Soil Biology & Biochemistry 43 (2011) 1118e1129
Contents lists available at ScienceDirect
Soil Biology & Biochemistry
journal homepage: www.elsevier.com/locate/soilbio
Review
Soil organic N - An under-rated player for C sequestration in soils?
Heike Knicker*
Instituto de Recursos Naturales y Agrobiología de Sevilla (IRNAS-CSIC), Avda. Reina Mercedes, 10, 41012 Sevilla, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 September 2010
Received in revised form
23 February 2011
Accepted 25 February 2011
Available online 11 March 2011
The availability of Soil Organic Nitrogen (SON) determines soil fertility and biomass production to a great
extent. SON also affects the amounts and turnover rates of the soil organic carbon (SOC) pools. Although
there is increasing awareness of the impact of the nitrogen (N) cycle on the carbon (C) cycle, the extent of
this interaction and the implications for soil organic matter (SOM) dynamics are still under debate.
Therefore, present knowledge about the inter-relationships of the soil cycles of C and N as well as current
ideas about SON stabilization are summarized in this paper in order to develop an advanced concept of
the role of N on C sequestration. Modeling global C-cycling, it was already recognized that SON and SOC
are closely coupled via biomass production and degradation. However, the narrow C/N ratio of mature
soil organic matter (SOM) shows further that the impact of SON on the refractory SOM is beyond that of
determining the size of the active cycling entities. It affects the quantity of the slow cycling pool and as
a major contributor it also determines its chemical composition. Although the chemical nature of SON is
still not very well understood, both improved classical wet chemical analyses and modern spectroscopic
techniques provide increasing evidence that almost the entire organic N in fire-unaffected soils is bound
in peptide-like compounds and to a lesser extent in amino sugars. This clearly points to the conclusion,
that such compounds have greater importance for SOM formation than previously assumed. Based on
published papers, I suggest that peptides even have a key function in the C-sequestration process.
Although the mechanisms involved in their medium and long-term stabilization are far from understood,
the immobilization of these biomolecules seems to determine the chemistry and functionality of the
slow cycling SOM fraction and even the potential of a soil to act as a C sink. Pyrogenic organic N, which
derives mostly from incomplete combustion of plant and litter peptides is another under-rated player in
soil organic matter preservation. In fire-prone regions, its formation represents a major N stabilization
mechanism, leading to the accumulation of heterocyclic aromatic N, the stability of which is still not
elaborated. The concept of peptide-like compounds as a key in SOM-sequestration implies that for an
improved evaluation of the potential of soils as C-sinks our research focus as to be directed to a better
understanding of their chemistry and of the mechanisms which are responsible for their resistance
against biochemical degradation in soils.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Soil organic matter
C and N humification
C-sink
N-cycle
Soil organic nitrogen
Soil peptides
1. Introduction
Presently, considerable attention is given to the potential of soils
to act as C sinks. This is reflected in the emphasis to determine
turnover rates, sizes and composition of their different C pools as
well as in developing and elucidation of hypotheses addressing the
mechanisms involved in C stabilization (Sollins et al., 1996; von
Lützow et al., 2006; Kleber et al., 2007). However, although it is
already recognized that SON and SOC are closely coupled via
biomass production and degradation and as such nitrogen has
* Tel.: þ34 954624711; fax: þ34 954624002.
E-mail address: [email protected].
0038-0717/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.soilbio.2011.02.020
a major impact on the global C and N-cycling (Gärdenäs et al., 2011),
up to now, its consideration as important factor in the shaping of
the recalcitrant SOM pool is still limited.
Nitrogen (N) is a major nutrient element controlling the cycling
of organic matter in the biosphere. As a limiting growth factor, the
concentration and availability of N in both its organic and inorganic
forms is closely related to biological productivity in terrestrial and
aquatic systems. The significance of this indirect impact on the C
cycle arises from the fact that alteration of the N availability is
related to changes in the amount of C, immobilized into newly
formed biomass, which subsequently affects both size and quality
of the SOM pools. This is of particular interest in the light of
increasing N input to soils by anthropogenic sources such as
fertilizers, N-oxides depositions from combustion processes or
ammonia from agricultural sources.
H. Knicker / Soil Biology & Biochemistry 43 (2011) 1118e1129
Once having entered the soil, N is not a static entity, but takes
part in a series of interconnected reactions that constitute the N
cycle. This cycle is determined by the competition for this nutrient
between plants, fungi and soil bacteria. Additionally, a part of the N
suffers a long-term sequestration by its transformation and incorporation into biologically refractory organic material. The pathways
of this incorporation, however, are still not understood and subject
of ongoing debate (Knicker, 2004; Rillig et al., 2007; Colman et al.,
2008; Davidson et al., 2008; Thorn and Cox, 2009). The importance
of increasing our knowledge in this field becomes even more urgent
if one bears in mind that all forms of organic N are connected to
a “C-backbone”. This implies that SON has not only an indirect but
also a direct impact on C cycling by determining the quality and
quantity of at least the N-containing part of the total soil organic
matter (SOM) pool.
Alteration of the SOM pools by changing the management or by
erosion, grazing or burning can in turn affect the N-cycling. Those
events alter the amount and quality of the input material and the
N-supply which has consequences for N-partitioning between the
different soil compartments as there are bioavailable N, biotically
immobilized N or otherwise sequestered N.
The described close interrelationship between C and N cycles
clearly demonstrates that a realistic evaluation of the potential of
soils as a C sink can only be achieved if we can answer the question
of the importance of SON for SOM formation. Accordingly, the
intention of the following review is to bring some light on this
aspect by mapping out present knowledge and views about the role
of SON sequestration in the C cycle. It includes the above mentioned
interrelationship between the C and the N cycles but goes beyond
the goal of increasing our knowledge for improving general circulation models (Gärdenäs et al., 2011) by emphasizing that the
chemistry of SON determines to a large part the composition of
the refractory SOM pool. This requires a better understanding of the
nature of SON, which is the reason why controversially discussed
mechanisms of SON formation are presented. Focusing on proteins
and peptides as the main SON source and showing their important
contribution to SOM, pathways presently suggested to be responsible for their stabilization are discussed.
2. SON as part of the N-cycle
2.1. The terrestrial N cycle and its short-circuit by amino acids or
peptides
The organic N in soils originates from living organisms where it
occurs mainly as peptides and amino acids (Friedel and Scheller,
2002). Only small amounts of N can be assigned to amino sugars,
nucleic acids, alkaloids or tetrapyrroles. Most of these compounds
enter the soil during litter fall. In fire-affected ecosystems, incomplete combustion transforms them into pyrogenic N-heterocyclic
structures (Knicker et al., 1996a, 2008; Knicker, 2010) which
subsequently are also incorporated into the SOM pool, although the
respective mechanisms are not yet understood. With respect to
biogenic N-containing residues on the other hand, it is well
established that their microbial decomposition results in amino
acids (by depolymerization) together with NH4þ, NO3, H2O and
CO2. Whereas the latter is for the most part released into the
atmosphere, the inorganic N compounds can easily be recycled for
the build-up of new biomass, which is associated with an immobilization of organic carbon. However, a considerable part of the
organic N escapes complete mineralization and the residues are
entering the refractory SOM pool where they will be sequestered
from both the N and C cycle. It was estimated that approximately
60 Tg N yr1 is accumulating in terrestrial systems, although there
is still considerable uncertainty about the correct values for N
1119
storage and the production of reactive N from N2 (Galloway et al.,
2004).
Although the traditional view of N-cycling in terrestrial
ecosystems has focused almost exclusively on primary production,
plant availability, and loss of inorganic N, there is ample evidence
that in some ecosystems, plants may “short circuit” the terrestrial N
cycle by direct uptake of amino acids from the dissolved organic
matter fraction (Neff et al., 2003). At least in some forest systems
organic N can represent a substantial fraction of the total plant N
uptake (Schimel and Bennett, 2004). It is reported that in soils
under temperate forests the N pool representing amino acids can
exceed inorganic N pools at low fertility sites (Rothstein, 2009).
Other studies have shown that in boreal forest systems, plants are
able to take up free amino acids at rates that equal or exceed those
of inorganic N (Näsholm et al., 1998; Lipson and Näsholm, 2001).
Only recently, evidence that even proteins can be used by plants
without assistance of other organisms has been presented
(Paungfoo-Lonhienne et al., 2008). The authors suggested that
roots access proteins by secreting proteolytic enzymes that digest
proteins at the root surfaces and possibly also in the apoplast of the
root cortex. Additionally, the authors reported that intact proteins
are taken up in root cells probably by endocytosis. However,
whereas many of the in situ observations of amino acid uptake by
plants have been made in northern or alpine ecosystems, the
evidence is mixed in other environments (Finzi and Berthrong,
2005). In some agricultural ecosystems it was demonstrated that
plants used for cropping are poor competitors for amino acids
(Owen and Jones, 2001; Xu et al., 2008).
In most environments, microbes strongly compete for uptake of
both inorganic and organic N (Kaye and Hart, 1997). It is well
established that this microbial N immobilization is an effective
mechanism of N retention in soils (Friedel and Scheller, 2002).
Application of in situ labeling techniques and measurements of the
15
N recovery in the biomass of competitive plant species, labeled
plants and microbes, indicated the use of the same N source. An
uptake of glycine was evidenced in temperate grasslands, (Bardgett
et al., 2003), but found that most of the N was sequestered by
microbes. Regardless of whether the N uptake occurs in plants or
microorganisms, the growth and death of the organisms ultimately
results in returning N into polymeric forms, which are not directly
bioavailable. Since depolymerization is needed to transform polymeric N to bioavailable dissolved organic nitrogen (DON) (Cookson
and Murphy, 2004), the depolymerization step can be seen as
a critical point in maintaining an active N cycle (Schimel and
Bennett, 2004).
2.2. The role of DON in N sequestration and the controversy about
its formation and nature
The formation of DON can result directly from microbial turnover and indirectly through generation of extracellular enzymes. A
further pathway, which is still subject of a controversy represents
the fast abiotic transformation of inorganic N into DON (Dail et al.,
2001) via the “ferrous wheel” mechanism (Davidson et al., 2003,
2008). According to this hypothesis, DON can be formed from
a physically or biologically mediated association of dissolved
organic matter (DOM) with nitrate after its reduction to nitrite by
reactive Fe(II) species. The required DOM compounds were suggested to be derived from phenols and lignin derivatives that form
aromatic N by nitration and nitrosation. An argument supporting
this pathway is that abiotic reactions were evidenced during
laboratory experiments (Thorn and Mikita, 1992; Potthast et al.,
1996). On the contrary, up to now, the respective products have
not been identified in natural DOM. Further arguments against this
hypothesis derive from alternative studies, assigning the quick
1120
H. Knicker / Soil Biology & Biochemistry 43 (2011) 1118e1129
disappearance of inorganic N during abiotic N-immobilization to an
interference of Fe during the nitrate determination (Colman et al.,
2007, 2008).
With respect to the structural composition of DON, there is still
a paucity of information. In Oa horizon leachates from forest soils in
Northern California about 48e74% of the total DON were recovered
as hydrolyzable amino acids, suggesting that proteins and peptides
were the main contributors (Yu et al., 2002). The contribution of
free amino acid/sugar was only about 2e11%. Similarly large
amounts of hydrolyzable amino acids from DON were reported
from the mor layer in a pine forest (Hagedorn et al., 2000) and from
the upper 10 cm of three Scottish soils (Paul and Williams, 2005).
Solid-state 15N NMR spectroscopy confirms a high peptide content
of DON (Michalzik and Matzner, 1999). The fact that almost all of
the 15N NMR intensity can be assigned to amide N and the
remaining intensity is attributable to free amino groups of amino
acids allows the conclusion that the non-hydrolyzable DON, is also
derived from peptideous structures. These structures may have
escaped identification after acid hydrolysis either due to their steric
nature or because hydrophobic domains protected them from
chemical degradation. Alternatively, they were protected by association with or encapsulation into hydrophobic DOM components,
as was suggested regarding peptideous material in a sapropel
(Knicker and Hatcher, 1997). In fire-affected ecosystems, on the
other hand, heterocyclic N can occur as it was demonstrated by the
solid-state 15N NMR spectra of DOM samples from the Everglades
(Maie et al., 2006b). Those structures derive tentatively from
charred litter and humic material that entered the soil system after
the fire event. After their oxidation, they turned into water soluble
compounds that were able to be leached and transported with the
soil solution. However, it has to be mentioned that solid-state 15N
NMR spectroscopy has been criticized to underestimate heterocyclic N in soil organic matter because i) the unfavorable nuclear
properties of 15N and inefficient cross polarization inhibits their
visibility (Smernik and Baldock, 2005), or due to ii) low sensitivity
of this technique (Leinweber et al., 2009). However, the high
consistency of the NMR spectra from 15N-enriched grass acquired
via direct excitation of the 15N nuclei and via cross polarization
(Fig. 1) and the fact that heterocyclic N is clearly visible in solidstate 15N NMR spectra of fire-affected SOM stand against those
arguments (Knicker, 2007).
There is evidence showing that DON can leach from ecosystems,
despite a high biotic demand for N (Perakis and Hedin, 2002). This
fact was suggested to point towards the existence of two pools. One
of them is labile and can quickly be used by plants and microorganisms and the other represents a recalcitrant fraction of the DON,
inaccessible for biological growth (Neff et al., 2003). According to
Neff et al. (2003), this distinction would place soluble amino acids
in the same general class of terrestrial N as NH4þ and NO3-, whereas
the recalcitrant DON would function in a manner similar to bulk
SOM. However, there is almost no information available either
about the decomposability of DON in soils or the mechanisms
involved in its stabilization. Even so, it can be assumed that DON
and DOC do not represent independent SOM pools but are closely
related, and determine the turnover of each other.
Studies at sites from the Nantahala Range of the Southern
Appalachian Mountains, NC, USA (Qualls et al., 2002), showed that
the average DOC/DON ratio (w/w) varied between 34 and 40 in the
O, A and B horizons and was about 20 in the C horizon. DOC/DON
ratios between 32 and 39 were reported for unfertilized forest
floors of the Harvard Forest, Massachusetts, USA (McDowell et al.,
2004). Nitrogen application decreased this ratio down to 10 to 22.
The change was attributed to increasing concentrations of amino
sugars and amino acids. In addition to hydroxyl and carboxyl
groups, these compounds also contain amino functions as possible
Fig. 1. Solid-state 15N NMR spectra of 15N-enriched grass char obtained after charring
the sample under oxic conditions for 3 min at 350 C. Spectra were acquired with the
a) Bloch Decay (direct excitation of the 15N nuclei) technique and b) after cross
polarization at the Lehrstuhl für Bodenkunde, TU-München, Germany according to the
parameters described in Knicker (2010).
sorption sites, such alterations of the DON pool are likely to also
have a considerable impact on the mobility of the bulk DOM and
thus on the potential of soils to sequester DOC in mineral-organic
matter associations.
3. Links between N and C immobilization: “microbial N
mining” versus “stoichiometric decomposition”
Since N is a limiting factor for biological productivity and
represents an important SOM constituent, the sequestration of
biogenic N is closely related to the formation and accumulation of
humic material. Thus, it was stated that the N cycle drives to some
extent the C cycle (Högberg, 2007). The issue of interacting N and C
cycles deserves an increasing attention because inputs of N to soils
due to elevated concentration of atmospheric nitric oxides as well
as an increase in ’industrial N-fixation’ for inorganic fertilizers now
exceed the N input from microbial N-fixation (Vitousek et al., 1997).
The deposition of anthropogenically fixed N has been discussed as
a means to explain part of the “missing sink” in the imbalance
between known CO2 emissions from fossil fuel combustion and
deforestation versus known CO2 accumulation in the atmosphere
(Vitousek et al., 1997). Estimates of how much terrestrial C storage
could result from N deposition range between 0.1 and 1.3 Pg C per
year. On the other hand, it was found that under elevated CO2,
plants in a California annual grassland increased their N uptake at
the direct expense of soil microbial activity and biomass N content
(Hu et al., 2001).
It has long been known that increased N uptake by plants will
increase their foliar biomass and the concentrations of ribulose-1,5bisphosphate carboxylase/oxygenase. The latter enhances the CO2
assimilation by photosynthesis, leading to higher production of
biomass which eventually forms litter which will need a longer
time for decomposition. It has been found that at places without N
limitation larger soil N availability decreases the decomposition
time of the litter, but at sites where N limits net primary production, N availability has no or an inconsistent effect on litter
decomposition (Hobbie and Vitousek, 2000).
Presently, literature reports about two main mechanisms to
explain the interrelationship between N availability and C storage
H. Knicker / Soil Biology & Biochemistry 43 (2011) 1118e1129
in ecosystems. The first, known as “microbial N mining”, suggests
that low N contents may increase litter decomposition as microbes
use labile substrates to acquire N from recalcitrant organic matter
(Craine et al., 2007). According to the authors, this “microbial N
mining” is consistently suppressed by high soil-N-supply or
substrate-N concentrations. Consequently, it will lead to predictions that ecosystem C storage would increase with greater N
availability. This could explain observed declines in decomposition
after N additions (Hagedorn et al., 2003; Wang et al., 2004).
Alternatively, the basic “stoichiometric decomposition” theory
predicts that ecosystems will store more C if increasing atmospheric CO2 leads to greater substrate C/N ratios. However, as soon
as the C/element ratios in food approach those of the consumers,
the C-use efficiency in food webs tends to increase, decreasing C
sequestration in soils (Hessen et al., 2004). Supporting this view,
Wang et al. (2004) found that addition of NO3 initially promoted C
mineralization of the plant material, but a lower long-term
cumulative C mineralization was found. This is in agreement with
other findings that large N concentrations promote carbohydrate
decomposition during the early stages, but may suppress lignin
decomposition in the long run (Berg and Ekbohm, 1991). Mechanisms for the suppressive effect of N on long-term decomposition
may include i) inhibition of lignin-degrading enzymes by low
molecular N compounds (Carreiro et al., 2000), or ii) production of
toxic or inhibitory products or iii) by N sequestration into biologically recalcitrant forms.
4. SON sequestration e still a subject of controversial
discussion
Major difficulties in the understanding and predicting of
organic-N dynamics and its effect on the C cycle originate from the
lack of understanding the mechanisms involved in SON
stabilization.
A classical but nowadays highly questioned concept represent
the depolymerizationerecondensation pathways (Stevenson,
1982). Here it is assumed that naturally occurring macromolecules are microbially degraded to oligomers and monomers, which
for the most part are further mineralized. However, a small fraction
of those compounds recombines by random condensation into
larger, insoluble and refractory structures, which survive a prolonged pedogenesis. In line with this pathway, an earlier publication suggested that humic material in soils is formed by
condensation of C]O groups of lignin with NH2-groups of
proteinaceous material to produce Schiff bases (Waksman and Iyer,
1932). Others propose abiotic condensation of phenolic or quinonic
structures with N-containing compounds (Theis, 1945;
Rinderknecht and Jurd, 1958; Piper and Posner, 1972; Ladd and
Butler, 1975). In accordance with the abiotic immobilization
a newer report, it was assumed that the above mentioned “ferrous
wheel” hypothesis can also occur without the intermediate DON
(Fitzhugh et al., 2003).
The formation of heterocyclic N during SON sequestration
received some recent support from X-ray photoelectron spectroscopy (Abe and Watanabe, 2004) which allowed the identification
between 3 and 20% of the total N as aromatic N in humic acids of
Japanese soils using deconvolution techniques. On the other hand,
in another study, the same signals were observed but solely
attributed to amides and ammonium groups (Monteil-Rivera et al.,
2000). This indicates the difficulties which are involved in an
unbiased assignment of SON-derived signals with this technique.
Comparatively, care has to be taken by using K-edge XANES,
although it was recently shown to allow reliable differentiation
between two p*-regions in spectra of unknown environmental
samples: 400 eV (pyridinic N and some nitrile N) and >400 eV
1121
(amide N, nitro N and pyrrolic N) (Leinweber et al., 2007). However,
the authors stated further that a quantitative estimate of the
proportions of amide N and pyrrolic N requires a separate determination of one of the compounds, which remains a challenge for
forthcoming studies. They assumed that “given the subtle variations in spectral features, even for compounds sharing very similar
structures, it is unlikely that the current N K-edge XANES can be
used exclusively to quantitatively determine the chemical composition of an environmental sample”.
The second classical pathway of soil organic matter stabilization
is the selective preservation pathway, first developed for sediments
(Hatcher et al., 1983) and later adapted to soil systems (Hatcher and
Spiker, 1988). According to it, refractory biopolymers resist a quick
biodegradation and preferentially accumulate, whereas other more
labile compounds are mineralized during the early stages of
humification (de Leeuw and Largeau, 1993). The finding that several
vascular plants, algae, and bacteria produce hydrolysis-resistant
biopolymers that are highly recalcitrant supports this hypothesis.
A more recent adaption of this concept to soil systems states
that not only chemical properties of biomolecules are responsible
for their stabilization but additional mechanisms can be active (von
Lützow et al., 2006) and may even be of more importance. On the
other hand, the fact that organic N is also stabilized in mineral-poor
environments such as Tangel humus or mor layers, composts, peats
or sapropels indicates that chemical recalcitrance still must be an
important player in N sequestration.
With respect to the chemical nature of this SON, recent NMR
studies gave strong evidence that in fire-unaffected soil systems,
proteinaceous material has the potential to be selectively preserved
and to dominate the SON pool (Knicker et al., 1993; Knicker, 2004).
Recalcitrant peptide-like compounds were shown to be ubiquitous
(Knicker and Hatcher, 1997) and to exist even in fossilized organic
matter rich deposits (del Río et al., 2004). Their preservation may
partially be caused by unfavorable conditions for microbial survival
and growth. This mechanism is certainly of great importance in
peats (Knicker et al., 2002) and buried soils that suffer from O2
depletion or in soils occurring in dry and cold or extremely warm
climatic zones but also in environments with extremely acid or
alkaline conditions. Additionally, SON may be physically protected
by incorporation within aggregates (Skjemstad et al., 1993), by
encapsulation into hydrophobic macromolecules (Knicker and
Hatcher, 1997), by sequestration in nanopores that are too small
for organisms or enzymes to enter and function (Mayer, 1994) or
due to the formation of organo-mineral-associates (Baldock and
Skjemstad, 2000). The latter alternative may be due to intercalation between the silicate sheets of clay minerals (Theng et al., 1986,
1992) or sorption of N-containing compounds in soil minerals
(Kaiser and Zech, 2000).
5. Possible mechanisms involved in the sequestration of
peptides and proteins in soil
5.1. Interaction with the mineral phase
As mentioned above, many studies have demonstrated the role
of peptides as important constituents of the recalcitrant SON fraction. Direct measurements of the chirality of several amino acids
isolated from grassland soils suggested that the age of their
proteins was within the range of several hundred years (Amelung
et al., 2006). However, they can be even older as it was indicated
by peptides found in soil layers on the Chinese loess plateau with an
age of more than 10 000 years (Curry et al., 1994) as well as in
sapropels and peats dated to approximately the same age or even
older (Knicker and Hatcher, 2001; Knicker et al., 2002). Fig. 2 shows
the respective solid-state 15N NMR solid-state spectra. They are
1122
H. Knicker / Soil Biology & Biochemistry 43 (2011) 1118e1129
-260
Atomic C/N
11.6
Clay (A horizon)
12.2
Sapropel (510 cm)
15.2
Peat (650-660 m)
compounds. This idea of an “onion” layering model is based on the
preferential accumulation of polar carboxyl and cationic amino
compounds by ligand and cation exchange on mineral surfaces. It
was suggested that a first a contact zone is formed in which
proteinaceous materials are particularly strongly associated to the
mineral phase. Upon adsorption the proteinaceous material may
unfold which increases its adhesive strength by adding hydrophobic interactions to electrostatic binding (Kleber et al., 2007). The
amphiphilic character of proteins and peptides could help to create
the next zone, referred to as the hydrophobic zone, which could
provide a suitable region for sorption of hydrophobic moieties
including further amphiphilic components. Sorbed to the hydrophilic exterior of this zone, organic molecules may form the kinetic
zone that is largely controlled by exchange kinetics. It was suggested that movement of organics into and out of this region has to
be regarded as a phase partitioning process. Although still under
debate, this hypothesis may be supported by the finding that the
weight of soil density fractions is negatively related to the thickness
of the organic layer around the mineral particle and that the age of
organic C increased with the weight of the fraction (Sollins et al.,
2006). The C/N ratio decreased from 66 in the fraction
<1.65 g cm3 to 12 in the fractions >2.0 g cm3, confirming the
importance of SON for the stabilization of SOM via mineraladsorption.
5.2. Formation of protein-tannin complexes
0
-100
-200
-300
-400 ppm
15
Fig. 2. Solid-state N NMR spectra of the clay fraction of the A horizon of a Paleosol
from the Chinese Loess Plateau demineralised with 10% hydrofluoric acid, peat derived
from a depths between 650 and 660 cm of the Grober Bolchow, Germany, and the
Sapropel of the Mangrove Lake, Bermuda (Knicker and Hatcher, 2001).
dominated by a signal at 260 ppm which is assignable to amide N
and reveals that peptide N constitutes most- if not all - of their SON.
In spite of the fact that more and more evidence is reported that
supports the importance of peptides as constituents of both the
labile and the recalcitrant SON pool, we still are far away from
understanding which factors are causing their degradation and
which mechanisms are responsible for their sequestration.
One mechanisms explaining how supposedly easily degradable
biomolecules may survive on a long-term scale in soil environments. is the “Mesopore-hypothesis”. It was developed based on
the finding that peptides adsorb strongly to clay particles (Wang
and Lee, 1993; Aufdenkampe et al., 2001; Ding and Henrichs,
2002). The adsorption may physically protect the peptides by
restricting the accessibility to degrading enzymes. This protection
is even increased if the small organic molecules are adsorbed into
mesopores <10 nm that are too small to permit the entry and
functioning of degrading enzymes (Mayer, 1994). This pathway is
supported by studies of the sorption behavior of amino acid
monomers and polymers onto fabricated mesoporous and nonporous alumina and silica that indicated that these compounds may
be strongly bound within mesopores and thereby undergo
a reduced desorption (Zimmerman et al., 2004). Larger proteins, on
the other hand, were excluded from the pores, indicating that the
mesopore protection hypothesis (Mayer, 1994) is indeed a feasible
mechanism for soil and sedimentary environments.
Alternatively, but also based on adsorption to mineral surfaces,
it was suggested that peptideous compounds may form a stable
inner organic layer around a mineral surface on which less polar
organics could sorb more readily than onto the highly charged
mineral surfaces (onion-layer hypothesis) (Sollins et al., 2006).
Concomitantly, this layer could serve as a reactive chemical
template for further coupling and cross-linking of organic
A further suggested mechanism that could be responsible for at
least part-time stabilization of proteins and peptides in soils is the
formation of protein-tannin complexes. Tannins are secondary
plant metabolites occurring in the bark and foliage of most plant
species. These polymers consist of flavan-3-ol units (condensed
tannins) or gallic acid monomers (hydrolyzable tannins). Proteintannin complexes can be formed in the soil during litter decomposition but also already during leaf senescence (Kuiters, 1990).
They are assumed to resist degradation by many microorganisms.
However, some ectomycorhizal fungi in coniferous forest soils are
able to mineralize tannin compounds (Northup et al., 1995). Other
studies using matrix-assisted laser desorption ionization time of
flight mass spectrometry (MALDI-TOF MS) and solution 13C NMR
spectroscopy, examining the chemical alteration of condensed
tannins of pine needles and willow leaves during litter degradation
(Behrens et al., 2002; Maie et al., 2002) could not confirm the high
refractory nature of condensed tannins. After eight weeks of incubation, no signals of condensed tannins were detected by MALDITOF MS. Low recoveries were also reported for tannins of Picea sp
and Kalmia angustifolia L. in a black spruce forest floor horizon
(Bradley et al., 2000). This may be explained either by fast degradation of the tannins or by hindrance of their extraction due to their
fast and tight binding to SOM, including proteins. Such proteintannin complexes were suggested to occur either by covalent, ionic
or hydrogen bonding (Zucker, 1983). The formation of covalent
bonding, however, seems unlikely since the phenols first have to be
transformed into quinones and semiquinones that are normally not
formed under the redox potentials found in plant cells and soils
(Van Sumere et al., 1975). This conclusion is supported by solidstate 15N NMR spectroscopy that failed to identify such condensation products (Knicker, 2004). However, up to now, ionic and
hydrogen bonding cannot be excluded. Additional possibilities are
hydrophobic interactions that were shown to play an important
role in protein-tannin complexation in laboratory studies (Luck
et al., 1994; Charlton et al., 1996). Nevertheless, summarizing the
present knowledge about the role of tannins during organic N and
in particular during peptide sequestration in soil, we are far from
a satisfactory understanding. This is partly because suitable
H. Knicker / Soil Biology & Biochemistry 43 (2011) 1118e1129
approaches to isolate and quantify tannins in degrading litter or
humic samples are still missing but also because this mechanism
for organic matter stabilization has been widely neglected during
the last years.
5.3. Encapsulation pathway
Since long-term survival of peptide-like compounds is also
observed in mineral-poor and tannin-free or depleted environments such as sapropels (Knicker et al., 1996b) and peats (Knicker
et al., 2002). In those environments, the survival of proteinaceous
material was explained with the encapsulation pathway (Knicker
and Hatcher, 1997). According to this conceptual model, peptides
that are connected to recalcitrant hydrophobic biopolymers will be
surrounded by these polymers, and therefore they will be selectively enriched during cell degradation, while other, more accessible compounds will be consumed by microorganisms (Junction
Pathway). Alternatively, during cell lysis proteins and peptides are
sandwiched between hydrophobic biopolymer layers (Sandwich
Pathway) and protected from hydrophilic enzymes that will not
have access to certain key structures of the proteinaceous material.
Only conditions that alter the steric configuration of the
biopolymer may liberate proteinaceous compounds, so they
become accessible for microbial activity. Although this mechanism
was first suggested for accumulation of organic matter derived
from algae surrounded by such a polyaromatic network, it is not
restricted to subaquatic systems. The mechanism is also possible in
terrestrial environments where hydrophobic or protective macrobiopolymers such as cell wall components of bacteria or waxes and
lignin in plant material occur. It was also suggested that proteins
can even extract into humic acids and that the complete hydrolysis
of such proteins is prevented by the encapsulating humic acid
(Zang et al., 2000).
5.4. Intrinsic stabilization of proteinaceous material
A further mechanism responsible for the protection of peptideous compounds could be related to their molecular nature. In
order to fulfill their function in the soil, molecules such as extracellular enzymes, surface-active agents or plant hormones must
have some properties that protect them from fast microbial
degradation. They may be methylated or derivatized at certain key
groups that are essential for reorganization by and functioning of
degrading enzymes. Alternatively, they may contain conformational restrictions and hydrophobic domains which efficiently
protect them within the soil. In a review, evaluating the role of
proteins in soil carbon and nitrogen storage, Rillig et al. (2007)
suggested that protection may be supported by domains forming
amyloid aggregates and fibrils that were rather stable against
chemical denaturation and enzymatic hydrolysis. Hydrophobins
secreted by ascomycetes and basidiomycetes (Wösten, 2001)
contain such hydrophobic and amyloid domains, and can make up
to 10% of the total cellular protein. They are involved in the mediation of attachment of the fungi to hydrophobic solids, provide
hydrophobicity to fruiting bodies and spores and help fungi to
escape aqueous environments (Wösten, 2001). Chaplins, which are
proteins excreted by Streptomycetes sp. fulfill comparable functions (Claessen et al., 2004). However, information about the
contribution of such proteins to SOM or their role for its stabilization is still not available.
The glycoprotein glomalin is another N-containing molecule
which is believed to give large contributions to SOM. Rather than
being exudated by arbuscular mycorrhiza (AM) it comprises a large
fraction of the hyphal walls of the AM fungi (Driver et al., 2005).
Therefore, it has been assumed that glomalin has its primary role in
1123
the living fungus, possibly as a structural component (Rillig et al.,
2007). In temperate climatic zones, the glomalin concentration
was reported to vary between 1 and 21 mg g1 in soil aggregates
(Wright et al., 1996).
More than 60 mg of glomalin per cm3 soil was found in
a chronosequence on Hawaii (Rillig et al., 2001). For tropical soils in
Costa Rica, 3e5% of the total soil C and N in the top 10 cm was
accounted for by glomalin (Lovelock et al., 2004). As a result of
a strong relationship between soil aggregate stability and glomalin
concentration (Wright and Upadhyaya, 1998), this glycoprotein has
been credited with enhanced ecosystem productivity due to
improved soil aeration, drainage and microbial activity (Lovelock
et al., 2004). It is further thought to significantly reduce SOM
degradation by protecting labile compounds within soil aggregates
and thus enhancing C sequestration in soil ecosystems (Wright and
Anderson, 2000).
On the other hand, other studies provided evidence that the
significance of glomalin may be overestimated, since other studies
evidenced that the common extraction technique may not eliminate all non-glomalin protein sources (Rosier et al., 2006). This
assumption is supported by solid-state 13C NMR spectroscopy
revealing that the current operationally defined “glomalin-related
soil protein fraction” is actually a mixture of humic acids and
proteinaceous material (Schindler et al., 2007). It was suggested
that if we intend to unveil the geochemistry of this biomolecule,
preparative techniques have to be found that are more specific for
glycoprotein. On the other hand, bearing in mind that this “glomalin-related soil protein fraction” is relatively persistent against
biological degradation (Rillig et al., 2001; Steinberg and Rillig,
2003) it can be concluded that the co-extracted proteinaceous
material must have properties that turn the material into a potential contributor to the refractory SOM pool.
Other mechanisms increasing the stability of proteins are
glycosylation, binding to biofilms or incorporation into microbial
biomass. Glycosylation, which is the covalent linkage of specific
oligosaccharides to specific amino acids, was demonstrated to
increase in vitro molecular stability against proteolytic enzymes.
However, the impact of this reaction on soil protein stabilization is
still far from being understood.
Biofilms contain living cells but also extracellular polymeric
substances including proteins (Omoike and Chorover, 2004).
Similar to glycosylation, the extent to which occlusion within biofilms can protect and stabilize protein N in soils is still unknown
(Rillig et al., 2007).
A further possibility to aggravate proteolysis represents the
introduction of D-amino acids as they occur for example in the
peptidoglucan skeleton of bacteria (Kimber et al., 1990; Amelung
and Zhang, 2001; Amelung et al., 2006). It was shown that the
rate of C mineralization from D-amino acids is initially less than
from the corresponding L-amino acids in a range of soils (Hopkins
et al., 1997; O’Dowd and Hopkins, 1998; O’Dowd et al., 1999). This
suggests that cell wall residues of bacteria comprise a considerable
fraction of the refractory SON pool in soils.
6. The quantitative role of proteinaceous and peptideous
material for the size of the refractory SOM pool
Although we are still lumbering in the dark with respect to the
mechanisms involved in the sequestration of soil peptideous/
proteinaceous material, there are many findings that support their
important role as SOM constituent. Applying the common method
for determining the amount of proteinaceous material in soil by hot
acid hydrolysis with HCl, Amelung et al. (2006) assigned between
22% and 46% of the soil N to hydrolyzable amino acids. This
accounted for 5e10% of the SOC. The remaining N was attributed to
1124
H. Knicker / Soil Biology & Biochemistry 43 (2011) 1118e1129
inorganic N, amino sugar N (up to 10% of the total N), the amino
acids arginine, histidine, cysteine and ’unknown N’. However, there
are reports saying that the commonly used HCl hydrolysis does not
hydrolyze all proteins or peptides in soils and sediments. Using for
example
thermochemolysis
with
tetramethylammonium
hydroxide, additional peptide derived amino acids were identified
that were not released neither by acid nor by alkaline hydrolysis
(Knicker and Hatcher, 1997; Knicker et al., 2001). The mechanisms
involved in their chemical protection are likely to be comparable to
those that are active in the protection against microbial degradation. Alternative to encapsulation into hydrophobic structures
(Knicker and Hatcher, 1997), reactions of released amino acids with
soil clay minerals or quantitative interference with mineral oxides
during digestion with HCl may contribute to the low recovery of
amino acids with the common approach (Cheng et al., 1975). In line
with this, it was observed that a significant portion of the soil N
which is non-hydrolyzable with 6 M HCl was composed of amino
acids bound to reactive surfaces found in peptides (Leinweber and
Schulten, 2000).
By applying a modified hydrolysis technique using methane
sulfonic acid (MSA) the amount of recovered soil amino acids
combined with amino sugars and NH4þ increased to such an extent
that they accounted for 80e93% of the soil N (Martens and
Loeffelmann, 2003). This is in line with the solid-state 15N NMR
spectroscopic data shown in Fig. 1 obtained from other soils unaffected by fire (Knicker, 2004). Martens and Loeffelmann (2003)
further proposed that this SON is bound in peptideous structures
rather than occurring in large intact proteins.
If we now consider that humified SOM has narrow C/N ratios (w/
w) varying between 20 and 10, the conclusion must be that these
peptideous structures account for a major fraction of the refractory
SOC. To demonstrate this, let us perform a rough estimation,
assuming that amino sugars represent a minor proportion of SON
and take the protein casein as a representative for proteinaceous
compounds. Its atomic C/N ratio of 4.1 indicates that in soils with an
atomic C/N ratio of 23.3 (corresponding to a w/w C/N ratio of 20)
approximately 17% of its total SOC is derived from peptides. In soils
having the atomic C/N ratio of 11.7 (corresponding to a w/w ratio of
10) the percentage figure will be 34%. By performing this calculation for the samples the spectra of which are shown in Fig. 2, 35, 34
and 27% of the total organic C in the loess clay, the sapropel and the
peat, respectively is derived from peptideous material. Considering
further that litter degradation and humification are associated with
declining C/N ratios, it becomes clear that relative to other
biomolecules, peptideous material does not only represent an
important SOM pool but is also enriched during SOM maturation.
A similar calculation can be made for soil separates fractionated
according to physical size or density. A number of studies (Baldock
et al., 1992; Christensen, 1992; Sollins et al., 2006) have shown that
such fractions have characteristic turnover times that commonly
increase with decreasing particle size and increasing density.
Concomitantly, their C/N ratios decrease, which indicates the
increasing importance of SON for the slowly cycling SOM pools.
Solid-state 15N NMR spectra of the fine particle size fractions of an A
horizon from a Luvisol under pasture in Southern Germany are
shown in Fig. 3. These data suggest that almost all the organic N even that in the small particle size fractions - is accounted for by N
with amide or peptide bonds. Contributions of heterocyclic N can
only contribute to the shoulders in the chemical shift region
between 220 an 240 ppm and are suggested to be derived from
aromatic amino acid moieties or nucleic acids remains. Similar
spectra were obtained from fractions of other soils which did not
have any documented fire history (Knicker et al., 2000). The atomic
C/N ratios of the respective samples indicate a contribution of
peptide-like material in terms of organic C of 27% in the fine sand
-260
C/N (atm)
Bulk
10.9
63 – 200 µm
15.1
14.0
6 – 20 µm
2 – 6 µm
10.2
0 – 2 µm
10.1
0
-100
-200
-300
-400 ppm
Fig. 3. Solid-state 15N NMR spectra of the HF-treated bulk material and the fine
particle size fractions of an A horizon of a Luvisol from Viehhausen, Southern Germany.
and up to 41% in the clay fraction. By performing a similar calculation for the density fractions isolated by Sollins et al. (2006),
peptide contributions increase from 5% of the organic C in the light
fraction <1.65 g cm3 to 29% in the heavy fraction >2.55 g cm3,
which underlines the important role of peptides for the formation
of organic matter e mineral associations. The intensity distributions of the solid-state 13C NMR spectra of the particle size fractions
from the German Luvisol are shown in Fig. 4. The contribution of
peptide C to the different chemical shift regions assignable to
compound classes can be calculated by using the weighted intensity distribution of a solid-state 13C NMR spectrum of casein.
Subtraction of the peptide contents allows the assignment of up to
35% of the organic C in the different fractions to O-alkyl C, tentatively derived from carbohydrates. The sum of O-aryl C and aryl C in
the difference spectrum is attributable to non-proteinaceous
aromatic C. Its content is highest in the coarse fraction but
decreases to 7% of the total organic C in the clay fraction, suggesting
a relatively low impact of this compound class for SOM stabilization
via interaction with the mineral phase.
Comparable calculations are made for material from an A/B
horizon (15e25 cm) of a Ferralsol from Southern Brazil published
by Dick et al. (2005). This soil has an atomic C/N ratio of 14 (Fig. 5).
Here, the estimated contribution of peptides is approximately 29%
of the total SOC, which provides evidence that peptides comprise
a considerable fraction of SOM in this subsoil. Most of the
H. Knicker / Soil Biology & Biochemistry 43 (2011) 1118e1129
31
28
23
15
14
6 5
1
3
Carboxyl C
3
relative intensity (%)
18
10
7
3
15
3
1
Carboxyl C
10
64
3
17
13
4
34
27
17
13
9
9
4 3
1
4
Carboxyl C
3
6
Aryl C
O-Aryl C
11
7
3
10
4
O-Alkyl C
2
Alkyl C
56
2
6
N-Alkyl
Aryl C
Alkyl C
O-Alkyl C
Peptide C: 33%
14
14
77
11
11
29
7 6
26
10
6
3
14
11
4
N-Alkyl
Aryl C
O-Aryl C
25
11
3
1
Alkyl C
O-Alkyl C
Clay
Peptide C: 41%
38
35
26
17
13
9
4
8
3
Carboxyl C
N-Alkyl
17
6
Carboxyl C
Peptide C: 40%
37
7
1
Alkyl C
O-Alkyl C
Fine silt
8
32
14
Middle silt
N-Alkyl
Aryl C
O-Aryl C
relative intensity (%)
34
8 7
6
O-Aryl C
Peptide C: 31%
37
16
14
Alkyl C
O-Alkyl C
Coarse silt
34
Carboxyl C
N-Alkyl
Aryl C
O-Aryl C
7
4
relative intensity (%)
87
16
11
7
11
Peptide C: 27%
Fine sand
relative intensity (%)
relative intensity (%)
Bulk soil
relative intensity (%)
Total C
Peptide C: 38%
Difference
1125
12
35
Aryl C
O-Aryl C
11
8
3
9
3
N-Alkyl
O-Alkyl C
Alkyl C
Fig. 4. Relative contribution of different C groups to the total organic C of the bulk material and the fine particle size fractions of an A horizon of a Luvisol from Viehhausen,
Southern Germany according to their solid-state 13C NMR spectra. The relative contribution of peptide C was obtained from the respective solid-state 13C spectrum (Knicker, 2000)
weighted with the calculated contribution of peptide C to the total C. The difference represents the C contribution remaining after subtracting the peptide C from the total C.
remaining intensity was attributed to carbohydrates (41%) which
may be closely associated to the peptide fractions as it would be the
case for microbial cell walls. Only 15% was assignable to nonproteinaceous aromatic C, indicating that the role of aromatic
structures in SOM stabilization in subsoils may be overestimated.
7. Stabilization of N by immobilization in pyrogenic organic
matter
In ecosystems that are frequently exposed to vegetation fires
alterations of size and quality of the SON and SOC pools must be
encountered if the C and N sequestration potential should be estimated. The response of the SOM content to fire depends on fire
intensity, type of vegetation, the fuel load as well as soil texture and
even slope (González-Pérez et al., 2004). High fire intensity can lead
to a complete destruction of the organic layer and the humic
material in the mineral topsoil (Fernández et al., 1997; Hernández
et al., 1997; Haslam et al., 1998; Knicker et al., 2006). After
moderate and prescribed fires, the alterations in SOM are minor
and sometimes even result in an increase of organic C and N due to
the input of partly charred organic material or litter from dead trees
(Almendros et al., 1988, 1990; Knicker et al., 2005).
During the charring of biomass, proteins are thermally decomposed via systematic and random depolymerization reactions and
the respective products are incorporated into the SOM pool.
Certainly, a part of the N will be lost due to volatilization but in
1126
H. Knicker / Soil Biology & Biochemistry 43 (2011) 1118e1129
13C
-260
NMR
Atomic C/N: 14
0
-100
-200
-300 -400 ppm
relative 13 C intensity %
15N
Intensity Distribution
43
41
AB
Peptide: 29%
Difference
18
17
15
13
12
8
6 6.6
3
Carboxyl C
Aryl C
2
6
5
2
O-Alkyl C
Alkyl C
N-Alkyl C
Fig. 5. a) Solid-state 15N NMR spectrum of HF-treated bulk soil from the AB horizon of a Ferrasol derived from Southern Brazil (Dick et al., 2005) and b) the relative distribution of
different C groups and peptide C in its solid-state 13C NMR spectrum.
several cases a decrease of the soil C/N ratio has been reported
(Almendros et al., 1984; Shindo et al., 1986; Shindo and Urabe,
1993), indicating that organic N products are formed that are
recalcitrant towards high temperatures. As constituent of the so
called “Black Carbon” (BC) fraction, they are expected to contribute
mainly to the storage of recalcitrant compounds (Knicker et al.,
2008). Whereas, BC is frequently included in global C-cycling
models (González-Pérez et al., 2004), the concept of this “Black
Nitrogen” (BN) as an important fraction of charcoal in soils was only
recently introduced (Knicker, 2007, 2010). In Gärdenäs et al. (2011)
it was discussed in the frame of general circulation models.
Considering the relatively low C/N ratios found in chars derived
from plant litter and humic material (Knicker et al., 1996a;
Almendros et al., 2003), the formation and stabilization of BN has
certainly the same potential importance for C sequestration as that
attributed to BC. Products formed during heating of proteins and
peptides are cyclic di- and oligopeptides (Reeves and Francis, 1998;
Meetani et al., 2003). At lower temperatures (200 Ce300 C), low
molecular weight heterocyclic compounds and above 500 C,
polynuclear N-containing aromatic structures have been observed
(Sharma et al., 2003). A further reaction, occurring during charring
of biomaterial is the Maillard reaction (Ikan et al., 1996). During this
process, ammonia or free amino groups of amino acids and amino
sugars react with carbonyl groups in sugar compounds to form
Schiff bases, which subsequently undergo rearrangements to
produce
dark-colored
melanoidins.
Such
heterocyclic-N
compounds were detected in fire-affected SOM (Knicker and
Skjemstad, 2000; Knicker et al., 2005; Maie et al., 2006a, 2006b),
but not in SOM modified solely by microorganisms (Knicker, 2004)
Comparable to the elucidation of the role of peptideous material for
the quality of SOM, the contribution of BN structures to the bulk
char was estimated to account for more than 50% of the organic C in
a recently examined grass char (Knicker et al., 2008) and at least
12% to the total BC pool of charred peat. Therefore, burning may
lead to a relative enrichment of organic N by transforming biomass
into pyrogenic material that is incorporated into the soil and alters
the long-term N availability for biomass production. Thus, apart
from a direct impact on the quality and quantity of the refractory
soil organic C pool, the formation and addition of BN to soils has
also an indirect impact on the dynamics of C cycling in soils.
8. Summary and conclusions
It is well accepted that the biochemical availability of N, both in
its organic and inorganic forms, determines the amount of C
immobilized in living biomass and litter which are the sources for
the SOM pool. Moreover, recent studies indicate that the N
accessibility for microorganisms affects the degradation pattern
and the turnover rates of the different soil organic C pools. Those
interactions between the C and N cycles are well recognized in
ecological literature but often remained neglected in models
describing the cycling of organic matter in soil. Most of the models
describing C and N dynamics in soils distinguish between the N and
C cycle, admitting the interactive character of the two cycles but
still consider them to act independently. However, such a view
underrates the important chemical premise which is that
compounds forming organic N are also contributing to the organic
C fraction. This literally direct connection between the C and N pool
implies that mechanisms affecting the N stabilization must have
a considerable impact on size and quality of the different organic C
pools, too (Fig. 6). Bearing in mind that in humified SOM with
narrow atomic C/N ratios < 23 N-containing organic compounds
are important constituents, their chemical properties must be of
high relevance for the mechanisms involved in SOM stabilization
and the respective degradation patterns.
With respect to the chemical nature of SON, there is accumulating evidence suggesting that peptideous material makes up the
dominating organic N form in soils unaffected by fire and that its
C
immobilization
N cycle
Biomass
C cycle
fragmentation
CO2
C
mobilization
N
immobilization
biopolymers
N
mobilization
depolymerization
DON/DOC
(amino acids
amino sugars)
mineralization
DIN
(NH4+, NO3-)
C and N sequestration by:
-
Sorption
Bridging by cations
Physical protection
Biofilm
- Steric hindrance
- Chirality
- Glycosylation
SOM ( including
peptides)
Fig. 6. Concept of the interrelationship of the C and N cycle in soils.
H. Knicker / Soil Biology & Biochemistry 43 (2011) 1118e1129
contribution to the SOC pool is increasing with increasing age of
this pool. Thus, in spite of being frequently classified as labile and
fast turning SOM components, peptides must represent more
important players within the SOM-sequestration process than
commonly assumed. With their amphiphilic character and the fact
that approximately two functional groups per amino acid unit can
be charged they are excellent candidates for sorptive interactions
with the soil mineral phase. Such interactions would even be
enforced if the peptide moiety occurs in association with carbohydrates as is the case for microbial cell wall residues. Their
increasing contribution to the organic matter of particle fractions
with decreasing size may thus be related to their preferential
adsorption to surfaces of mineral oxides. In calcareous soils, Ca2þ
ions are likely to be involved as bridging elements. On the other
hand, the fact that peptides also make up the dominant N form in
peat deposits indicates that apart from physical protection via
mineral interactions other or additional mechanisms must aggravate their degradation potential. Most likely steric hindrance
(Knicker and Lüdemann, 1995), encapsulation (Knicker and
Hatcher, 1997), chirality (Kimber et al., 1990; Amelung and Zhang,
2001; Amelung et al., 2006) or protective molecular properties
(Rillig et al., 2007) contribute to their recalcitrance.
Summarizing the observations and considerations discussed in
the present work, one can conclude that not only SON but
in particular peptide-like materials are indeed under-rated players
in the C sequestration in soils. The information of this review clearly
points to the conclusion that they are an important soil organic
matter fraction, but how they affect SOM or even soil functionality
is still in the dark. Therefore, directing the research effort towards
an understanding on the relationship between peptide chemistry
and SOM functionality may be a future important step for
improving our knowledge of SOM formation and its role of SOM as
a C sink.
Acknowledgment
Dr. F.J. González-Vila is gratefully acknowledged for helpful
discussion and suggestions. Dr. S.I. Nilsson, Dr. G. Almendros,
Dr. D.W. Hopkins and Dr. J.P. Shimel are thanked for critical proof
reading and their constructive comments. I also want to thank the
anonymous reviewers for their helpful suggestions. Financial
support was obtained from the Ministerio de Ciencia e Innovación
(Spain) and the Deutsche Forschungsgesellschaft (DFG) (Germany).
References
Abe, T., Watanabe, A., 2004. X-ray photoelectron spectroscopy of nitrogen functional groups in soil humic acids. Soil Science 169, 35e43.
Almendros, G., Polo, A., Lobo, M.C., Ibanez, J., 1984. Contribución al estudio de la
influencia de los incendios forestales en las caracteristicas de la materia
orgánica del suelo: tranformationes del humus por ignición en condiciones
controladas de laboratorio. Revue d’Écologie et de Biologie du Sol 21, 145e160.
Almendros, G., Martín, F., González-Vila, F.J., 1988. Effects of fire on humic and lipid
fractions in a Dystric Xerochrept in Spain. Geoderma 42, 115e127.
Almendros, G., González-Vila, F.J., Martín, F., 1990. Fire-induced transformation of
soil organic matter from an oak forest. An experimental approach to the effects
of fire on humic substances. Soil Science 149, 158e168.
Almendros, G., Knicker, H., González Vila, J.F., 2003. Rearrangement of carbon and
nitrogen forms in peat after progressive thermal oxidation as determined by
solid-state 13C- and 15N-NMR spectroscopy. Organic Geochemistry 34,
1559e1568.
Amelung, W., Zhang, X., 2001. Determination of amino acid enantiomers in soils.
Soil Biology and Biochemistry 33, 553e562.
Amelung, W., Zhang, X., Flach, K.W., 2006. Amino acids in grassland soils: climatic
effects on concentrations and chirality. Geoderma 130, 207e217.
Aufdenkampe, A.K., Hedges, J.I., Richey, J.E., Krusche, A.V., Llerena, C.A., 2001.
Sorptive fractionation of dissolved organic nitrogen and amino acids onto fine
sediments within the Amazon Basin. Limnology and Oceanography 46,
19211935.
1127
Baldock, J.A., Skjemstad, J.O., 2000. Role of soil matrix and minerals in protecting
natural organic materials against biological attack. Organic Geochemistry 31,
697e710.
Baldock, J.A., Oades, J.M., Waters, A.G., Peng, X., Vassallo, A.M., Wilson, M.A., 1992.
Aspects of the chemical structure of soil organic materials as revealed by solidstate 13C NMR spectroscopy. Biogeochemistry 16, 1e42.
Bardgett, R.D., Streeter, T.C., Bol, R., 2003. Soil microbes compete effectively with
plants for organic-nitrogen inputs to temperate grasslands. Ecology 84,
1277e1287.
Behrens, A., Maie, N., Knicker, H., Kögel-Knabner, I., 2002. MALDI-TOF mass spectrometry and PDS fragmentation as means for the analysis of condensed
tannins in plant leaves and needles. Phytochemistry 62, 1159e1170.
Berg, B., Ekbohm, G., 1991. Litter mass loss rates and decomposition patterns in
some needle and leaf litter types. Long-term decomposition in a Scots pine
forest VII. Canadian Journal of Botany 69, 1449e1456.
Bradley, R.L., Titus, B.D., Preston, C.P., 2000. Changes to mineral N cycling and
microbial communities in black spruce humus after additions of (NH4)2SO4 and
condensed tannins extracted from Kalima angustifolia and balsam fir. Soil
Biology and Biochemistry 32, 1227e1240.
Carreiro, M.M., Sinsabaugh, R.L., Repert, D.A., Parkhurst, D.F., 2000. Microbial
enzyme shifts explain litter decay responses to stimulated nitrogen deposition.
Ecology 81, 2359e2365.
Charlton, A.J., Baxter, N.J., Lilley, T.H., Haslam, E., Mc Donald, C.J., Williamson, M.P.,
1996. Tannin interactions with a full-length human salivary proline-rich protein
display a stronger affinity than with single proline-rich repeats. FEBS Letters
382, 289e292.
Cheng, C.N., Shufeldt, R.C., Stevenson, F.J., 1975. Amino acid analysis of soils and
sediments: extraction and desalting. Soil Biology and Biochemistry 7, 143e151.
Christensen, B.T., 1992. Physical fractionation of soil and organic matter in primary
particle size and density separates. Advances in Soil Science 20, 1e90.
Claessen, D., Stokroos, L., Deelstra, H.J., Penninga, N.A., Bormann, C., Salas, J.A.,
Dijkhuizen, L., Wösten, H.A.B., 2004. The formation of the rodlet layer of
streptomycetes is the result of the interplay between rodlins and chaplins.
Molecular Microbiology 53, 433e443.
Colman, B.P., Fierer, N., Schimel, J.P., 2007. Abiotic nitrate incorporation in soil: it is
real? Biogeochemistry 84, 161e169.
Colman, B., Fierer, N., Schimel, J., 2008. Abiotic nitrate incorporation, anaerobic
microsites, and the ferrous wheel. Biogeochemistry 91, 223e227.
Cookson, W.R., Murphy, D.V., 2004. Quantifying the contribution of dissolved
organic matter to soil nitrogen cycling using 15N isotopic pool dilution. Soil
Biology and Biochemistry 36, 2097e2100.
Craine, J.M., Morrow, C., Fierer, N., 2007. Microbial nitrogen limitation increases
decomposition. Ecology 88, 2105e2113.
Curry, G.B., Theng, B.K.G., Zheng, H., 1994. Amino acid distribution in a LoessPalaeosol sequence near Luochuan, loess plateau, China. Organic Geochemistry
22, 287e298.
Dail, D.B., Davidson, E.A., Chorover, J., 2001. Rapid abiotic transformation of nitrate
in an acid forest soil. Biogeochemistry 54, 131e146.
Davidson, E.A., Chorover, J., Dail, D.B., 2003. A mechanism of abiotic immobilization
of nitrate in forest ecosystems: the ferrous wheel hypothesis. Global Change
Biology 9, 228e236.
Davidson, E.A., Dail, D., Chorover, J., 2008. Iron interference in the quantification of
nitrate in soil extracts and its effect on hypothesized abiotic immobilization of
nitrate. Biogeochemistry 90, 65e73.
de Leeuw, J.W., Largeau, C., 1993. A review of macromolecular organic compounds
that comprise living organisms and their role in kerogen, coal, and petroleum
formation. In: Engel, M.H., Macko, S.A. (Eds.), Organic Geochemistry. Plenum
Press, New York, N.Y, pp. 23e72.
del Río, J.C., Olivella, M.A., Knicker, H., de las Heras, F.X.C., 2004. Preservation of
peptide moieties in three Spanish sulfur-rich tertiary kerogens. Organic
Geochemistry 35, 993e999.
Dick, D.P., Goncalves, C.N., Dalmolin, R.D., Knicker, H., Klamt, E., Kögel-Knabner, I.,
Somoes, M.L., Martin-Neto, L., 2005. Characteristics of soil organic matter in
top- and subsoils of different Brazilian Ferralsols under native vegetation.
Geoderma 124, 319e333.
Ding, X., Henrichs, S.M., 2002. Adsorption and desorption of proteins and polyamino acids by clay minerals and marine sediments. Marine Chemistry 77,
225e237.
Driver, J.D., Holben, W.E., Rillig, M.C., 2005. Characterization of glomalin as a hyphal
wall component of arbuscular mycorrhizal fungi. Soil Biology and Biochemistry
37, 101e106.
Fernández, I., Cabaneiro, A., Carballas, T., 1997. Organic matter changes immediately
after a wildfire in an Atlantic forest soil and comparison with laboratory soil
heating. Soil Biology and Biochemistry 29, 1e11.
Finzi, A.C., Berthrong, S.T., 2005. The uptake of amino acids by microbes and trees in
three cold-temperate forests. Ecology 86, 3345e3353.
Fitzhugh, R.D., Lovett, G.M., Venterea, R.T., 2003. Biotic and abiotic immobilization
of ammonium, nitrite, and nitrate in soils developed under different tree
species in the Catskill Mountains, New York, USA. Global Change Biology 9,
1591e1601.
Friedel, J.K., Scheller, E., 2002. Composition of hydrolysable amino acids in soil
organic matter and soil microbial biomass. Soil Biology and Biochemistry 34,
315e325.
Galloway, J.N., Dentener, F.J., Capone, D.G., Boyer, E.W., Howarth, R.W.,
Seitzinger, S.P., Asner, G.P., Cleveland, C.C., Green, P.A., Holland, E.A., Karl, D.M.,
1128
H. Knicker / Soil Biology & Biochemistry 43 (2011) 1118e1129
Michaels, A.F., Porter, J.H., Townsend, A.R., Vörösmarty, C.J., 2004. Nitrogen
cycles: past, present, and future. Biogeochemistry 70, 153e226.
Gärdenäs, A.I., Ågren, G.I., Bird, J.A., Clarholm, M., Hallin, S., Ineson, P., Kätterer, T.,
Knicker, H., Nilsson, S.I., Näsholm, T., Ogle, S., Paustian, K., Persson, T.,
Stendahl, J., 2011. Knowledge gaps in soil carbon and nitrogen interactions From molecular to global scale. Soil Biology and Biochemistry 43, 702e717.
González-Pérez, J.A., González-Vila, F.J., Almendros, G., Knicker, H., 2004. The effect of
fire on soil organic matter - a review. Environmental International 30, 855e870.
Hagedorn, F., Schleppi, P., Waldner, P., Flühler, H., 2000. Export of dissolved organic
carbon and nitrogen from Gleysol dominated catchments e the significance of
water flow paths. Biogeochemistry 50, 137e161.
Hagedorn, F., Spinnler, D., Siegwolf, R., 2003. Increased N deposition retards
mineralization of old soil organic matter. Soil Biology and Biochemistry 35,
1683e1692.
Haslam, S.F.I., Chudek, J.A., Goldspink, C.R., Hopkins, D.W., 1998. Organic matter
accumulation following fires in a moorland soil chronosequence. Global Change
Biology 4, 305e313.
Hatcher, P.G., Spiker, E.C., 1988. Selective degradation of plant biomolecules. In:
Frimmel, F.H., Christman, R.F. (Eds.), Humic Substances and Their Role in the
Environment. Wiley, New York, pp. 59e74.
Hatcher, P.G., Spiker, E.C., Szeverenyi, N.M., Maciel, G.E., 1983. Selective preservation
and origin of petroleum-forming aquatic kerogen. Nature 305, 498e501.
Hernández, T., García, C., Reinhardt, I., 1997. Short-term effect of wildfire on the
chemical, biochemical and microbiological properties of Mediterranean pine
forest soils. Biology and Fertility of Soils 25, 109e116.
Hessen, D.O., Agren, G.I., Anderson, T.R., Elser, J.J., de Ruiter, P.C., 2004. Carbon
sequestration in ecosystems, the role of stoichiometry. Ecology 85, 1179e1192.
Hobbie, S.E., Vitousek, P.M., 2000. Nutrient limitation of decomposition in Hawaiian
forests. Ecology 81, 1867e1877.
Högberg, P., 2007. Nitrogen impacts on forest carbon. Nature 447, 781e782.
Hopkins, D.W., Odowd, R.W., Shiel, R.S., 1997. Comparison of D- and L-amino acid
metabolism in soils with differing microbial biomass and activity. Soil Biology &
Biochemistry 29, R1eR2, vol. 29, pg 23, 1997.
Hu, S., Chapin III, F.S., Firestone, M.K., Field, C.B., Chiariello, N.R., 2001. Nitrogen
limitation of microbial decomposition in a grassland under elevated CO2.
Nature 409, 188e190.
Ikan, R., Rubinsztain, Y., Nissenbaum, A., Kaplan, I.R., 1996. Geochemical aspects of
the Maillard reaction. In: Ikan, R. (Ed.), The Maillard Reaction; Consequences for
the Chemical and Life Sciences. John Wiley & Sons, Chichester, pp. 1e25.
Kaiser, K., Zech, W., 2000. Sorption of dissolved organic nitrogen by acid subsoil
horizons and individual mineral phases. European Journal of Soil Science 51,
403e411.
Kaye, J.P., Hart, S.C., 1997. Competition for nitrogen between plants and soil
microorganisms. Trends in Ecology & Evolution 12, 139e143.
Kimber, R.W.L., Nannipieri, P., Ceccanti, B., 1990. The degree of racemization of
amino acids released by hydrolysis of humic-protein complexes: implications
for age assessment. Soil Biology and Biochemistry 22, 181e185.
Kleber, M., Sollins, P., Sutton, R., 2007. A conceptual model of organo-mineral
interactions in soils: self-assembly of organic molecular fragments into zonal
structures on mineral surfaces. Biogeochemistry 85, 9e24.
Knicker, H., Hatcher, P.G., 1997. Survival of protein in an organic-rich sediment possible protection by encapsulation in organic matter. Naturwissenschaften
84, 231e234.
Knicker, H., Hatcher, P.G., 2001. Sequestration of organic nitrogen in the sapropel
from Mangrove Lake, Bermuda. Organic Geochemistry 32, 733e744.
Knicker, H., Lüdemann, H.-D., 1995. N-15 and C-13 CPMAS and solution NMR studies
of N-15 enriched plant material during 600 days of microbial degradation.
Organic Geochemistry 23, 329e341.
Knicker, H., Skjemstad, J.O., 2000. Nature of organic carbon and nitrogen in physically protected organic matter of some Australian soils as revealed by solidstate 13C and 15N NMR spectroscopy. Australian Journal of Soil Research,
113e127.
Knicker, H., Fründ, R., Lüdemann, H.-D., 1993. The chemical nature of nitrogen in soil
organic matter. Naturwissenschaften 80, 219e221.
Knicker, H., Almendros, G., González-Vila, F.J., Martín, F., Lüdemann, H.-D., 1996a.
13
C- and 15N-NMR spectroscopic examination of the transformation of organic
nitrogen in plant biomass during thermal treatment. Soil Biology and
Biochemistry 28, 1053e1060.
Knicker, H., Scaroni, A.W., Hatcher, P.G., 1996b. 13C and 15N NMR spectroscopic
investigation on the formation of fossil algal residues. Organic Geochemistry 24,
661e669.
Knicker, H., Schmidt, M.W.I., Kögel-Knabner, I., 2000. Immobilization of peptides in
fine particle size separates of soils as revealed by NMR spectroscopy. Soil
Biology and Biochemistry 32, 241e252.
Knicker, H., del Río, J.C., Hatcher, P.G., Minard, R.D., 2001. Identification of protein
remnants in insoluble geopolymers using TMAH thermochemolysis/GC-MS.
Organic Geochemistry 32, 397e409.
Knicker, H., Hatcher, P.G., González-Vila, F.J., 2002. Formation of heteroaromatic
nitrogen after prolonged humification of vascular plant remains as revealed by
nuclear magnetic resonance spectroscopy. Journal of Environmental Quality 31,
444e449.
Knicker, H., González-Vila, F.J., Polvillo, O., González, J.A., Almendros, G., 2005. Fireinduced transformation of C- and N- forms in different organic soil fractions
from a Dystric Cambisol under a Mediterranean pine forest (Pinus pinaster). Soil
Biology and Biochemistry 37, 701e718.
Knicker, H., Almendros, G., González-Vila, F.J., González-Pérez, J.A., Polvillo, O., 2006.
Characteristic alterations of quantity and quality of soil organic matter caused
by forest fires in continental Mediterranean ecosystems: a solid-state 13C NMR
study. European Journal of Soil Science 57, 558e569.
Knicker, H., Hilscher, A., González-Vila, F.J., Almendros, G., 2008. A new conceptual
model for the structural properties of char produced during vegetation fires.
Organic Geochemistry 39, 935e939.
Knicker, H., 2000. Biogenic nitrogen in soils as revealed by solid-state carbon-13
and nitrogen-15 nuclear magnetic resonance spectroscopy. Journal of Environmental Quality 29, 715e723.
Knicker, H., 2004. Stabilization of N-compounds in soil and organic matter rich
sediments - What is the difference? Marine Chemistry 92, 167e195.
Knicker, H., 2007. Vegetation fires and burnings, how does char input affect the
nature and stability of soil organic nitrogen and carbon? - a review. Biogeochemistry 85, 91e118.
Knicker, H., 2010. “Black nitrogen” - an important fraction in determining the
recalcitrance of charcoal. Organic Geochemistry 41, 947e950.
Kuiters, A.T., 1990. Role of phenolic substances from decomposing forest litter in
plant-soil interactions. Acta Botanica Neerlandica 39, 329e348.
Ladd, J.N., Butler, J.H.A., 1975. Humus-enzyme systems and synthetic organic
polymer-enzyme analogs. In: Paul, E.A., McLaren, A.D. (Eds.), Soil Biochemistry.
Marcel Dekker, New York, pp. 143e194.
Leinweber, P., Schulten, H.-R., 2000. Nonhydrolyzable forms of soil organic
nitrogen: extractability and composition. Journal of Plant Nutrition and Soil
Science 163, 433e439.
Leinweber, P., Kruse, J., Walley, F.L., Gillespie, A., Eckhardt, K.-U., Blyth, R.I.R.,
Regier, T., 2007. Nitrogen K-edge XANES - an overview of reference compounds
used to identify ’unknown’ organic nitrogen in environmental samples. Journal
of Synchrotron Radiation 14, 500e511.
Leinweber, P., Walley, F., Kruse, J., Jandl, G., Eckhardt, K.-U., Blyth, R.I.R., Regier, T.,
2009. Cultivation affects soil organic nitrogen: pyrolysis- mass spectrometry
and nitrogen K-edge XANES spectroscopy evidence. Soil Science Society of
America Journal 73, 82e92.
Lipson, D., Näsholm, T., 2001. The unexpected versatility of plants: organic nitrogen
use and availability in terrestrial ecosystems. Oecologia 128, 305e316.
Lovelock, C.E., Wright, S.F., Clark, D.A., Ruess, R.W., 2004. Soil stocks of glomalin
produced by arbuscular mycorrhizal fungi across a tropical rain forest landscape. Journal of Ecology 92, 278e287.
Luck, G., Liao, H., Murray, N.J., Grimmer, H.R., Warminski, E.E., Williamson, M.P.,
Lilley, T.H., Haslam, E., 1994. Polyphenols, astringency and proline-rich proteins.
Phytochemistry 37, 357e371.
Maie, N., Behrens, A., Knicker, H., Kögel-Knabner, I., 2002. Changes in the structure
and protein binding ability of condensed tannins during decomposition of fresh
plant material. Soil Biology and Biochemistry 35, 577e589.
Maie, N., Knicker, H., Watanabe, A., Kimura, M., 2006a. Heterocyclic N in the highly
humified humic acids extracted from the subsoil of paddy fields and surface
ando soils. Organic Geochemistry 37, 12e19.
Maie, N., Parish, K.J., Watanabe, A., Knicker, H., Benner, R., Abe, T., Kaiser, K., Jaffé, R.,
2006b. Chemical characteristics of dissolved organic nitrogen in an oligotrophic
subtropical coastal ecosystem. Geochimica et Cosmochimica Acta 70, 4491e4506.
Martens, D.A., Loeffelmann, K.L., 2003. Soil amino acid composition quantified by
acid hydrolysis and anion chromatography-pulsed amperometry. Journal of
Agricultural and Food Chemistry 51, 6521e6529.
Mayer, L.M., 1994. Surface area control of organic carbon accumulation in continental shelf sediments. Geochimica et Cosmochimica Acta 58, 1271e1284.
McDowell, W.H., Magill, A.H., Aitkenhead-Peterson, J.A., Aber, J.D., Merriam, J.L.,
Kaushal, S.S., 2004. Effects of chronic nitrogen amendment on dissolved organic
matter and inorganic nitrogen in soil solution. Forest Ecology and Management
196, 29e41.
Meetani, M.A., Basile, F., Voorhees, K.J., 2003. Investigation of pyrolysis residues of
poly(amino acids) using matrix assisted laser desorption ionization-time of flightmass spectrometry. Journal of Analytical and Applied Pyrolysis 68/69, 101e113.
Michalzik, B., Matzner, E., 1999. Dynamics of dissolved organic nitrogen and carbon
in a Central European Norway spruce ecosystem. European Journal of Soil
Science 50, 579e590.
Monteil-Rivera, F., Brouwer, E.B., Masset, S., Deslandes, Y., Dumonceau, J., 2000.
Combination of X-ray photoelectron and solid-state 13C nuclear magnetic
resonance spectroscopy in the structural characterisation of humic acids.
Analytica Chemica Acta 424, 243e255.
Näsholm, T., Nordin, A., Ekblad, A., Giesler, R., Högberg, M., Högberg, P., 1998. Boreal
forest plants take up organic nitrogen. Nature 392, 914e917.
Neff, J.C., Chapin III, F.S., Vitousek, P.M., 2003. Breaks in the cycle: dissolved organic
nitrogen in terrestrial ecosystems. Frontiers in Ecology and the Environment 1,
205e211.
Northup, R.R., Yu, Z., Dahlgren, R.A., Vogt, K., 1995. Polyphenol control of nitrogen
release from pine litter. Nature 377, 227e228.
O’Dowd, R.W., Hopkins, D.W., 1998. Mineralization of carbon from D- and L-amino
acids and D-glucose in two contrasting soils. Soil Biology & Biochemistry 30,
2009e2016.
O’Dowd, R.W., Barraclough, D., Hopkins, D.W., 1999. Nitrogen and carbon mineralization in soil amended with D- and L-leucine. Soil Biology & Biochemistry 31,
1573e1578.
Omoike, A., Chorover, J., 2004. Spectroscopic Study of extracellular polymeric
substances from Bacillus subtilis: aqueous chemistry and adsorption effects.
Biomacromolecules 5, 1219e1230.
H. Knicker / Soil Biology & Biochemistry 43 (2011) 1118e1129
Owen, A.G., Jones, D.L., 2001. Competition for amino acids between wheat roots and
rhizosphere microorganisms and the role of amino acids in plant N acquisition.
Soil Biology and Biochemistry 33, 651e657.
Paul, J.P., Williams, B.L., 2005. Contribution of a-amino N to extractable organic
nitrogen (DON) in three soil types from the Scottish uplands. Soil Biology and
Biochemistry 37, 801e803.
Paungfoo-Lonhienne, C., Lonhienne, T.G.A., Rentsch, D., Robinson, N., Christie, M.,
Webb, R.I., Gamage, H.K., Carroll, B.J., Schenk, P.M., Schmidt, S., 2008. Plants can
use protein as a nitrogen source without assistance from other organisms.
Proceedings of the National Academy of Sciences 105, 4524e4529.
Perakis, S.S., Hedin, L.O., 2002. Nitrogen loss from unpolluted South American
forests mainly via dissolved organic compounds. Nature 415, 416e419.
Piper, T.J., Posner, A.M., 1972. Humic acid nitrogen. Plant and Soils 36, 595e598.
Potthast, A., Schiene, R., Fischer, K., 1996. Structural investigations of N-modified
lignins by 15N-NMR spectroscopy and possible pathways for formation of
nitrogen containing compounds related to lignin. Holzforschung 50, 554e562.
Qualls, R.G., Haines, B.L., Swank, W.T., Tyler, S.W., 2002. Retention of soluble organic
nutrients by a forested ecosystem. Biogeochemistry 61, 135e171.
Reeves, J.B., Francis, B.A., 1998. Pyrolysis-gas chromatography for the analysis of
proteins: with emphasis and forages. In: Stankiewicz, B.A., van Bergen, P.F.
(Eds.), Nitrogen-containing Macromolecules in the Bio- and Geoshpere. American Chemical Society, Washington, DC, pp. 47e62.
Rillig, M.C., Wright, S.F., Nichols, K.A., Schmidt, W.F., Torn, M.S., 2001. Large
contribution of arbuscular mycorrhizal fungi to soil carbon pools in tropical
forest soils. Plant and Soil 233, 167e177.
Rillig, M.C., Caldwell, B.A., Wösten, H.A.B., Sollins, P., 2007. Role of proteins in soil
carbon and nitrogen storage: controls on persistence. Biogeochemistry 85, 25e44.
Rinderknecht, H., Jurd, L., 1958. A novel non-enzymatic browning reaction. Nature
181, 1268e1269.
Rosier, C.L., Hoye, A.T., Rillig, M.C., 2006. Glomalin-related soil protein: assessment
of current detection and quantification tools. Soil Biology and Biochemistry 38,
2205e2211.
Rothstein, D., 2009. Soil amino-acid availability across a temperate-forest fertility
gradient. Biogeochemistry.
Schimel, J.P., Bennett, J., 2004. Nitrogen mineralization: challenges of a changing
paradigm. Ecology 85, 591e602.
Schindler, F.V., Mercer, E.J., Rice, J.A., 2007. Chemical characteristics of glomalinrelated soil protein (GRSP) extracted from soils of varying organic matter
content. Soil Biology and Biochemistry 39, 320e329.
Sharma, R.K., Chan, W.G., Seeman, J.I., Hajaligol, M.R., 2003. Formation of low
molecular weight heterocycles and polycyclic aromatic compounds (PACs) in
the pyrolysis of a-amino acids. Journal of Analytical and Applied Pyrolysis 66,
97e121.
Shindo, H., Urabe, M.,1993. Changes in the humus composition of volcanic ash soils by
heating at various temperatures. Soil Science and Plant Nutrition 39, 189e192.
Shindo, H., Matsui, Y., Higashi, T., 1986. A possible source of humic acids in volcanic
ash soils in Japan - Charred residue of Miscanthus sinensis. Soil Science 141,
84e87.
Skjemstad, J.O., Janik, L.J., Head, M.J., McClure, S.G., 1993. High energy ultraviolet
photo-oxidation: a novel technique for studying physically protected organic
matter in clay- and silt-sized aggregates. Journal of Soil Science 44, 485e499.
Smernik, R., Baldock, J., 2005. Does solid-state 15N NMR spectroscopy detect all soil
organic nitrogen? Biogeochemistry 75, 507e528.
Sollins, P., Homann, P., Caldwell, B.A., 1996. Stabilization and destabilization of soil
organic matter: mechanisms and controls. Geoderma 74, 65e105.
Sollins, P., Swanston, C., Kleber, M., Filley, T., Kramer, M., Crow, S., Caldwell, B.A.,
Lajtha, K., Bowden, R., 2006. Organic C and N stabilization in a forest soil:
evidence from sequential density fractionation. Soil Biology and Biochemistry
38, 3313e3324.
1129
Steinberg, P.D., Rillig, M.C., 2003. Differential decomposition of arbuscular mycorrhizal fungal hyphae and glomalin. Soil Biology and Biochemistry 35, 191e194.
Stevenson, F.J., 1982. Humus Chemistry. Wiley, New York, N.Y.
Theis, E.R., 1945. The collagen-quinone reaction. I. Fixation and thermolability as
a function of pH values. Journal of Biological Chemistry 157, 23e33.
Theng, B.K.G., Churchman, G.J., Newman, R.H., 1986. The occurrence of interlayer
clay-organic complexes in two New Zealand soils. Soil Science 142, 262e266.
Theng, B.K.G., Tate, K.R., Becker-Heidmann, P., 1992. Towards establishing the age,
location, and identity of the inert soil organic matter of a spodosol. Zeitschrift
für Pflanzenernährung und Bodenkunde 155, 181e184.
Thorn, K.A., Cox, L.G., 2009. N-15 NMR spectra of naturally abundant nitrogen in soil
and aquatic natural organic matter samples of the International Humic
Substances Society. Organic Geochemistry 40, 484e499.
Thorn, K.A., Mikita, M.A., 1992. Ammonia fixation by humic substances: a nitrogen15 and carbon-13 NMR study. The Science of the Total Environment 113, 67e87.
Van Sumere, C.F., Albrecht, J., Dedonder, A., DePooter, H., Pé, I., 1975. Plant proteins
and phenolics. In: Harborne, J.B., Van Sumere, C.F. (Eds.), Annual Proceedings of
the Phytochemical Society, pp. 211e264.
Vitousek, P.M., Aber, J.D., Howarth, R.W., Likens, G.E., Matson, P.A., Schindler, D.W.,
Schlesinger, W.H., Tilman, D.G., 1997. Human alteration of the global nitrogen
cycle: sources and consequences. Ecological Applications 7, 737e750.
von Lützow, M., Kögel-Knabner, I., Ekschmitt, K., Matzner, E., Guggenberger, G.,
Marschner, B., Flessa, H., 2006. Stabilization of organic matter in temperate
soils: mechanisms and their relevance under different soil conditions a review. European Journal of Soil Sciences 57, 426e445.
Waksman, S.A., Iyer, N.R.N., 1932. Contribution to our knowledge of chemical nature
and origin of humus. Soil Science 34, 43e69.
Wang, X.-C., Lee, C., 1993. Adsorption and desorption of aliphatic amines, amino
acids and acetate by clay minerals and marine sediments. Marine Chemistry 44,
1e23.
Wang, W.J., Baldock, J.A., Dalal, R.C., Moody, P.W., 2004. Decomposition dynamics of
plant materials in relation to nitrogen availability and biochemistry determined
by NMR and wet-chemical analysis. Soil Biology and Biochemistry 36,
2045e2058.
Wösten, H.A.B., 2001. Hydrophobins: multipurpose proteins. Annual Review of
Microbiology 55, 625e646.
Wright, S.F., Anderson, R.L., 2000. Aggregate stability and glomalin in alternative
crop rotations for the central Great Plains. Biology and Fertility of Soils 31,
249e253.
Wright, S.F., Upadhyaya, A., 1998. A survey of soils for aggregate stability and glomalin, a glycoprotein produced by hyphae of arbuscular mycorrhizal fungi.
Plant and Soil 198, 97e107.
Wright, S.F., Franke-Snyder, M., Morton, J.B., Upadhyaya, A., 1996. Time-course study
and partial characterization of a protein on hyphae of arbuscular mycorrhizal
fungi during active colonization of roots. Plant and Soil 181, 193e203.
Xu, X., Stange, C., Richter, A., Wanek, W., Kuzyakov, Y., 2008. Light affects competition for inorganic and organic nitrogen between maize and rhizosphere
microorganisms. Plant and Soil 304, 59e72.
Yu, Z., Zhang, Q., Kraus, T.E.C., Dahlgren, R.A., Anastasio, C., Zasoski, R.J., 2002.
Contribution of amino compounds to dissolved organic nitrogen in forest soils.
Biogeochemistry 61, 173e198.
Zang, X., van Heemst, D.H., Dria, K.J., Hatcher, P.G., 2000. Encapsulation of protein in
humic acid from a histosol as an explanation for the occurrence of organic
nitrogen in soil and sediment. Organic Geochemistry 31, 679e695.
Zimmerman, A.R., Goyne, K.W., Chorover, J., Komarneni, S., Brantley, S.L., 2004.
Mineral mesopore effects on nitrogenous organic matter adsorption. Organic
Geochemistry 35, 355e375.
Zucker, W.V., 1983. Tannins: does structure determine function? An ecological
perspective. The American Naturalist 121, 335e365.