Published May, 2000

Biogenic Nitrogen in Soils as Revealed by Solid-State Carbon-13 and Nitrogen-15

Nuclear Magnetic Resonance Spectroscopy
Heike Knicker*
ABSTRACT
Solid-state nuclear magnetic resonance (NMR) spectroscopy represents a valuable nondestructive alternative to common chemolytic
and thermolytic analytical approaches for characterizing the formation
of humified organic N from biogenic precursors in soils. In this review,
recent studies using solid-state TiN NMR spectroscopy for the examination of the fate of biogenic N in soils are summarized. From their
results it can be assumed that most of the N occurs as peptide-like
structures. Heterocyclic aromatic-N was not identified to a large extent
in naturally humified material but was observed in spectra obtained
from a humic acid of a soil incubated with 15N-labeled trinitrotoluene.
The dominance of amide-N in humified organic N is supported by
the application of dipolar dephasing (DD) solid-state 13C NMR spectroscopy. This technique can be used to estimate the relative content
of N-substituted aliphatic carbons and thus, to calculate the relative
contribution of peptides to the total C and N content of a sample.
Applying this technique to degraded plant and algal material and to
a humic fraction obtained from a natural soil indicates that peptides
comprise more than 80% of the total organic N in the examined
samples. The solid-state 1SN NMR spectrum of a clay fraction of the
Chinese Loess Plateau (age: 10 000 yr) reveals that some peptide-like
material can survive prolonged pedogenesis. Several explanations for
the protection of these commonly thought labile compounds are available and discussed.

OLID-STATE NMR spectroscopy, in particular solidstate 13C NMR, has long been recognized as a powerful tool for examining and characterizing the chemical
structure of soil organic matter (Friind et al., 1994; Kogel-Knabner, 1997; Preston, 1996; Preston et al., 1987,
1994; Wilson, 1987). Solid-state 15N NMR spectroscopy
has only recently been introduced to soil science, possiH. Knicker, Lehrstuhl fur Bodenkunde, Technische Universitat
Miinchen, 85350 Freising-Weihenstephan, Germany. Received
19 Jan. 1999. *Corresponding author (knicker@pollux.edv.agrar.
tu-muenchen.de).
Published in J. Environ. Qual. 29:715-723 (2000).

bly due to particular sensitivity problems not encountered in 13C NMR. The low sensitivity of this technique
may be the reason that most applications of 15N NMR
for characterizing soil organic N are performed with
15
N-enriched plant incubates and soils incubated with
15
N-enriched compounds (Almendros et al., 1991; Benzing-Purdie et al., 1986,1992; Cheshire et al., 1990; Clinton et al., 1996; Preston et al., 1986; Zhuo et al., 1992),
15
N-enriched melanoidins (Benzing-Purdie and Ripmeester, 1983), and model polymers (Preston et al.,
1982). To the best of the author's knowledge, Preston
et al. (1986) presented the first solid-state 15N NMR
spectrum of humic material at natural 15N levels from
a peat.
This review summarizes some recent results of studies
using solid-state 15N NMR spectroscopy for investigating
the chemical structure of humified biogenic N. It is further intended to introduce the reader to DD solid-state
13
C NMR spectroscopy as a possible tool for estimating
the relative contribution of peptide-like structures to
humified material. A comparison of the peptide content,
calculated from such experiments with that obtained via
solid-state 15N NMR spectroscopy, was performed to
test the reproducibility of the latter.
MATERIALS AND METHODS
Sample Material
Nitrogen-15-enriched wheat (Tritium aestivum L.: 17 atom
% 15N) (Knicker and Ludemann, 1995) and 15N-enriched algal
residues from a mixed algal culture (Knicker et al., 1996b)
were mixed with quartz sand (6:100) and kept at 25C at 60%
water3 holding capacity. The samples were inoculated with
1 cm of an aqueous extract from a natural compost. After
distinct periods of incubation the residual organic material
Abbreviations: NMR, nuclear magnetic resonance; DD, dipolar dephasing; CPMAS, cross-polarization magic angle spinning; SPE, single
pulse excitation.

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J. ENVIRON.QUAL., VOL. 29, MAY-JUNE

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was mechanically separated from the quartz sand and the

quartz washedseveral times with triply distilled waterin order
to separate the residual organic fraction. This washwas combined with the mechanicallyseparatedsolid parts and then lyophilized.
Humicmaterial with natural lSN abundancewas extracted
from the A horizon of a Chromo-CalcicCambisol (forest)
close to GOttingen,Germany
by mixing 20 g of the soil with
60 g 0.5 Maqueoussodium hydroxide (FrOnd and LiJdemann,
1991).Aftersonification of the dispersion for 5 rain, the mixture was immediatelycentrifuged until the supernatant liquid
becamefree of solid material. The extraction process was
repeated four times. The supernatant liquid was dialyzed
against distilled water and subsequentlyfreeze dried.
The samplematerial of the Santa BabaraBasin was supplied
by Dr. T. Filly (PennsylvaniaState University) and the clay
fraction of the Chinese Loess Plateau was donated by Dr.
B.K.G. Theng (Landcare Research, NewZealand). To increase the sensitivity of these twosamplesfor solid-state
NMRspectroscopy their organic material was enriched by
reducingthe mineral matter content after extraction with 10%
hydrofluoric acid according to the method described by
Schmidtet al. (1997).

excitation (SPE) ~N NMR

experiments, a pulse delay of
s was used. Between500 and 6000 scans were accumulated.
A line broadening of 150 to 200 Hz was applied before Fourier transformation.
SOLID-STATE
CARBON-13
NUCLEAR
MAGNETIC RESONANCE
SPECTROSCOPY ON NITROGENCONTAINING PRECURSORS OF SOIL
ORGANIC MATTER
In biogenic precursors most of the N is bound in
peptides and aminoacids, resulting in typical signals in
distinct chemical shift regions of a solid-state 13C NMR
spectrum as it is shownfor casein in Fig. 1A. Like most
solid-state ~3C and ~N NMRspectra of soils and soilrelated samples, this spectrum was obtained with the
CPMAStechnique (Schaefer and Stejskal,
1976)
which the magnetization is transferred from the
spin-system to that of the ~3C isotope with subsequent
detection of the 13C magnetization. Compared with a
direct observation of 13C with the SPEpulse sequence,
this technique increases the sensitivity of 13C nuclei for
solid-state NMRspectroscopy and greatly reduces the
measurement time needed to receive spectra with acceptable signal-to-noise
ratios. In the solid-state
CPMAS13C NMRspectrum of casein, the C carbon of
all amino acids except glycine (42 ppm) contributes
the signal in the chemical shift region between 60 and
45 ppm(Kalinowski et al., 1984). Another signal, indicative for N-substituted C, is found at 173 ppm and is
assigned to amide carbons. The carbons of the aliphatic
side chain of aminoacids contribute to the intensity in
the chemical shift region between 45 ppm and 0 ppm.
The signals between 140 ppmand 110 ppm are attributed to the unsubstituted aromatic C occurring in phenylalanine, thyrosine, thrypthophan, and histidine. The
phenolic C of thyrosine is observed at 156 ppm.
In the solid-state CPMAS
13C NMRspectra of algal
material that was degraded for 2 mounder aerobic conditions (Knicker et al., 1996b) (Fig. 1B), signals
deriving from peptides and amino acids are overlapped
by resonance lines originating from carbons of other
biomolecules. Signals of carbohydrates, carbons linked
in ether bonds, and C substituted by hydroxyl groups
appear in the chemical shift region between 110 and
60 ppm. Resonance lines of unsaturated C in aliphatic
structures mayadd to the signal intensity in the chemical
shift region between 160 and 110 ppm. Signal intensity
of carboxyl groups in fatty acids and esters is observed
in the chemical shift region between 185 and 160 ppm.

Nuclear Magnetic Resonance Spectroscopy

The solid-state 13CNMR
spectra were obtained on a Bruker
DSX200 (Karlsruhe, Germany)operating at a frequency
50.3 MHzusing zirconium rotors of 7 mmODwith KEL-Fcaps. The cross- polarization magic angle spinning (CPMAS)
technique (Schaefer and Stejskal, 1976) was applied during
magic-anglespinning of the rotor at 6.8 kHz. A ramped~Hpulse was used during contact time in order to circumvent
spin modulation of Hartmann-Hahnconditions (Peersen et
al., 1993). A contact time of 1 ms and a 90 ~H-pulsewidth
of 4.3 t~s wereusedfor all spectra. The~3C-chemical
shifts were
calibrated to tetramethylsilane (=0 ppm). Between10 000 and
60 000 scans were accumulatedusing a pulse delay of 400 ms
(FrOndet al., 1989). Prior to Fourier transformation, a line
broadeningof 0 to 20 Hzwas applied. Relative C distribution
wasdeterminedby integration of signal intensity in the various
chemicalshift regions, given in Table 1, via an integration
routine supplied with the instrument software.
The DDexperiments (Wilson, 1987) were performed
applying the rampedcross-polarization technique (Peersen et
al., 1993)and inserting a DDdelay (tdd = 68 ~S) during which
the ~Hdecouplerwasturned off betweenthe cross-polarization
and acquisition portions of the CPMAS
pulse sequence. A 180
refocusingpulse also wasinserted at 1/2 tdd to assist phasing.
The solid-state CPMAS
15N NMR
spectra were obtained
on a Bruker DMX
400 operating at 40.56 MHzand applying
a contact time of 0.7 ms, a 90 pulse widthof 5 p.s, a pulse
delay of 150 ms, and a line broadening of 200 Hz. Between
1.5 million and 3 million scans were accumulatedat a magicangle spinning speedof 5.5 kHz.The chemicalshift is referenced to the nitromethane scale (=0 ppm)and was adjusted
with ~Nlabeled glycine (-347.6 ppm). For the single pulse
Table 1. Relative intensity distribution
Sample
Casein
Degraded algae
Wheat(58 d)
Wheat(4 yr)
NaOH-extract
(forest soil)

CarbonyI-C
(220/185 ppm)

of the solid-state
Carboxyl]Amide-C
(1851160 ppm)

CPMAS NMRspectra shown in Figure 1.

Aromatic-C
(1601110 ppm)

O-AlkyI-C
(110/60 ppm)

OCH3,N-Aliphatic-C
(60/45 ppm)

AlkyI-C
(45/0 ppm)

0
0
1
1

22
12
5
7

10
11
12
17

8
19
63
40

19
10
8
12

41
48
11
23

10

10

43

12

22

KN1CKER:BIOGENICN IN SOILS AS REVEALED

BY 13C AND1SN NMR

Between 45 and 0 ppm one can find contributions of

their aliphatic chains.
The overlapping becomes even more pronounced in
a solid-state
CPMAS13C NMRspectrum of degraded
plant residues, due to the presence of lignin. As shown
in Fig. 1C, 1D and 1E, in the spectra of wheat incubated
for 58 d (Knicker et al., 1996a)and for 4 yr, respectively,
under water saturation conditions, contribution of lignin-derived C can be found over the whole chemical
shift region between 220 and 0 ppm. Methoxyl-substituted carbons in lignin result in a narrow line peaking
at 56 ppm and may overlap those deriving from
N-substituted aliphatic carbons. Contributions of the
aromatic core of lignin occur in the chemical shift region
between 160 and 110 ppm. The fact that signals of
CPMAS

DD-CPMAS

Casein

DegradedAlgae

Degraded Wheat
58 days

Degraded Wheat
4yearn

Llgnin

NaOH- Extract
(forest soil)

/~

3~0
2~)0 1~)0
0
pprn
ppm
Fig. 1. Solid-state 13Cnuclear magnetic resonance (NMR)spectra
casein, degradedalgae and wheat, lignin, and a sodiumhydroxide
extract of a forest soil (Cromo-CalcicCambisol)obtained without
an interruption delay tDi) (A-F) and a tDi) = 68 (K-L). The
dipolar dephasing (DD)--cross- polarization magic angle spinning
(CPMAS)NMR
spectra are slightly enlarged in order to be able
to recognize the peaks.

717

O-substituted carbons ovedap signals of N-substituted

carbons makes it difficult to obtain information about
the nature of nitrogen-containing compoundsin the heterogeneous mixture of biogenic residues by the single
use of solid-state 13C NMRspectra obtained with the
standard cross-polarization technique.
Dipolar Dephasing Carbon-13 Nuclear Magnetic
Resonance Spectroscopy
Applying a technique known as DD solid-state
CPMASNMRspectroscopy allows a first estimation
about the contribution of amino acids and peptides to
the overall C content of a heterogeneous mixture. This
technique takes advantage of the different behavior of
carbons exhibiting strong or weakdipolar coupling with
hydrogens during a CPMASNMRexperiment.
In a DDexperiment, the proton-decoupling is interrupted for a certain time delay (interruption delay
tad). In the spectrum the carbons, effected by strong
proton dipolar coupling, exhibit a loss of intensity with
increasing tdd. Such carbons (e.g., aliphatic carbons) are
bound to amino groups. Carbons without directly
attached hydrogens and carbons with high molecular
motion levels (i.e., in terminal methyl groups, methoxyl
groups, and long aliphatic chains) experience weak proton dipolar coupling. In contrast to signals of carbons
with strong dipolar interactions, their signals will still
be visible in a spectrum obtained with tad > 68
The solid-state
DD CPMAS13C NMRspectrum of
casein (Fig. 1G) obtained with tdd = 68 IXS shows no
signal intensity between 60 and 40 ppm, indicating that
this signal originates from carbons with efficient dipolar
coupling, as it is expected for aliphatic C substituted
with amino groups. The signals between 40 and 30 ppm
exhibit the same fate, allowing an assignment to unsaturated carbons in short aliphatic chains. The resonance
between 20 and 0 ppm derives from mobile terminal
methyl carbons with weak dipolar interactions. While
the signal around 116 ppmin the aromatic region has
completely vanished, some intensity is observable for
the peaks at 129, 136, and 156 ppm, indicating the presence of some nonprotonated aromatic carbons. The
strong signal intensity in the chemical shift region between 185 and 160 ppm is expected for carboxylic
carbons.
In the solid-state
DDCPMAS13C NMRspectrum of
lignin (Fig. 1K) obtained with tdd = 68 IXS, signals of the
propanyl side chains and the intensity between 140 and
110 ppm(unsubstituted aromatic C) experiencing strong
dipolar interactions with neighboring protons, vanish.
However, the signals assignable to O-substituted aromatic or quaternary aromatic C are still visible. In contrast to that of the casein, the DD CPMAS13C NMR
spectrum of lignin showsa strong signal at 56 ppm, which
is typical for mobile methoxyl carbons. This particular
behavior of methoxylic C during a DD experiment
allows a distinction between signal intensity deriving
from these groups and those originating from N-substituted aliphatic C in a heterogeneous mixture such as
plant incubates and soils. For example, in the solid-state

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J. ENVIRON.QUAL., VOL. 29, MAY-JUNE

2000

DD CPMAS13C NMRspectra of the wheat incubates

(Fig. lI, 1J), the signal between 60 and 45 ppmshows
that methoxyl groups, most probably from lignin, are
present.
The DD-experiments on a number of model compounds and humic substances have shown that the ~ignal
intensity decays with respect to the interruption delay
tad according to Eq. [1] (Wilson, 1987).
I(tdd) = IA(tad) IB(td~) = IA(0) (e xp - ~/2 D~z)
+ IB(0) (exp t~ /D~)

[1]
whereI(t~d) is the total signal intensity determined in a
specific chemical shift region at the dephasing time t~a.
IA(0) is the initial signal intensity of carbons experiencing strong dipolar interactions and I~(0) is that of carbons with weak dipolar coupling. D~. and D~ represent
D~ their corresponding time constants for DD.
Applying this equation to the signal intensity decay
in the chemical shift region between 60 and 45 ppmof
the DD CPMAS13C NMRspectra of casein and the
degraded algae, obtained from a set of experiments with
increasing t~, a D~of 24 Ixs was calculated for N-substituted aliphatic carbons. Respectively, for that of the
solid-state
DD CPMAS
~3C NMRspectra of the lignin
a D~ of 44 p~s was determined for the methoxyl C of
lignin. Applying Eq. [1] with D~ = 24 Ixs and D~ = 44
Ixs to the intensity decay determined from the DD
CPMAS13C NMRspectra of the degraded wheat samples, a relative contribution of N-substituted aliphatic
C to the total signal intensity of 2.3% and 5.3% was
calculated (Tables 1 and 2).
Under the approximation that casein represents a
chemical composition comparable with other proteins,
further calculations can be performed with these results.
As shownin Table 1, the relative intensity in the chemical shift region between 185 and 160 ppmin the solidstate CPMAS13C NMRspectrum of casein comprise
approximately 1.2 times of that determined for the region between 60 and 45 ppm, approximately half of that
between 45 and 0 ppm, and 22%of the total C content.
These ratios can be used for the estimation of peptide
content in a heterogeneous mixture from a solid-state
CPMAS~3C NMRspectrum.
Applying these calculations to the solid-state CPMAS
~C NMRspectrum of the degraded algal material, almost all of the signal intensity between 185 and 160

ppmcan be assigned to amide-C [12% of total C (Ct)].

Thus, approximately half of the signal intensity in the
aliphatic region between 45 and 0 ppmis attributed to
the chain-C of peptides (24% of Ct). The remaining
intensity in this region may be assigned to long chain
aliphatic structures.
Such compounds were found to
form the refractory biopolymer of some algae, the algaenan, which is expected to survive extended sediment
diagenesis (Hatcher et al., 1983).
Considering further that in the spectrum of casein the
amide region comprises 22%of the total signal intensity,
the relative contribution of peptides to Ct comprises
55%, demonstrating that more than half of the total
organic C is formed by peptide-like material.
In a further calculation, the relative contribution of
amide-N (Nv) in percent of total N (Nt) shall be estimated using the ratio of the total C intensity I0 to the
intensity originating from amide-C (Ip) and the atomic
C/N ratio of the sample according to Eq. [2]:
Uv = [(C/N)/(Io/Io)] 100
[2]
From integration of the spectrum of the degraded algae,
a Io/Ip = 8.3 was obtained (Table 2). For the degraded
algae an atomic C/N ratio of 7 was measured (Knicker
et al., 1996b). According to Eq. [2], 84%of Nt is found
in amide functional groups (Table 2). A comparable
calculation is performed for the wheat samples. These
calculations reveal a relative increase of peptide-C from
13%of the Ct in the sample after 58 d of incubation to
29%in the sample after 4 yr of incubation. This is in
accordance with the strong decrease of the atomic
C/N ratio from 30.2 to 8.3, respectively, with increasing
incubation time. However, the low C/N ratio of the
wheat incubate after 4 yr is not completely explained
with the relative increase in peptide-N, as it is revealed
from the calculation of the relative contribution of amide-N to Nt. Only 53%of Nt are assignable to amideN. This indicates that during prolonged incubation some
of the N was chemically transformed from mainly amide-N to other N-containing compounds.
Performing these calculations for a sodium hydroxide
extract of a forest soil (Chromo-CalcicCambisol) (Fig.
1F, 1L), approximately 72%to the intensity in the chemical shift region between 60 and 45 ppmof the solidstate CPMAS~3C NMRspectrum was determined to
originate from N-aliphatic carbons (Table 2). The estimated relative contribution of peptide-C to C~ of 36%

Table 2. Relative contribution of amides to the total nitrogen (Nt) content of degraded biogenic precursors (Knicker al. , 199 6a,b)
and a sodium hydroxide extract of a forest soil (Chromo-Calcic Cambisol) (Friind al. , 199 4).
Sample

C/N

IA~of I0~:

Degradedalgae
Degradedwheat (58 days)
Degradedwheat (4 years)
NaOH-extract
(forest soil)

7.0
30.2
8.3
8.5

10.0
2.3
5.3
8.6

I~ of I0

Ip~l of I0

I0/I~

Peptide-C/Ct#

12.0
2.8
6.4
10.3

8.3
35.7
15.6
9.7

55
13
29
39

%
0
5.7
6.7
3.4

Amide-NJN~
(calculated)
%
84
85
53
88

The intensities of N-substituted carbonsof the solid-state CPMAS

~C NMR
spectra as they were determinedaccordingto Equation[1].
Total signal intensity of the solid-state ~C NMR
spectrum
The intensities of OCH~
of the solid-state CPMAS
~C NMR
spectra as they were determinedaccording to Equation [1].
Calculatedfrom1Amultiplied with 1.2 accordingto the relative intensity distribution of the CPMAS
~3C NMR
spectrumof casein.
Estimated fromlp divided by the relative intensity between185 and 160 ppmof the solid-state CPMAS
~3C NMR
spectrumof casein.
"~" Determinedfrom the SPEt~N NMRspectrumshownin Fig. 2.

Amide-N/Nt
(~SN NMR)
84
88
82 (60~~)
82

KNICKER:BIOGENICN IN SOILS AS REVEALED

BY ]3C AND]SN NMR

strongly indicates that peptides comprise a considerable

fraction in humic material.

719

CPMAS ~N:NMR

Nitrogen-15 Nuclear Magnetic Resonance

Spectroscopy on Degraded Algae
and Degraded Wheat
As mentioned above, solid-state ]3C NMRspectroscopy is a powerful technique to characterize the chemical composition of the C fraction of a heterogeneous
organic mixture. Using solid-state
DD CPMAS]3C
NMRspectroscopy can even result in a first estimation
of the relative contribution of peptides to degraded biogenie material. However, for the investigation of the
chemical transformation of biogenic N occurring during
humification, solid-state
CPMAS15N NMRspectroscopy can provide more detailed data.
Applying this technique to casein with natural lSN
levels results in a spectrum that is dominatedby a signal
between -220 and -285 ppm, peaking at -260 ppm
(Fig. 2A). It is assigned to amide-Nand comprises more
than 80%of the total I~N signal intensity of the spectrum. A further signal is identified at -347 ppmand
originates from amino groups of terminal amino acids.
These signals also are observed in the solid-state
CPMAS15N NMRspectra of ~SN-enriched undegraded
algae and wheat (Fig. 2B-C). Further, less intense signals that were not seen in the spectrum of casein, due
to its low signal-to-noise ratio, are observable between
-285 and -325 ppm and between -145 and -230 ppm.
The first can be assigned to NH2or NR2of guanidines
or the additional amino groups of some basic amino
acids. The shoulder between -145 and -230 ppm can
derive from the N in Position 9 of purines, indoles,
imidazoles (e.g., histidine), or pyrrole-like compounds.
The signals denoted by asterisks are spinning sidebands
of the amide signal. They arise due to incomplete removal of the chemical anisotropy by insufficient high
magic-angle spinning of the sample at higher magnetic
field strength.
The solid-state CPMAS
~5N NMRspectra of the 15Nenriched degraded algae (Fig. 2D) (Knicker et al.,
1996b) and the wheat incubates (Fig. 2E, 2F) (Knicker
et al., 1996a) showa similar feature to those obtained
from undegraded biogenic precursors. The additional
signal between 25 and -25 ppmin the solid-state lSN
NMRspectrum of the wheat incubated for 4 yr and that
between -350 and -375 ppm in the ~SN NMRof the
degraded algae reveal the presence of inorganic nitrate
and ammonium,respectively,
formed during ammonification and nitrification.
Comparablewith the spectrum
of casein they are dominated by the peak at -260 ppm,
showing that even after prolonged microbial degradation amide-N is the main form of organic N and comprises between 80%and 90% of the total intensity of
the spectrum. However, one has to bear in mind that
in these spectra N in acetylated amino sugars, lactams,
unsubstituted pyrroles, indoles, and carbazoles also may
contribute to the downfield shoulder (-145 to -230
ppm) of the main signal (Witanowski et al., 1993).
the degraded algae and the wheat incubated for 58 d,

Casein

Fresh Algae

Fresh Wheat

Degraded Algae

Degraded Wheat
58 days

4 years

(SPE)4 years

-100

-200 -300 -400

ppm
Fig. 2. Solid-state cross-polarization magic angle spinning (CPMAS)
I~N nuclear magnetic resonance (NMR)spectra of casein (A), undegraded algae (B) and wheat (C), and degradedalgae (D) and
(E, F). For comparison, the single pulse excitation (SPE) ~N NMR
of the wheat incubated for 4 yr (G) is shown.

the relative signal intensity of amide-Nis in accordance

to the amide-N content calculated from the DDexperiments, as discussed above. This is consistent with results
of previous comparative studies using solution and solidstate CPMAS~N NMRspectroscopy of soluble humic

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J. ENVIRON.QUAL., VOL. 29, MAY-JUNE

2000

fractions of plant incubates showing that solid-state

CPMAS~SN NMRspectroscopy can be used as a quantitative means for determining the chemical structure
of organic N during humification (Knicker and Ltidemann, 1995).
An exception to this trend were the results from wheat
degraded for 4 yr. In this case, the amide-Ncontent of
82% (Table 2) obtained from the solid-state
CPMAS
15N NMR
spectrum is muchhigher than it was calculated
with Eq. [2]. The solid-state CPMAS
15N NMRspectrum
seems not to reveal the true N-contribution of different
N-containing compounds in this sample. This may be
explained by underestimation of N that is not in direct
vicinity of IH nuclei. Due to their missing or weakcoupling to the ~H spin system their signal may not occur
or may be diminished in a solid-state CPMAS
~SN NMR
spectrum. Such N can be bound in six-membered aromatic ring structures, imines, or nitriles, structures which
are commonlysuggested to represent stabilized soil organic N formed during humification (Anderson et al.,
1989; Flaig et al., 1975; Kelly and Stevenson, 1996;
Schnitzer, 1985; Schulten and Schnitzer, 1993). Signals
of these compoundsare expected to occur in the chemical shift region between -25 and -150 ppm, but remained unidentified in previously accumulated solidstate CPMAS15N NMRspectra of humified organic
matter formed via biogenic humification of biological
resources (Bedrock et al., 1998; Clinton et al., 1996;
Knicker and Liidemann, 1995; Knicker et al., 1997;
Knicker et al., 1996b). On the other hand, a weak and
broad signal is observed in the chemical shift region
between -25 and -90 ppm in the solid-state
CPMAS
~SN NMRspectrum of a humic acid of a soil incubated
with ~SN-labeled trinitrotoluene (Knicker et al., 1999a)
(Fig. 3A). A solid-state
~SN NMRspectrum obtained

from the same sample but with SPE (Fig. 3B), revealed
no major intensity loss due to cross-polarization. During
this experiment, also knownas Bloch Decay, the ~N spin
system is directly polarized. Comparablewith solution
NMR
spectroscopy, the signal intensity distribution in
such spectra is expected to give a true reflection of the
chemical composition of the examined samples, under
the premise that saturation effects are avoided. This
result indicates that in spite of its weakinteraction with
the ~H spin system such N in humic material may still
be detectable via CPMAS~SN NMRspectroscopy.
To test whether the wheat incubated for 4 yr contains
N-containing heterocyclic
six-membered aromatic
structures or imines the sample was subjected to solidstate SPE ~SNNMR.In the spectrum shown in Fig. 2G,
signals of amides and free amino groups are observed.
Resonance lines between -25 and -150 ppm cannot
be distinguished from the noise, showing that heterocyclic six-memberedaromatic N, imines, and nitriles are
not formed to a larger extent during the humification
of wheat residues. Thus, the presence of such compounds cannot explain the contradicting results for the
amide-Ncontent of the sample obtained via solid-state
CPMASt~N NMRand DD CPMAS~3C NMRspectroscopy. A possible explanation maybe the high concentration of nitrate and ammonium,revealed by the intense
signals around -3 ppm and -358 ppm, respectively,
in the SPE aSN NMRspectrum of this sample. Their
underestimation in the CPMAS~SN NMRspectrum can
be explained with their high mobility and their weak
interactions with the 1H spin system resulting in inefficient cross- polarization. This experiment supports earlier assumptions that with the commonparameter setup used for the identification of organic forms in soils
and soil-related samples, inorganic N forms may not be
quantitatively determined (Knicker, 1993; Knicker and
Ltidemann, 1995). Integration
of the SPE aSN NMR
spectrum reveals that in this sample, inorganic N comprises more than 30% of the N fraction. The amide-N
content (60%) obtained from this spectrum is comparable with that obtained via calculation from DDCPMAS
~3C NMRspectrum. It can be concluded that, in spite
of underestimation of inorganic N, the solid-state
CPMAS~N NMRspectrum of the wheat incubated
for 4 yr gives a reasonable reflection of the chemical
composition of its organic N forms.
Nitrogen-15 Nuclear Magnetic Resonance
Spectra of Natural Soils

260
ppm
Fig. 3. Solid-state cross-polarization magic angle spinning (CPMAS)
(A) and single pulse excitation (SPE) (B) lSN nuclear magnetic
resonance (NMR)spectra of a humic acid of a soil incubated with
lSN-trinitrotoluene (Knickeret al., 1999a).

The solid-state
CPMASI~N NMRspectrum of the
NaOH-extract of a Chromo-Calcic Cambisol at natural
~N abundance is shown in Fig. 4A. It is surprisingly
similar to those obtained from undegraded soil organic
matter precursors and their decomposedresidues. Comparable spectra were obtained from other natural soils
and their humicfractions (Knicker et al., 1993). Integration of the spectrum revealed that the intensity in the
amide-N region comprises approximately 82% (Table
2). This value is in accordance with that calculated via
Eqs. [1] and [2], demonstrating that this solid-state

KNICKER:BIOGENICN IN SOILS AS REVEALED

BY ~3C AND~SN NMR

CPMAS
15N NMRspectrum gives a fairly correct reflection of the chemical composition of the N compounds
in these samples.
Comparable with the solid-state 15N NMRspectra of
the incubated wheat, no signals are observed in the
chemical shift region of six-memberedheterocyclic aromatic N or imines. Considering the low signal/noise ratio, the broad lineshape of the resonances, and the shoulder in the region of pyrroles, heterocyclic aromatic N
does not contribute more than 10%of the total N signal
intensity. It can be concluded that such compoundsdo
not accumulate to detectable levels during soil organic
matter formation.
The dominance of the amide signal also is observed
in solid-state
CPMAS15N NMRspectra of a marine
sediment from the Santa Babara Basin (USA) (Fig. 4B),
the Torreblanca peat (Spain) (Knicker et al., 1996a)
and an algal sapropel from Mangrove Lake (Bermuda)
(Knicker and Hatcher, 1997). Thus, the persistence
amide functional groups during maturation is not limited to well-aerated soils but also can be observed in
environments with reducing conditions.
POSSIBLE
MECHANISMS LEADING TO
THE SURVIVAL OF SOME PEPTIDES
DURING HUMIFICATION
One possibility for explanation of the high proportion
of amide functional groups in soils and recent sediments
could be a continuous de novo synthesis of microbial
proteins from N released during the breakdown of decaying organic material. Such proteinaceous materials
are commonly thought to be completely hydrolyzed
after treatment with hot 6 M HCI. Subjecting soils
(Knicker and K6gel-Knabner,1998; Siebert et al., 1998)
and organic-rich sediments (Knicker and Hatcher, 1997)
to hot acid hydrolysis with subsequent 15N NMR
spectroscopic examination of the extraction residue revealed
that at least someof the amides, but also somealiphatic
amines, survive this harsh treatment. Whenthe newer
technique of thermochemolysis with tetramethylammonium hydroxide (TMAH) was applied to the HC1hydrolysis residues, products comparable with those obtained after TMAH
thermochemolyis of albumin were
identified, supporting the assumption that some peptides remained in the residue after HCI hydrolysis
(Knicker and Hatcher, 1997; Siebert et al., 1998). From
these results it was concluded that the commonchemical
tests for the identification of nitrogenous compounds
used in previous studies did not detect all proteinaceous
components, perhaps because they rely on extraction
of protein and amino acids into solution and some of
these compoundsare tightly bound to a nonextractable
phase (Knicker and Hatcher, 1997).
Another explanation for the high amide content in
humified material may be the protection of proteinaceous material during humification via adsorption onto
the mineral phase. The potential implication of the fine
silt and clay fraction in protecting peptides from microbial or chemical degradation was recently demonstrated
with solid state CPMAS15N NMRspectra of the fine

721

particle size fractions of different soils (Knicker et al.,

1999b; K6gel-Knabner et al., 1996). Comparable with
the solid-state
CPMAS
lSN NMRspectrum of the clay
fraction of the 10 000-yr-old Loess Plateau in China(Fig.
4C), most of their signal intensity is observed in the
chemical shift region assigned to amide-N. No signals
of six-memberedheterocyclic aromatic N or imines were
identified, which could confirm the catalytic involvement of clay minerals in the formation of Maillard products (Hedges, 1988).
As aforementioned, peptide-like material also was
identified in solid-state
CPMAS~5N NMRspectra of
clay-flee plant incubates and the algal sapropel of Mangrove Lake with a mineral content of less than 10%
w/w(Knicker et al., 1996b). These studies strongly suggest the existence of additional processes other than
mineral adsorption and protection, which are responsible for the survival of peptide-like structures. A possible
explanation for the survival of such structures in these
samples maybe their association with refractory biomolecules. Evidence for this was recently obtained from
solid-state CPMAS
lSN NMRspectra of fungal melanins
(Knicker et al., 1995) and the refractory biopolymer
algae, the algaenan (Knicker et al., 1996b). The latter
was suggested to protect labile organic compoundsfrom
CPMAS15N-NMR

NaOH
- Extract

(fore~tA

soil)

SantaBarbaraBasin

(28-40cm
Age: 10 000yij
I

-100 -200 -300 -400 -500

ppm
Fig. 4. Solid-state cross-polarization magic angle spinning (CPMAS)
lSN nuclear magnetic resonance (NMR)spectra of a sodiumhydroxide extract of a forest soil (Cromo-CalcicCambisol) (A), marine
sedimentof Santa BabaraBasin (California) (B), and a clay fraction
of the Chinese Loess Plateau (C).

722

J. ENVIRON. QUAL., VOL. 29, MAY-JUNE 2000

enzymatic attack during sediment diagenesis by encapsulation in their hydrophobic network (Knicker and
Hatcher, 1997). It also was considered that parts of the
algal cell walls are involved in the protection and that
labile compounds may become sandwiched between algaenan layers. Although algae may not present a major
fraction of soil biota, comparable structures may be
present in the cell walls of soil bacteria.
Summarizing the results concerning the structure of
immobilized organic N in soils obtained via solid-state
15
N NMR spectroscopy, it can be assumed that the formation of heterocyclic aromatic N is of less importance
in soil organic N stabilization than formerly thought.
Although several solid-state 15N NMR spectroscopic
studies clearly indicated that some peptide-like structures can resist microbial degradation over prolonged
humification both in soils and sediments, at this point
of the research, the results do not confirm a specific
pathway for their survival. Much work is still necessary
to clarify the question of how some peptide-like structures of biogenic precursors of soil organic matter resist
both microbial and chemical degradation.

MARTENS: MANAGEMENT AND CROP RESIDUE INFLUENCE SOIL AGGREGATE STABILITY

723