field strength, number of transients, and sample quantities used in these experiments. The percentage of naturally abundant 15N nuclei actually observed in CP/MAS experiments of humic substances is a question that still needs to be addressed.
Amide and heterocyclic nitrogens were observed in a natural abundance CP/MAS 15N NMR spectrum of the SRFA, and amide nitrogens were observed in the LFA (K.A. Thorn, 1998, unpublished data). These spectra suggest that potential sites for N-nitrosation in the fulvic and humic acids may include secondary amide nitrogens, heterocyclic nitrogens such as indoles, and the free amino nitrogens in amino sugars and amino acids.
15N NMR Spectra of Nitrosated Suwannee River Fulvic Acid
The ACOUSTIC 15N NMR spectrum (NOE retained) of SRFA reacted with 15N-labeled hydroxylamine at pH 6 and room temperature is shown in Fig. 4a
. The major peak occurs from 430 to 330 ppm. Oximes of ketones occur from 390 to 330 ppm. The downfield shoulder from 430 to 390 ppm represents the monoxime derivatives of quinones, in tautomeric equilibrium with their corresponding nitrosophenol forms (35).
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(Dioximes of quinones overlap with oximes of ketones; however, under the mild reaction conditions employed, formation of dioximes with quinones is unlikely.) Other peaks in the spectrum correspond with Beckmann reaction products of the initial oxime derivatives. These are diminished compared with the samples reacted with hydroxylamine at pH 5 under reflux conditions reported previously (35). Also compared with the spectrum reported previously, resolution between the nitrosophenol and oxime peaks is greater when the oximation is performed at room temperature and pH 6.
The major peak in the ACOUSTIC 15N NMR spectrum (NOE retained) of SRFA nitrosated at pH 3 with labeled NO-2 occurs from 430 to 340 ppm, again representing oximes from 390 to 340 ppm and nitrosophenols from 430 to 390 ppm (Fig. 4b). The oximes arise from nitrosation of activated methylene and methyl groups, and the nitrosophenols from nitrosation of phenolic moieties (Reactions [3]–[8]). The peak from 280 to 225 ppm, with maximum at 247 ppm, corresponds with nitrile N, resulting from Beckmann fragmentations of the ketoximes and quinone monoximes (35, 54–57).
Oximes likely to undergo fragmentation to nitriles are those adjacent to quaternary carbon centers, because of the stability of the carbonium ion that is cleaved. A DEPT 15N NMR spectrum recorded on the sample (not shown) indicated that the nitrogens from 120 to 90 ppm in Fig. 4b are bonded to two protons and therefore correspond with primary amides, while the nitrogens from 180 to 120 ppm are singly protonated. The primary amides (120–90 ppm) are assumed to result from hydrolysis of the nitriles. The peak from 150 to 120 ppm represents secondary amides, from Beckmann rearrangements of oximes.
The assignment of the peak from about 180 to 150 ppm, centered at 167 ppm, is uncertain. In spectra of hydroxylamine derivatized samples, the peak was assigned as hydroxamic acid N, resulting from the reaction of hydroxylamine with esters or amides in the humic substances (35). As discussed below, hydroxylamine appears to be a side product of the reaction of NaNO2 with weak acids at acidic pH. It is possible that some of this hydroxylamine reacted with the fulvic acid to form hydroxamic acids. Imides are another possible assignment for these nitrogens.
Three reagent peaks occur at 376, 105, and 20 ppm in Fig. 4b. The peak at 376 ppm corresponds with nitric acid, resulting from the decomposition of nitrous acid (20):
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The peak at 105 ppm appears to be hydroxylamine. This peak occurred in all 15N NMR spectra of nitrosated model phenol and activated methylene compounds and nitrosated humic and fulvic acids prior to dialysis. The 15N NMR spectrum of a reaction blank in which acetic acid was added to an aqueous solution of Na15NO2 (30 mg Na15NO2, 200 mL glacial acetic acid, 2 mL H2O; pH 3.3) also exhibited an hydroxylamine peak at 81 ppm along with the nitrous acid peak (376 ppm). Thus the hydroxylamine is produced independently of any nitrosation reactions. It is interesting to note that Van Hecke et al. (58) provided evidence that hydroxylamine may be formed directly out of NO-3, although their studies were conducted in the pH range from 7.5 to 8.1. The peak at 20.7 ppm corresponds with NH3. The NH3 may arise from an aminolysis or hydrolysis reaction of the primary amides.
Since nitric acid, hydroxylamine, and NH3 are formed as byproducts of the nitrosation reactions, the question arises, to what extent do these three reagents go on to react with the fulvic acid? In other words, can nitration, oximation, or NH3 fixation occur as secondary reactions? At this point, we can only offer the opinion that the kinetics of the nitrosation and Beckmann reactions are faster than any potential reactions from these other three reagents and that the major peaks in the 15N NMR spectra result from nitrosations. In one instance, for example, acquisition of the 15N NMR spectrum of SRFA was commenced within 20 min of nitrosation at pH 3, and the major resonances were visible within 10 min of acquisition. This assumption will have to be confirmed in future experiments.
The spectrum of the fulvic acid nitrosated at pH 6 (Fig. 4c) differs from the spectrum at pH 3 (Fig. 4b) in two respects. First, the nitrosophenol peak is shifted downfield, so that the peak maximum now occurs at 438 ppm. Secondly, an additional peak is present from 315 to 295 ppm, centered at 306 ppm. The identity of this peak is uncertain. The chemical shift range is characteristic of pyridine-like, imine, and azoxybenzene nitrogens. Several Beckmann reactions of ketoximes may result in the formation of pyridines, isoquinolines, dihydroisoquinolines, and 1
-pyrrolines, all of which would occur in this region (35). Alternatively, a transnitrosation reaction may result in the formation of indophenol N (43):
A related condensation of phenols with nitroso groups can lead to the formation of phenoxazinone compounds (41).
As one might expect, the lesser signal/noise ratio of the spectrum of the sample nitrosated at pH 6 than the spectrum at pH 3 (Fig. 4b and 4c) indicates that less nitrosation has occurred at the higher pH.
The question of whether the activated carbons undergoing nitrosation to form ketoximes could be further differentiated as methylenes or methyls was addressed through methylation analysis of the SRFA. In previous work with humic substances, including Suwannee River and Armadale fulvic acids, single-pulse 13C NMR spectra indicated that significant methylation of activated carbons occurred upon reaction of samples with sodium hydride and methyl iodide (50). Since these activated carbons may include the same substrate sites that undergo nitrosation, the methylation reaction was examined in more detail using 13C NMR subspectral editing techniques. A combination of quaternary C (APT) and DEPTGL 13C NMR spectra were recorded on SRFA sequentially methylated with unlabeled diazomethane and methyl iodide and sodium hydride (K.A. Thorn, 1998, unpublished data). A decrease in methylene carbons concomitant with the appearance of quaternary aliphatic carbons was observed, consistent with methylation of activated methylenes:
Confirmation of activated methylenes as the predominant sites for C methylation provides support that these are also the sites for nitrosation.
N-nitroso groups were not observed in the nitrosated SRFA, nor in the Laurentian and Armadale fulvic acid samples discussed next, samples all with elemental N contents <1% (Table 1). Whereas secondary amines and amides form stable N-nitroso groups upon nitrosation, primary amines and amides usually decompose to N gas and a variety of products (Reactions [10]–[16]). It is possible that nitrosation of primary amines or amides occurred in the samples. However, these reactions would not be detected by 15N NMR because of the escape of the N2 gas (Reactions [13]–[15]). One previous report documented the nitrosative deamination of a peat fulvic acid (59).
The possibility that aromatic nitroso compounds can be further oxidized to aromatic nitro compounds in the presence of excess nitrous acid was stated above (Reaction [9]). If nitro groups are present in the nitrosated fulvic acid, they would occur in the range from 380 to 350 ppm and would thus overlap with the ketoxime nitrogens. In this regard, it is interesting to note that Azhar et al. (60) reported formation of nitro- and nitrosonaphthol upon incubation of naphthol with NO-2 in both sterilized and unsterilized soils. However, in their work, the amounts of NO-2 used would not have lead to excess concentrations of nitrous acid in the soil solutions. Assignments for the 15N NMR spectra are summarized in Table 2
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180 to 90 ppm are diminished in the LFA and just about absent in the Armadale FA compared with SRFA. In the spectrum of the LFA at pH 6, as with SRFA, the nitrosophenol peak is shifted downfield and another peak centered at 306 ppm appears. The concentration of NaNO2 used in the dilute nitrosation of the LFA was 1/200 that of the samples shown in Fig. 5b and 5c. The overall similarity between the spectra of the samples nitrosated at dilute and high concentrations of NaNO2 suggests that high concentrations of NaNO2 do not lead to reaction products that are artifacts.
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90 to 220 ppm represents other secondary and tertiary reaction products. These latter signals are enhanced in the DEPT spectra. The DEPT spectra show only nitrogens directly bonded to protons. In Fig. 6b, all nitrogens bonded to protons are in positive phase. In Fig. 6c, nitrogens bonded to one proton are positive and nitrogens bonded to two protons are inverted. Thus, nitrogens from 90 to 112 ppm can be assigned as doubly protonated (primary amides) and nitrogens from 112 to 180 ppm as singly protonated. Lactams and secondary amides, from Beckmann rearrangements of oximes, would occur in the range from 112 to 133 ppm. As already stated, the primary amides most likely arise from hydrolysis of nitriles. The most interesting feature in the ACOUSTIC spectrum of the nitrosated peat humic acid is the presence of the peak from 530 to 564 ppm, with maximum at 540 ppm, corresponding with N-nitroso nitrogens. These may include N-nitrosamides, from nitrosation of the naturally abundant secondary amide nitrogens in the peat humic acid, or N-nitrosamines, from nitrosation of side chains of amino acids such as tryptophan, indole type nitrogens, or other secondary amines that may occur in the fulvic acid (Reactions [12] and [16]). The observation of N-nitrosation in the peat humic acid but not the Suwannee, Laurentian, or Armadale fulvic acids is consistent with the greater elemental N content of the former (3.74%).
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90–180 ppm) compared with the ACOUSTIC spectrum. Although the potential exists for overlap between the labeled nitrogens from 90 to 200 ppm representing the Beckmann reaction products and the naturally abundant nitrogens from 0 to 200 ppm observed in Fig. 3, because of the much greater concentration of labeled nitrogens, it is unlikely the natural abundant nitrogens contribute to the intensities of the labeled N peaks. The CP/MAS spectrum of the nitrosated peat exhibits the same peaks as the humic acid, although the relative intensities of the peaks differ. For example, the nitrile peak is diminished in intensity with respect to the amide and oxime peaks compared with the humic acid. The nitrosophenol shoulder (390–420 ppm) is not as clearly resolved in the whole peat compared with the humic acid. Most interestingly, the intensity of the peak at 540 ppm indicates that significantly more N-nitrosation has occurred in the whole peat than in the peat humic acid. This may reflect a contribution from nitrosation of the fulvic acid or other N-containing constituents in the whole peat. Future investigations into the nitrosation chemistry of whole soils should consider the occurrence of N-nitrosation reactions.
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Since demethoxylation may be a significant reaction upon nitrosation of humic substances, 13C NMR spectra were recorded on the IHSS soil humic acid before and after reaction with NO-2 to determine if the demethoxylation could be observed spectroscopically. The signal intensity of the methoxyl peak in the unreacted humic acid was greater in polarization transfer spectra than in single-pulse continuous decoupled spectra, and so DEPTGL spectra are shown (Fig. 8) . The methoxyl peak occurs at 56 ppm in the unreacted sample. It is clearly diminished in the nitrosated sample, thus confirming the demethoxylation reaction reported in earlier studies. Demethoxylation was also observed in the nitrosated peat humic acid.
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The reaction of NO-2 with humic substances is one of several potential pathways for the abiotic incorporation of low molecular weight N compounds into soil organic matter. Ammonia, hydroxylamine, and amino acids have all been shown to react chemically with humic substances in laboratory studies. The extent that these reactions occur in soil ecosystems is still unknown. These N compounds react with humic substances to form products that in principle should be distinguishable spectroscopically from N immobilized by soil microbes. Solid state 15N NMR spectra of whole soils in which labeled N in the form of NH3 or NO-3 has been taken up by soil bacteria and fungi and converted into protein and other biochemical constituents would presumably be characterized by sharp amide bands and amino sugars (61, 62). The heterocyclic N resonances diagnostic of humic substances reacted with NH3 or amino acids are diminished, and the oxime and nitrosophenol resonances diagnostic of oximated or nitrosated samples absent. With 15N NMR spectra of nitrosated organic matter now in hand, it should be possible to conduct soil microcosm experiments to confirm nitrosation of organic matter under nitrifying or denitrifying conditions, and differentiate, at least partially, between pathways of microbial immobilization vs. abiotic incorporation into organic matter.
Thus far, oximes, nitrosophenols, and N-nitroso groups have not been observed in the natural abundance 15N NMR spectra of soil organic matter fractions reported in the literature. What implications this has for the significance of nitrosation reactions in soils must remain the subject for future investigations. For now the possibility may be considered that these nitrogens occur in soil organic matter fractions at concentrations below the detection limit of NMR, and that the use of 15N-labeled NH3 or NO-3 amended to soils under documented nitrifying or denitrifying conditions may be necessary to confirm nitrosation of soil organic matter. Alternatively, under genuine nitrifying or denitrifying conditions, oximes or nitrosophenols are formed only as transitory species, subject to attack by successively produced NO-2.
Given the potential combination of reactions of NH3, hydroxylamine, amino acids, and NO-2 with humic substances, the abiotic pathways of trace N gas formation in soils may be complex. The application of 15N NMR in conjunction with high resolution isotope ratio mass spectrometry holds promise for elucidating these mechanisms in greater detail.1; 2; 3; 4; 5; 6; 7; 8; 9; 10; 11; 12; 13; 14; 15; 16; 17; 18; 19; 20; 21; 22; 23; 24; 25; 26; 27; 28; 29; 30; 31; 32; 33; 34; 35; 36; 37; 38; 39; 40; 41; 42; 43; 44; 45; 46; 47; 48; 49; 50; 51; 52; 53; 54; 55; 56; 57; 58; 59; 60; 61; 62; 63; 64; 65
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ACKNOWLEDGMENTS |
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Received for publication January 11, 1999.
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-naphthol in soil suspensions. J. Agric. Food Chem. 1989;37:262-266.This article has been cited by other articles:
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  A. W. Gillespie, F. L. Walley, R. E. Farrell, P. Leinweber, A. Schlichting, K.-U. Eckhardt, T. Z. Regier, and R. I. R. Blyth Profiling Rhizosphere Chemistry: Evidence from Carbon and Nitrogen K-Edge XANES and Pyrolysis-FIMS Soil Sci. Soc. Am. J., October 21, 2009; 73(6): 2002 - 2012. [Abstract] [Full Text] [PDF]
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D. C. Olk Improved Analytical Techniques for Carbohydrates, Amino Compounds, and Phenols: Tools for Understanding Soil Processes Soil Sci. Soc. Am. J., October 30, 2008; 72(6): 1672 - 1682. [Abstract] [Full Text] [PDF]
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