Artigo 9 Knicker 2000 RMN 13C e 15N

8/9/2019 Artigo 9 Knicker 2000 RMN 13C e 15N

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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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716

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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718


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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720


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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722

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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Artigo 9 Knicker 2000 RMN 13C e 15N