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Biogenic Nitrogen in Soils asRevealed by Solid-State Carbon-13
Nuclear Magnetic
Resonance
Spectroscopy
Heike
Knicker*
ABSTRACT
Solid-state nuclear magnetic resonance (N MR ) spectroscopy repre-
sents
a valuable nondestructive alternative to common chemolytic
and
thermolytic analytical
approachesfor
characterizing
the
formation
of
hum ified organic
N
from biogenic p recursors
in
soils.
In
this review,
recent
studies using solid-state
T i
N NMR
spectroscopy
for the
exami-
nation 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 m aterial
but was
observed
in
spectra obtained
from
ahumic acidof asoil incubated with
15
N-labeled trinitrotoluene.
Th e
dominance
of
amide-N
in
humified organic
N is
supported
by
the
application
of
dipolardephasing(DD)
solid state
13
C N M R
spec-
troscopy. This technique can be used to estimate the relative content
of N-substituted aliphatic carbons andthus, to calculatetherelative
contribution
of peptides to the total C and N content of a sample.
App lying this technique to d egraded plant and algal material and to
a
hum ic fraction obtained from
a
natural soil indicates that peptides
comprise more than 80 of the total organic N in the examined
1S
bly due to
particular
sensitiv
teredin
1 3
C
N M R .
The low
s
m ay be the reason that most a
fo r
characterizing
soil organic
15
N-enriched plant
incubates
15
N-enriched
compounds (Alm
ing-Purdie et al.,
1986,1992; C
ton
et
al., 1996;
Preston et
al.
15
N-enriched melanoidins (B
meester,
1983),
nd model
1982).To the best of the auth
et al. (1986) presented the
spectrum of
humic mater ia l
a
peat.
This
review summarizes
som
using
solid-state
15
N NMR
spec
the chemicalstructureof
hum
ther
intendedto introducethe
Published May, 2000
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J. ENVIRON.QUAL., VOL. 29, MAY-JUNE000
was mechanically separated from the quartz sand and the
quartz washedeveral times with triply distilled water n order
to separate the residual organic fraction. This washwas com-
bined with the mechanically eparated solid parts and then ly-
ophilized.
Humicmaterial with natural lSN abundancewas extracted
from the A horizon of a Chromo-CalcicCambisol (forest)
close to GOttingen,Germanyy mixing 20 g of the soil with
60 g 0.5 Maqueous sodium hydroxide (FrOnd and LiJdemann,
1991).After onification of the dispersion for 5 rain, the mix-
ture was immediately entrifuged until the supernatant liquid
became ree of solid material. The extraction process was
repeated four times. The supernatant liquid was dialyzed
against distilled water and subsequently reeze 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 in-
crease the sensitivity of these twosamples or solid-state
NMRpectroscopy their organic material was enriched by
reducing he mineral matter content after extraction with 10%
hydrofluoric acid according to the method described by
Schmidt t al. (1997).
excitation (SPE) ~N NMRxp
s was used. Between500 and
A line broadening of 150 to 2
rier transformation.
SOLID-STATE CAR
MAGNETIC
SPECTROSCOPY
CONTAINING PREC
ORGANIC
In biogenic precursors
peptides and aminoacids, res
distinct chemical shift regi
spectrum as it is shownfor
solid-state ~3C and ~N NM
related samples, this spec
CPMASechnique (Schaefer
which the magnetization is
spin-system to that of the
detection of the 13C magn
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KN1CKER:BIOGENICN IN SOILS AS REVEALED Y 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 CPMAS 3C NMR pectrum 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 lig-
nin-derived C can be found over the whole chemical
shift region between 220 and 0 ppm. Methoxyl-substi-
tuted 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
O-substituted carbons ovedap
carbons makes it difficult to
the nature of nitrogen-contain
erogeneous mixture of biogeni
use of solid-state 13C NMR
standard cross-polarization te
Dipolar Dephasing Carbon
Resonance Spe
Applying a technique kn
CPMASNMR pectroscopy allo
about the contribution of am
the overall C content of a he
technique takes advantage of
carbons exhibiting strong or
hydrogens during a CPMA
In a DD xperiment, the pro
rupted for a certain time del
tad). In the spectrum the car
proton dipolar coupling, exhib
increasing tdd. Such carbons (e
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J. ENVIRON.QUAL., VOL. 29, MAY-JUNE000
DD CPMAS
3C
NMR pectra of the wheat incubates
(Fig. lI, 1J), the signal between 60 and 45 ppm shows
that methoxyl groups, most probably from lignin, are
present.
The DD-experiments on a number of model com-
pounds 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]
where 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 experienc-
ing strong dipolar interactions and I~(0) is that of car-
bons 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 ppm of
the DD CPMAS 3C NMR pectra of casein and the
degraded algae, obtained from a set of experiments with
ppm can be assigned to amide
Thus, approximately half of t
aliphatic region between 45
the chain-C of peptides (24
intensity in this region may
aliphatic structures. Such
form the refractory biopolyme
nan, which is expected to s
diagenesis (Hatcher et al., 19
Considering further that in
amide region comprises 22%of
the relative contribution of
55%, demonstrating that mo
organic C is formed by peptid
In a further calculation, th
amide-N (Nv) in percent of to
mated using the ratio of the
intensity originating from a
C/N ratio of the sample acco
U
v
= [ C/N)/ Io
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KNICKER: BIOGENICN IN SOILS AS REVEALED Y ]3C AND SN NMR
strongly indicates that peptides comprise a considerable
fraction in humic material.
Nitrogen-15 Nuclear Magnetic Resonance
Spectroscopy on Degraded Algae
and Degraded Wheat
As mentioned above, solid-state ]3C NMR pectros-
copy is a powerful technique to characterize the chemi-
cal composition of the C fraction of a heterogeneous
organic mixture. Using solid-state DD CPMAS 3C
NMRpectroscopy can even result in a first estimation
of the relative contribution of peptides to degraded bio-
genie material. However, for the investigation of the
chemical transformation of biogenic N occurring during
humification, solid-state CPMAS 5N NMR pectros-
copy can provide more detailed data.
Applying this technique to casein with natural lSN
levels results in a spectrum hat 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 spec-
CPMAS ~
Casein
Fresh Algae
B
Fresh Wheat
C
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J. ENVIRON.QUAL., VOL. 29, MAY-JUNE000
fractions of plant incubates showing that solid-state
CPMAS SN NMR pectroscopy can be used as a quan-
titative means for determining the chemical structure
of organic N during humification (Knicker and Ltide-
mann, 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 NMRpectrum is muchhigher than it was calculated
with Eq. [2]. The solid-state CPMAS5N NMRpectrum
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 o their missing or weakcou-
pling to the ~H spin system their signal may not occur
or may be diminished in a solid-state CPMASSN NMR
spectrum. Such N can be bound in six-membered aro-
matic ring structures, imines, or nitriles, structures which
are commonly uggested to represent stabilized soil or-
ganic N formed during humification (Anderson et al.,
1989; Flaig et al., 1975; Kelly and Stevenson, 1996;
Schnitzer, 1985; Schulten and Schnitzer, 1993). Signals
from the same sample but with
no major intensity loss due to
this experiment, also knowna
system is directly polarized.
NMRpectroscopy, the signal
such spectra is expected to gi
chemical composition of the
the premise that saturation
result indicates that in spite
the ~H spin system such N in
be detectable via CPMAS S
To test whether the wheat i
N-containing heterocyclic
structures or imines the samp
state SPE ~SNNMR. n the sp
signals of amides and free a
Resonance lines between -
be distinguished from the no
cyclic six-memberedaromatic N
not formed to a larger exten
of wheat residues. Thus, th
pounds cannot explain the con
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KNICKER: BIOGENICN IN SOILS AS REVEALED Y ~3C AND~SN NMR
CPMAS5N NMRpectrum gives a fairly correct reflec-
tion of the chemical composition of the N compounds
in these samples.
Comparable with the solid-state 15N NMRpectra of
the incubated wheat, no signals are observed in the
chemical shift region of six-memberedheterocyclic aro-
matic N or imines. Considering the low signal/noise ra-
tio, the broad lineshape of the resonances, and the shoul-
der 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 CPMAS 5N NMR pectra 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 lim-
ited to well-aerated soils but also can be observed in
environments with reducing conditions.
particle size fractions of differe
1999b; K6gel-Knabner et al.,
the solid-state CPMASSN NM
fraction of the 10 000-yr-old Loe
4C), most of their signal intens
chemical shift region assigned
of six-memberedheterocyclic ar
identified, which could confirm
ment of clay minerals in the for
ucts (Hedges, 1988).
As aforementioned, peptide-li
identified in solid-state CP
clay-flee plant incubates and th
grove Lake with a mineral con
w/w Knicker et al., 1996b). The
gest the existence of additiona
mineral adsorption and protectio
ble for the survival of peptide-li
explanation for the survival of
samples maybe their association
lecules. Evidence for this was
solid-state CPMASSN NMRpec
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J.
ENVIRON. QUAL.,
VOL. 29,
MAY-JUNE 2000
enzymaticattack d uring sediment diagenesisb yencap-
sulation in their hydrophobic ne twork (Knicker and
Hatcher ,1997). It also was considered that parts of the
algal cell
walls
are involved in the protection and tha t
labile compounds
m ay
become sandwiched be tween
a l-
gaenan layers. Although algae may not present a major
fraction of soil biota, comparable structures may be
present
in the
cell walls
o f
soil bacteria.
Summariz ing
the results concerning the structure of
immobilized organic N in soils obtained via solid-state
15
N
NMR spectroscopy, i t can be assumed that the for-
mation
of
heterocyclic aromatic
N is of
less importance
in soil organic
N
stabilization than formerly thought.
Although several solid-state
15
N N M R spectroscopic
studies clearly indicated that some peptide-like struc-
tures can resist microbial degradation over prolonged
humification both
in
soils
an d
sediments ,
a t
this point
of the research, the results do not confirm a specific
pathway for
their survival. M uch w ork
i s
still necessary
to clarify the question of howsome
peptide-like struc-
tures of biogenic precursors of soil organic m atte r resist
both microbial and chemical degradation.
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MARTENS:MANAGEMENT AND CROP RESIDUE INFLUENCE SOIL AGGREGATE STA
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