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Soil Biology & Biochemistry 43 (2011) 1118e1129

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Soil Biology & Biochemistry

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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

Article history:Received 9 September 2010Received in revised form23 February 2011Accepted 25 February 2011Available online 11 March 2011

Keywords:Soil organic matterC and N humificationC-sinkN-cycleSoil organic nitrogenSoil peptides

* Tel.: þ34 954624711; fax: þ34 954624002.E-mail address: knicker@irnase.csic.es.

0038-0717/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.soilbio.2011.02.020

a b s t r a c t

The availability of Soil Organic Nitrogen (SON) determines soil fertility and biomass production to a greatextent. SON also affects the amounts and turnover rates of the soil organic carbon (SOC) pools. Althoughthere is increasing awareness of the impact of the nitrogen (N) cycle on the carbon (C) cycle, the extent ofthis 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 currentideas about SON stabilization are summarized in this paper in order to develop an advanced concept ofthe role of N on C sequestration. Modeling global C-cycling, it was already recognized that SON and SOCare closely coupled via biomass production and degradation. However, the narrow C/N ratio of maturesoil organic matter (SOM) shows further that the impact of SON on the refractory SOM is beyond that ofdetermining the size of the active cycling entities. It affects the quantity of the slow cycling pool and asa major contributor it also determines its chemical composition. Although the chemical nature of SON isstill not very well understood, both improved classical wet chemical analyses and modern spectroscopictechniques provide increasing evidence that almost the entire organic N in fire-unaffected soils is boundin 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 onpublished 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 theslow cycling SOM fraction and even the potential of a soil to act as a C sink. Pyrogenic organic N, whichderives mostly from incomplete combustion of plant and litter peptides is another under-rated player insoil organic matter preservation. In fire-prone regions, its formation represents a major N stabilizationmechanism, leading to the accumulation of heterocyclic aromatic N, the stability of which is still notelaborated. The concept of peptide-like compounds as a key in SOM-sequestration implies that for animproved evaluation of the potential of soils as C-sinks our research focus as to be directed to a betterunderstanding of their chemistry and of the mechanisms which are responsible for their resistanceagainst biochemical degradation in soils.

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1. Introduction

Presently, considerable attention is given to the potential of soilsto act as C sinks. This is reflected in the emphasis to determineturnover rates, sizes and composition of their different C pools aswell as in developing and elucidation of hypotheses addressing themechanisms involved in C stabilization (Sollins et al., 1996; vonLützow et al., 2006; Kleber et al., 2007). However, although it isalready recognized that SON and SOC are closely coupled viabiomass production and degradation and as such nitrogen has

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amajor impact on the global C and N-cycling (Gärdenäs et al., 2011),up to now, its consideration as important factor in the shaping ofthe recalcitrant SOM pool is still limited.

Nitrogen (N) is a major nutrient element controlling the cyclingof organic matter in the biosphere. As a limiting growth factor, theconcentration and availability of N in both its organic and inorganicforms is closely related to biological productivity in terrestrial andaquatic systems. The significance of this indirect impact on the Ccycle arises from the fact that alteration of the N availability isrelated to changes in the amount of C, immobilized into newlyformed biomass, which subsequently affects both size and qualityof the SOM pools. This is of particular interest in the light ofincreasing N input to soils by anthropogenic sources such asfertilizers, N-oxides depositions from combustion processes orammonia from agricultural sources.

H. Knicker / Soil Biology & Biochemistry 43 (2011) 1118e1129 1119

Once having entered the soil, N is not a static entity, but takespart in a series of interconnected reactions that constitute the Ncycle. This cycle is determined by the competition for this nutrientbetween plants, fungi and soil bacteria. Additionally, a part of the Nsuffers a long-term sequestration by its transformation and incor-poration into biologically refractory organic material. The pathwaysof this incorporation, however, are still not understood and subjectof ongoing debate (Knicker, 2004; Rillig et al., 2007; Colman et al.,2008; Davidson et al., 2008; Thorn and Cox, 2009). The importanceof increasing our knowledge in this field becomes evenmore urgentif one bears in mind that all forms of organic N are connected toa “C-backbone”. This implies that SON has not only an indirect butalso a direct impact on C cycling by determining the quality andquantity of at least the N-containing part of the total soil organicmatter (SOM) pool.

Alteration of the SOM pools by changing the management or byerosion, grazing or burning can in turn affect the N-cycling. Thoseevents alter the amount and quality of the input material and theN-supply which has consequences for N-partitioning between thedifferent soil compartments as there are bioavailable N, bioticallyimmobilized N or otherwise sequestered N.

The described close interrelationship between C and N cyclesclearly demonstrates that a realistic evaluation of the potential ofsoils as a C sink can only be achieved if we can answer the questionof the importance of SON for SOM formation. Accordingly, theintention of the following review is to bring some light on thisaspect bymapping out present knowledge and views about the roleof SON sequestration in the C cycle. It includes the abovementionedinterrelationship between the C and the N cycles but goes beyondthe goal of increasing our knowledge for improving general circu-lation models (Gärdenäs et al., 2011) by emphasizing that thechemistry of SON determines to a large part the composition ofthe refractory SOM pool. This requires a better understanding of thenature of SON, which is the reason why controversially discussedmechanisms of SON formation are presented. Focusing on proteinsand peptides as the main SON source and showing their importantcontribution to SOM, pathways presently suggested to be respon-sible 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 orpeptides

The organic N in soils originates from living organisms where itoccurs 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 compoundsenter the soil during litter fall. In fire-affected ecosystems, incom-plete combustion transforms them into pyrogenic N-heterocyclicstructures (Knicker et al., 1996a, 2008; Knicker, 2010) whichsubsequently are also incorporated into the SOM pool, although therespective mechanisms are not yet understood. With respect tobiogenic N-containing residues on the other hand, it is wellestablished that their microbial decomposition results in aminoacids (by depolymerization) together with NH4

þ, NO3�, H2O and

CO2. Whereas the latter is for the most part released into theatmosphere, the inorganic N compounds can easily be recycled forthe build-up of new biomass, which is associated with an immo-bilization of organic carbon. However, a considerable part of theorganic N escapes complete mineralization and the residues areentering the refractory SOM pool where they will be sequesteredfrom both the N and C cycle. It was estimated that approximately60 Tg N yr�1 is accumulating in terrestrial systems, although thereis still considerable uncertainty about the correct values for N

storage and the production of reactive N from N2 (Galloway et al.,2004).

Although the traditional view of N-cycling in terrestrialecosystems has focused almost exclusively on primary production,plant availability, and loss of inorganic N, there is ample evidencethat in some ecosystems, plants may “short circuit” the terrestrial Ncycle by direct uptake of amino acids from the dissolved organicmatter fraction (Neff et al., 2003). At least in some forest systemsorganic N can represent a substantial fraction of the total plant Nuptake (Schimel and Bennett, 2004). It is reported that in soilsunder temperate forests the N pool representing amino acids canexceed inorganic N pools at low fertility sites (Rothstein, 2009).Other studies have shown that in boreal forest systems, plants areable to take up free amino acids at rates that equal or exceed thoseof inorganic N (Näsholm et al., 1998; Lipson and Näsholm, 2001).Only recently, evidence that even proteins can be used by plantswithout assistance of other organisms has been presented(Paungfoo-Lonhienne et al., 2008). The authors suggested thatroots access proteins by secreting proteolytic enzymes that digestproteins at the root surfaces and possibly also in the apoplast of theroot cortex. Additionally, the authors reported that intact proteinsare taken up in root cells probably by endocytosis. However,whereas many of the in situ observations of amino acid uptake byplants have been made in northern or alpine ecosystems, theevidence is mixed in other environments (Finzi and Berthrong,2005). In some agricultural ecosystems it was demonstrated thatplants 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 ofboth inorganic and organic N (Kaye and Hart, 1997). It is wellestablished that this microbial N immobilization is an effectivemechanism of N retention in soils (Friedel and Scheller, 2002).Application of in situ labeling techniques and measurements of the15N recovery in the biomass of competitive plant species, labeledplants and microbes, indicated the use of the same N source. Anuptake of glycine was evidenced in temperate grasslands, (Bardgettet al., 2003), but found that most of the N was sequestered bymicrobes. Regardless of whether the N uptake occurs in plants ormicroorganisms, the growth and death of the organisms ultimatelyresults in returning N into polymeric forms, which are not directlybioavailable. Since depolymerization is needed to transform poly-meric N to bioavailable dissolved organic nitrogen (DON) (Cooksonand Murphy, 2004), the depolymerization step can be seen asa critical point in maintaining an active N cycle (Schimel andBennett, 2004).

2.2. The role of DON in N sequestration and the controversy aboutits formation and nature

The formation of DON can result directly from microbial turn-over and indirectly through generation of extracellular enzymes. Afurther pathway, which is still subject of a controversy representsthe 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 froma physically or biologically mediated association of dissolvedorganic matter (DOM) with nitrate after its reduction to nitrite byreactive Fe(II) species. The required DOM compounds were sug-gested to be derived from phenols and lignin derivatives that formaromatic N by nitration and nitrosation. An argument supportingthis pathway is that abiotic reactions were evidenced duringlaboratory experiments (Thorn and Mikita, 1992; Potthast et al.,1996). On the contrary, up to now, the respective products havenot been identified in natural DOM. Further arguments against thishypothesis derive from alternative studies, assigning the quick

Fig. 1. Solid-state 15N NMR spectra of 15N-enriched grass char obtained after charringthe sample under oxic conditions for 3 min at 350 �C. Spectra were acquired with thea) Bloch Decay (direct excitation of the 15N nuclei) technique and b) after crosspolarization at the Lehrstuhl für Bodenkunde, TU-München, Germany according to theparameters described in Knicker (2010).

H. Knicker / Soil Biology & Biochemistry 43 (2011) 1118e11291120

disappearance of inorganic N during abiotic N-immobilization to aninterference of Fe during the nitrate determination (Colman et al.,2007, 2008).

With respect to the structural composition of DON, there is stilla paucity of information. In Oa horizon leachates from forest soils inNorthern California about 48e74% of the total DON were recoveredas hydrolyzable amino acids, suggesting that proteins and peptideswere the main contributors (Yu et al., 2002). The contribution offree amino acid/sugar was only about 2e11%. Similarly largeamounts of hydrolyzable amino acids from DON were reportedfrom the mor layer in a pine forest (Hagedorn et al., 2000) and fromthe upper 10 cm of three Scottish soils (Paul and Williams, 2005).Solid-state 15N NMR spectroscopy confirms a high peptide contentof DON (Michalzik and Matzner, 1999). The fact that almost all ofthe 15N NMR intensity can be assigned to amide N and theremaining intensity is attributable to free amino groups of aminoacids allows the conclusion that the non-hydrolyzable DON, is alsoderived from peptideous structures. These structures may haveescaped identification after acid hydrolysis either due to their stericnature or because hydrophobic domains protected them fromchemical degradation. Alternatively, they were protected by asso-ciation 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 theother hand, heterocyclic N can occur as it was demonstrated by thesolid-state 15N NMR spectra of DOM samples from the Everglades(Maie et al., 2006b). Those structures derive tentatively fromcharred litter and humic material that entered the soil system afterthe fire event. After their oxidation, they turned into water solublecompounds that were able to be leached and transported with thesoil solution. However, it has to be mentioned that solid-state 15NNMR spectroscopy has been criticized to underestimate heterocy-clic N in soil organic matter because i) the unfavorable nuclearproperties of 15N and inefficient cross polarization inhibits theirvisibility (Smernik and Baldock, 2005), or due to ii) low sensitivityof this technique (Leinweber et al., 2009). However, the highconsistency of the NMR spectra from 15N-enriched grass acquiredvia direct excitation of the 15N nuclei and via cross polarization(Fig. 1) and the fact that heterocyclic N is clearly visible in solid-state 15N NMR spectra of fire-affected SOM stand against thosearguments (Knicker, 2007).

There is evidence showing that DON can leach from ecosystems,despite a high biotic demand for N (Perakis and Hedin, 2002). Thisfact was suggested to point towards the existence of two pools. Oneof them is labile and can quickly be used by plants and microor-ganisms and the other represents a recalcitrant fraction of the DON,inaccessible for biological growth (Neff et al., 2003). According toNeff et al. (2003), this distinction would place soluble amino acidsin the same general class of terrestrial N as NH4

þ and NO3-, whereasthe recalcitrant DON would function in a manner similar to bulkSOM. However, there is almost no information available eitherabout the decomposability of DON in soils or the mechanismsinvolved in its stabilization. Even so, it can be assumed that DONand DOC do not represent independent SOM pools but are closelyrelated, and determine the turnover of each other.

Studies at sites from the Nantahala Range of the SouthernAppalachian Mountains, NC, USA (Qualls et al., 2002), showed thatthe average DOC/DON ratio (w/w) varied between 34 and 40 in theO, A and B horizons and was about 20 in the C horizon. DOC/DONratios between 32 and 39 were reported for unfertilized forestfloors 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 aminosugars and amino acids. In addition to hydroxyl and carboxylgroups, these compounds also contain amino functions as possible

sorption sites, such alterations of the DON pool are likely to alsohave a considerable impact on the mobility of the bulk DOM andthus on the potential of soils to sequester DOC in mineral-organicmatter associations.

3. Links between N and C immobilization: “microbial Nmining” versus “stoichiometric decomposition”

Since N is a limiting factor for biological productivity andrepresents an important SOM constituent, the sequestration ofbiogenic N is closely related to the formation and accumulation ofhumic material. Thus, it was stated that the N cycle drives to someextent the C cycle (Högberg, 2007). The issue of interacting N and Ccycles deserves an increasing attention because inputs of N to soilsdue to elevated concentration of atmospheric nitric oxides as wellas an increase in ’industrial N-fixation’ for inorganic fertilizers nowexceed the N input frommicrobial N-fixation (Vitousek et al., 1997).The deposition of anthropogenically fixed N has been discussed asa means to explain part of the “missing sink” in the imbalancebetween known CO2 emissions from fossil fuel combustion anddeforestation versus known CO2 accumulation in the atmosphere(Vitousek et al., 1997). Estimates of how much terrestrial C storagecould result from N deposition range between 0.1 and 1.3 Pg C peryear. On the other hand, it was found that under elevated CO2,plants in a California annual grassland increased their N uptake atthe 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 willincrease their foliar biomass and the concentrations of ribulose-1,5-bisphosphate carboxylase/oxygenase. The latter enhances the CO2

assimilation by photosynthesis, leading to higher production ofbiomass which eventually forms litter which will need a longertime for decomposition. It has been found that at places without Nlimitation larger soil N availability decreases the decompositiontime of the litter, but at sites where N limits net primary produc-tion, N availability has no or an inconsistent effect on litterdecomposition (Hobbie and Vitousek, 2000).

Presently, literature reports about two main mechanisms toexplain the interrelationship between N availability and C storage

H. Knicker / Soil Biology & Biochemistry 43 (2011) 1118e1129 1121

in ecosystems. The first, known as “microbial N mining”, suggeststhat low N contents may increase litter decomposition as microbesuse labile substrates to acquire N from recalcitrant organic matter(Craine et al., 2007). According to the authors, this “microbial Nmining” is consistently suppressed by high soil-N-supply orsubstrate-N concentrations. Consequently, it will lead to predic-tions that ecosystem C storage would increase with greater Navailability. This could explain observed declines in decompositionafter N additions (Hagedorn et al., 2003; Wang et al., 2004).Alternatively, the basic “stoichiometric decomposition” theorypredicts that ecosystems will store more C if increasing atmo-spheric CO2 leads to greater substrate C/N ratios. However, as soonas the C/element ratios in food approach those of the consumers,the C-use efficiency in food webs tends to increase, decreasing Csequestration in soils (Hessen et al., 2004). Supporting this view,Wang et al. (2004) found that addition of NO3

� initially promoted Cmineralization of the plant material, but a lower long-termcumulative C mineralization was found. This is in agreement withother findings that large N concentrations promote carbohydratedecomposition during the early stages, but may suppress lignindecomposition in the long run (Berg and Ekbohm, 1991). Mecha-nisms for the suppressive effect of N on long-term decompositionmay include i) inhibition of lignin-degrading enzymes by lowmolecular N compounds (Carreiro et al., 2000), or ii) production oftoxic or inhibitory products or iii) by N sequestration into biologi-cally recalcitrant forms.

4. SON sequestration e still a subject of controversialdiscussion

Major difficulties in the understanding and predicting oforganic-N dynamics and its effect on the C cycle originate from thelack of understanding the mechanisms involved in SONstabilization.

A classical but nowadays highly questioned concept representthe depolymerizationerecondensation pathways (Stevenson,1982). Here it is assumed that naturally occurring macromole-cules are microbially degraded to oligomers and monomers, whichfor the most part are further mineralized. However, a small fractionof those compounds recombines by random condensation intolarger, insoluble and refractory structures, which survive a pro-longed pedogenesis. In line with this pathway, an earlier publica-tion suggested that humic material in soils is formed bycondensation of C]O groups of lignin with NH2-groups ofproteinaceous material to produce Schiff bases (Waksman and Iyer,1932). Others propose abiotic condensation of phenolic or quinonicstructures with N-containing compounds (Theis, 1945;Rinderknecht and Jurd, 1958; Piper and Posner, 1972; Ladd andButler, 1975). In accordance with the abiotic immobilizationa newer report, it was assumed that the above mentioned “ferrouswheel” hypothesis can also occur without the intermediate DON(Fitzhugh et al., 2003).

The formation of heterocyclic N during SON sequestrationreceived some recent support from X-ray photoelectron spectros-copy (Abe and Watanabe, 2004) which allowed the identificationbetween 3 and 20% of the total N as aromatic N in humic acids ofJapanese soils using deconvolution techniques. On the other hand,in another study, the same signals were observed but solelyattributed to amides and ammonium groups (Monteil-Rivera et al.,2000). This indicates the difficulties which are involved in anunbiased 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 differentiationbetween two p*-regions in spectra of unknown environmentalsamples: �400 eV (pyridinic N and some nitrile N) and >400 eV

(amide N, nitro N and pyrrolic N) (Leinweber et al., 2007). However,the authors stated further that a quantitative estimate of theproportions of amide N and pyrrolic N requires a separate deter-mination of one of the compounds, which remains a challenge forforthcoming studies. They assumed that “given the subtle varia-tions in spectral features, even for compounds sharing very similarstructures, it is unlikely that the current N K-edge XANES can beused exclusively to quantitatively determine the chemical compo-sition of an environmental sample”.

The second classical pathway of soil organic matter stabilizationis the selective preservation pathway, first developed for sediments(Hatcher et al., 1983) and later adapted to soil systems (Hatcher andSpiker, 1988). According to it, refractory biopolymers resist a quickbiodegradation and preferentially accumulate, whereas other morelabile compounds are mineralized during the early stages ofhumification (de Leeuwand Largeau,1993). The finding that severalvascular plants, algae, and bacteria produce hydrolysis-resistantbiopolymers that are highly recalcitrant supports this hypothesis.

A more recent adaption of this concept to soil systems statesthat not only chemical properties of biomolecules are responsiblefor their stabilization but additional mechanisms can be active (vonLützow et al., 2006) and may even be of more importance. On theother hand, the fact that organic N is also stabilized in mineral-poorenvironments such as Tangel humus or mor layers, composts, peatsor sapropels indicates that chemical recalcitrance still must be animportant player in N sequestration.

With respect to the chemical nature of this SON, recent NMRstudies gave strong evidence that in fire-unaffected soil systems,proteinaceousmaterial has the potential to be selectively preservedand 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 organicmatter rich deposits (del Río et al., 2004). Their preservation maypartially be caused by unfavorable conditions for microbial survivaland growth. This mechanism is certainly of great importance inpeats (Knicker et al., 2002) and buried soils that suffer from O2depletion or in soils occurring in dry and cold or extremely warmclimatic zones but also in environments with extremely acid oralkaline conditions. Additionally, SON may be physically protectedby incorporation within aggregates (Skjemstad et al., 1993), byencapsulation into hydrophobic macromolecules (Knicker andHatcher, 1997), by sequestration in nanopores that are too smallfor organisms or enzymes to enter and function (Mayer, 1994) ordue to the formation of organo-mineral-associates (Baldock andSkjemstad, 2000). The latter alternative may be due to intercala-tion 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 ofpeptides and proteins in soil

5.1. Interaction with the mineral phase

As mentioned above, many studies have demonstrated the roleof peptides as important constituents of the recalcitrant SON frac-tion. Direct measurements of the chirality of several amino acidsisolated from grassland soils suggested that the age of theirproteins was within the range of several hundred years (Amelunget al., 2006). However, they can be even older as it was indicatedby peptides found in soil layers on the Chinese loess plateauwith anage of more than 10 000 years (Curry et al., 1994) as well as insapropels and peats dated to approximately the same age or evenolder (Knicker and Hatcher, 2001; Knicker et al., 2002). Fig. 2 showsthe respective solid-state 15N NMR solid-state spectra. They are

Peat (650-660 m)

mpp004-001-0 -200 -300

11.6

12.2

15.2

Sapropel (510 cm)

Clay (A horizon)

Atomic C/N-260

Fig. 2. Solid-state 15N NMR spectra of the clay fraction of the A horizon of a Paleosolfrom the Chinese Loess Plateau demineralised with 10% hydrofluoric acid, peat derivedfrom a depths between 650 and 660 cm of the Grober Bolchow, Germany, and theSapropel of the Mangrove Lake, Bermuda (Knicker and Hatcher, 2001).

H. Knicker / Soil Biology & Biochemistry 43 (2011) 1118e11291122

dominated by a signal at �260 ppmwhich is assignable to amide Nand reveals that peptide N constitutes most- if not all - of their SON.In spite of the fact that more and more evidence is reported thatsupports the importance of peptides as constituents of both thelabile and the recalcitrant SON pool, we still are far away fromunderstanding which factors are causing their degradation andwhich mechanisms are responsible for their sequestration.

One mechanisms explaining how supposedly easily degradablebiomolecules may survive on a long-term scale in soil environ-ments. is the “Mesopore-hypothesis”. It was developed based onthe finding that peptides adsorb strongly to clay particles (Wangand Lee, 1993; Aufdenkampe et al., 2001; Ding and Henrichs,2002). The adsorption may physically protect the peptides byrestricting the accessibility to degrading enzymes. This protectionis even increased if the small organic molecules are adsorbed intomesopores <10 nm that are too small to permit the entry andfunctioning of degrading enzymes (Mayer, 1994). This pathway issupported by studies of the sorption behavior of amino acidmonomers and polymers onto fabricated mesoporous and non-porous alumina and silica that indicated that these compoundsmaybe strongly bound within mesopores and thereby undergoa reduced desorption (Zimmerman et al., 2004). Larger proteins, onthe other hand, were excluded from the pores, indicating that themesopore protection hypothesis (Mayer, 1994) is indeed a feasiblemechanism for soil and sedimentary environments.

Alternatively, but also based on adsorption to mineral surfaces,it was suggested that peptideous compounds may form a stableinner organic layer around a mineral surface on which less polarorganics could sorb more readily than onto the highly chargedmineral surfaces (onion-layer hypothesis) (Sollins et al., 2006).Concomitantly, this layer could serve as a reactive chemicaltemplate for further coupling and cross-linking of organic

compounds. This idea of an “onion” layering model is based on thepreferential accumulation of polar carboxyl and cationic aminocompounds by ligand and cation exchange on mineral surfaces. Itwas suggested that a first a contact zone is formed in whichproteinaceous materials are particularly strongly associated to themineral phase. Upon adsorption the proteinaceous material mayunfold which increases its adhesive strength by adding hydro-phobic interactions to electrostatic binding (Kleber et al., 2007). Theamphiphilic character of proteins and peptides could help to createthe next zone, referred to as the hydrophobic zone, which couldprovide a suitable region for sorption of hydrophobic moietiesincluding further amphiphilic components. Sorbed to the hydro-philic exterior of this zone, organic molecules may form the kineticzone that is largely controlled by exchange kinetics. It was sug-gested that movement of organics into and out of this region has tobe regarded as a phase partitioning process. Although still underdebate, this hypothesis may be supported by the finding that theweight of soil density fractions is negatively related to the thicknessof the organic layer around the mineral particle and that the age oforganic 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 cm�3 to 12 in the fractions >2.0 g cm�3, confirming theimportance of SON for the stabilization of SOM via mineral-adsorption.

5.2. Formation of protein-tannin complexes

A further suggested mechanism that could be responsible for atleast part-time stabilization of proteins and peptides in soils is theformation of protein-tannin complexes. Tannins are secondaryplant metabolites occurring in the bark and foliage of most plantspecies. These polymers consist of flavan-3-ol units (condensedtannins) or gallic acid monomers (hydrolyzable tannins). Protein-tannin complexes can be formed in the soil during litter decom-position 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 areable to mineralize tannin compounds (Northup et al., 1995). Otherstudies using matrix-assisted laser desorption ionization time offlight mass spectrometry (MALDI-TOF MS) and solution 13C NMRspectroscopy, examining the chemical alteration of condensedtannins of pine needles and willow leaves during litter degradation(Behrens et al., 2002; Maie et al., 2002) could not confirm the highrefractory nature of condensed tannins. After eight weeks of incu-bation, no signals of condensed tannins were detected by MALDI-TOF MS. Low recoveries were also reported for tannins of Picea spand Kalmia angustifolia L. in a black spruce forest floor horizon(Bradley et al., 2000). This may be explained either by fast degra-dation of the tannins or by hindrance of their extraction due to theirfast and tight binding to SOM, including proteins. Such protein-tannin complexes were suggested to occur either by covalent, ionicor hydrogen bonding (Zucker, 1983). The formation of covalentbonding, however, seems unlikely since the phenols first have to betransformed into quinones and semiquinones that are normally notformed under the redox potentials found in plant cells and soils(Van Sumere et al., 1975). This conclusion is supported by solid-state 15N NMR spectroscopy that failed to identify such condensa-tion products (Knicker, 2004). However, up to now, ionic andhydrogen bonding cannot be excluded. Additional possibilities arehydrophobic interactions that were shown to play an importantrole in protein-tannin complexation in laboratory studies (Lucket al., 1994; Charlton et al., 1996). Nevertheless, summarizing thepresent knowledge about the role of tannins during organic N andin particular during peptide sequestration in soil, we are far froma satisfactory understanding. This is partly because suitable

H. Knicker / Soil Biology & Biochemistry 43 (2011) 1118e1129 1123

approaches to isolate and quantify tannins in degrading litter orhumic samples are still missing but also because this mechanismfor organic matter stabilization has been widely neglected duringthe last years.

5.3. Encapsulation pathway

Since long-term survival of peptide-like compounds is alsoobserved in mineral-poor and tannin-free or depleted environ-ments such as sapropels (Knicker et al., 1996b) and peats (Knickeret al., 2002). In those environments, the survival of proteinaceousmaterial was explained with the encapsulation pathway (Knickerand Hatcher, 1997). According to this conceptual model, peptidesthat are connected to recalcitrant hydrophobic biopolymers will besurrounded by these polymers, and therefore they will be selec-tively enriched during cell degradation, while other, more acces-sible compounds will be consumed by microorganisms (JunctionPathway). Alternatively, during cell lysis proteins and peptides aresandwiched between hydrophobic biopolymer layers (SandwichPathway) and protected from hydrophilic enzymes that will nothave access to certain key structures of the proteinaceous material.Only conditions that alter the steric configuration of thebiopolymer may liberate proteinaceous compounds, so theybecome accessible for microbial activity. Although this mechanismwas first suggested for accumulation of organic matter derivedfrom algae surrounded by such a polyaromatic network, it is notrestricted to subaquatic systems. The mechanism is also possible interrestrial environments where hydrophobic or protective macro-biopolymers such as cell wall components of bacteria or waxes andlignin in plant material occur. It was also suggested that proteinscan even extract into humic acids and that the complete hydrolysisof 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 peptid-eous compounds could be related to their molecular nature. Inorder to fulfill their function in the soil, molecules such as extra-cellular enzymes, surface-active agents or plant hormones musthave some properties that protect them from fast microbialdegradation. They may be methylated or derivatized at certain keygroups that are essential for reorganization by and functioning ofdegrading enzymes. Alternatively, they may contain conforma-tional restrictions and hydrophobic domains which efficientlyprotect them within the soil. In a review, evaluating the role ofproteins in soil carbon and nitrogen storage, Rillig et al. (2007)suggested that protection may be supported by domains formingamyloid aggregates and fibrils that were rather stable againstchemical denaturation and enzymatic hydrolysis. Hydrophobinssecreted by ascomycetes and basidiomycetes (Wösten, 2001)contain such hydrophobic and amyloid domains, and can make upto 10% of the total cellular protein. They are involved in the medi-ation of attachment of the fungi to hydrophobic solids, providehydrophobicity to fruiting bodies and spores and help fungi toescape aqueous environments (Wösten, 2001). Chaplins, which areproteins excreted by Streptomycetes sp. fulfill comparable func-tions (Claessen et al., 2004). However, information about thecontribution of such proteins to SOM or their role for its stabiliza-tion is still not available.

The glycoprotein glomalin is another N-containing moleculewhich is believed to give large contributions to SOM. Rather thanbeing exudated by arbuscular mycorrhiza (AM) it comprises a largefraction of the hyphal walls of the AM fungi (Driver et al., 2005).Therefore, it has been assumed that glomalin has its primary role in

the living fungus, possibly as a structural component (Rillig et al.,2007). In temperate climatic zones, the glomalin concentrationwas reported to vary between 1 and 21 mg g�1 in soil aggregates(Wright et al., 1996).

More than 60 mg of glomalin per cm�3 soil was found ina chronosequence on Hawaii (Rillig et al., 2001). For tropical soils inCosta Rica, 3e5% of the total soil C and N in the top 10 cm wasaccounted for by glomalin (Lovelock et al., 2004). As a result ofa strong relationship between soil aggregate stability and glomalinconcentration (Wright and Upadhyaya, 1998), this glycoprotein hasbeen credited with enhanced ecosystem productivity due toimproved soil aeration, drainage and microbial activity (Lovelocket al., 2004). It is further thought to significantly reduce SOMdegradation by protecting labile compounds within soil aggregatesand thus enhancing C sequestration in soil ecosystems (Wright andAnderson, 2000).

On the other hand, other studies provided evidence that thesignificance of glomalin may be overestimated, since other studiesevidenced that the common extraction technique may not elimi-nate all non-glomalin protein sources (Rosier et al., 2006). Thisassumption is supported by solid-state 13C NMR spectroscopyrevealing that the current operationally defined “glomalin-relatedsoil protein fraction” is actually a mixture of humic acids andproteinaceous material (Schindler et al., 2007). It was suggestedthat if we intend to unveil the geochemistry of this biomolecule,preparative techniques have to be found that are more specific forglycoprotein. On the other hand, bearing in mind that this “glo-malin-related soil protein fraction” is relatively persistent againstbiological degradation (Rillig et al., 2001; Steinberg and Rillig,2003) it can be concluded that the co-extracted proteinaceousmaterial must have properties that turn the material into a poten-tial contributor to the refractory SOM pool.

Other mechanisms increasing the stability of proteins areglycosylation, binding to biofilms or incorporation into microbialbiomass. Glycosylation, which is the covalent linkage of specificoligosaccharides to specific amino acids, was demonstrated toincrease in vitro molecular stability against proteolytic enzymes.However, the impact of this reaction on soil protein stabilization isstill far from being understood.

Biofilms contain living cells but also extracellular polymericsubstances including proteins (Omoike and Chorover, 2004).Similar to glycosylation, the extent to which occlusion within bio-films can protect and stabilize protein N in soils is still unknown(Rillig et al., 2007).

A further possibility to aggravate proteolysis represents theintroduction of D-amino acids as they occur for example in thepeptidoglucan skeleton of bacteria (Kimber et al., 1990; Amelungand Zhang, 2001; Amelung et al., 2006). It was shown that therate of C mineralization from D-amino acids is initially less thanfrom the corresponding L-amino acids in a range of soils (Hopkinset al., 1997; O’Dowd and Hopkins, 1998; O’Dowd et al., 1999). Thissuggests that cell wall residues of bacteria comprise a considerablefraction of the refractory SON pool in soils.

6. The quantitative role of proteinaceous and peptideousmaterial for the size of the refractory SOM pool

Although we are still lumbering in the dark with respect to themechanisms involved in the sequestration of soil peptideous/proteinaceous material, there are many findings that support theirimportant role as SOM constituent. Applying the common methodfor determining the amount of proteinaceous material in soil by hotacid hydrolysis with HCl, Amelung et al. (2006) assigned between22% and 46% of the soil N to hydrolyzable amino acids. Thisaccounted for 5e10% of the SOC. The remaining N was attributed to

0 – 2 µm

2 – 6 µm

63 – 200 µm

mpp004-001-0 -200 -300

Bulk

C/N (atm)

10.9

15.1

10.2

10.1

-260

6 – 20 µm

14.0

Fig. 3. Solid-state 15N NMR spectra of the HF-treated bulk material and the fineparticle size fractions of an A horizon of a Luvisol from Viehhausen, Southern Germany.

H. Knicker / Soil Biology & Biochemistry 43 (2011) 1118e11291124

inorganic N, amino sugar N (up to 10% of the total N), the aminoacids arginine, histidine, cysteine and ’unknown N’. However, thereare reports saying that the commonly used HCl hydrolysis does nothydrolyze all proteins or peptides in soils and sediments. Using forexample thermochemolysis with tetramethylammoniumhydroxide, additional peptide derived amino acids were identifiedthat were not released neither by acid nor by alkaline hydrolysis(Knicker and Hatcher, 1997; Knicker et al., 2001). The mechanismsinvolved in their chemical protection are likely to be comparable tothose that are active in the protection against microbial degrada-tion. Alternative to encapsulation into hydrophobic structures(Knicker and Hatcher, 1997), reactions of released amino acids withsoil clay minerals or quantitative interference with mineral oxidesduring digestion with HCl may contribute to the low recovery ofamino acids with the common approach (Cheng et al., 1975). In linewith this, it was observed that a significant portion of the soil Nwhich is non-hydrolyzable with 6 M HCl was composed of aminoacids bound to reactive surfaces found in peptides (Leinweber andSchulten, 2000).

By applying a modified hydrolysis technique using methanesulfonic acid (MSA) the amount of recovered soil amino acidscombined with amino sugars and NH4

þ increased to such an extentthat they accounted for 80e93% of the soil N (Martens andLoeffelmann, 2003). This is in line with the solid-state 15N NMRspectroscopic data shown in Fig. 1 obtained from other soils unaf-fected by fire (Knicker, 2004). Martens and Loeffelmann (2003)further proposed that this SON is bound in peptideous structuresrather than occurring in large intact proteins.

If we nowconsider that humified SOMhas narrow C/N ratios (w/w) varying between 20 and 10, the conclusion must be that thesepeptideous structures account for a major fraction of the refractorySOC. To demonstrate this, let us perform a rough estimation,assuming that amino sugars represent a minor proportion of SONand take the protein casein as a representative for proteinaceouscompounds. Its atomic C/N ratio of 4.1 indicates that in soils with anatomic 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 soilshaving the atomic C/N ratio of 11.7 (corresponding to a w/w ratio of10) the percentage figure will be 34%. By performing this calcula-tion for the samples the spectra of which are shown in Fig. 2, 35, 34and 27% of the total organic C in the loess clay, the sapropel and thepeat, respectively is derived from peptideous material. Consideringfurther that litter degradation and humification are associated withdeclining C/N ratios, it becomes clear that relative to otherbiomolecules, peptideous material does not only represent animportant SOM pool but is also enriched during SOM maturation.

A similar calculation can be made for soil separates fractionatedaccording to physical size or density. A number of studies (Baldocket al., 1992; Christensen, 1992; Sollins et al., 2006) have shown thatsuch fractions have characteristic turnover times that commonlyincrease with decreasing particle size and increasing density.Concomitantly, their C/N ratios decrease, which indicates theincreasing importance of SON for the slowly cycling SOM pools.Solid-state 15N NMR spectra of the fine particle size fractions of an Ahorizon from a Luvisol under pasture in Southern Germany areshown in Fig. 3. These data suggest that almost all the organic N -even that in the small particle size fractions - is accounted for by Nwith amide or peptide bonds. Contributions of heterocyclic N canonly contribute to the shoulders in the chemical shift regionbetween �220 an �240 ppm and are suggested to be derived fromaromatic amino acid moieties or nucleic acids remains. Similarspectra were obtained from fractions of other soils which did nothave any documented fire history (Knicker et al., 2000). The atomicC/N ratios of the respective samples indicate a contribution ofpeptide-like material in terms of organic C of 27% in the fine sand

and up to 41% in the clay fraction. By performing a similar calcu-lation for the density fractions isolated by Sollins et al. (2006),peptide contributions increase from 5% of the organic C in the lightfraction <1.65 g cm�3 to 29% in the heavy fraction >2.55 g cm�3,which underlines the important role of peptides for the formationof organic matter e mineral associations. The intensity distribu-tions of the solid-state 13C NMR spectra of the particle size fractionsfrom the German Luvisol are shown in Fig. 4. The contribution ofpeptide C to the different chemical shift regions assignable tocompound classes can be calculated by using the weighted inten-sity distribution of a solid-state 13C NMR spectrum of casein.Subtraction of the peptide contents allows the assignment of up to35% of the organic C in the different fractions to O-alkyl C, tenta-tively derived from carbohydrates. The sum of O-aryl C and aryl C inthe difference spectrum is attributable to non-proteinaceousaromatic C. Its content is highest in the coarse fraction butdecreases to 7% of the total organic C in the clay fraction, suggestinga relatively low impact of this compound class for SOM stabilizationvia interaction with the mineral phase.

Comparable calculations are made for material from an A/Bhorizon (15e25 cm) of a Ferralsol from Southern Brazil publishedby 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 comprisea considerable fraction of SOM in this subsoil. Most of the

Bulk soil

Carboxyl C

O-Aryl CAryl C

O-Alkyl C

N-Alkyl

Alkyl C

15

6

14

31

11

23

8

13 3

7

16

7 511

28

47

rela

tive

inte

nsity

(%)

Fine sand

14

7

16

34

1117

61 2 2

511

8 6

14

32

6 6

rela

tive

inte

nsity

(%)

Coarse silt

10 8

18

37

10

17

71 3 3

6

13

37

15

34

4 4

rela

tive

inte

nsity

(%)

Middle silt

14

7

14

29

10

25

7

1 3 36

14

7 611

26

4

11

rela

tive

inte

nsity

(%)

Fine silt

13

49

37

11

27

9

13 3

7

17

4 3 6

34

4

10

rela

tive

inte

nsity

(%)

Total CPeptide C: 38%Difference

Clay

13

38

38

11

26

9

1 3 38

17

4 2 5

35

39

rela

tive

inte

nsity

(%)

Carboxyl C

O-Aryl CAryl C

O-Alkyl C

N-Alkyl

Alkyl C

Carboxyl C

O-Aryl CAryl C

O-Alkyl C

N-Alkyl

Alkyl C

Carboxyl C

O-Aryl CAryl C

O-Alkyl C

N-Alkyl

Alkyl C

Carboxyl C

O-Aryl CAryl C

O-Alkyl C

N-Alkyl

Alkyl C

Carboxyl C

O-Aryl CAryl C

O-Alkyl C

N-Alkyl

Alkyl C

Peptide C: 27%

Peptide C: 31% Peptide C: 33%

Peptide C: 40% Peptide C: 41%

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.

H. Knicker / Soil Biology & Biochemistry 43 (2011) 1118e1129 1125

remaining intensity was attributed to carbohydrates (41%) whichmay be closely associated to the peptide fractions as it would be thecase for microbial cell walls. Only 15% was assignable to non-proteinaceous aromatic C, indicating that the role of aromaticstructures in SOM stabilization in subsoils may be overestimated.

7. Stabilization of N by immobilization in pyrogenic organicmatter

In ecosystems that are frequently exposed to vegetation firesalterations of size and quality of the SON and SOC pools must beencountered if the C and N sequestration potential should be esti-mated. The response of the SOM content to fire depends on fire

intensity, type of vegetation, the fuel load as well as soil texture andeven slope (González-Pérez et al., 2004). High fire intensity can leadto a complete destruction of the organic layer and the humicmaterial in the mineral topsoil (Fernández et al., 1997; Hernándezet al., 1997; Haslam et al., 1998; Knicker et al., 2006). Aftermoderate and prescribed fires, the alterations in SOM are minorand sometimes even result in an increase of organic C and N due tothe 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 decom-posed via systematic and random depolymerization reactions andthe respective products are incorporated into the SOM pool.Certainly, a part of the N will be lost due to volatilization but in

138

17

63

126.6

15

41

25

43

18

62

Carboxyl C

Aryl C

O-Alkyl C

N-Alkyl C

Alkyl C

re

la

tiv

e13C

in

ten

sity

%

AB

Peptide: 29%

Difference

0 -100 -400 ppm-200 -300

-26015N NMR

13C Intensity Distribution

Atomic C/N: 14

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 ofdifferent C groups and peptide C in its solid-state 13C NMR spectrum.

DON/DOC

(amino acids

amino sugars)

DIN

(NH4

+, NO

3

-

)

N

immobilization

Biomass

biopolymers

fragmentation

C and N sequestration by:

C

immobilization

C

mobilization

CO2

N

mobilization

SOM ( including

peptides)

depolymerization

mineralization

N cycle

C cycle

- Steric hindrance

- Chirality

- Glycosylation

- Sorption

- Bridging by cations

- Physical protection

- Biofilm

Fig. 6. Concept of the interrelationship of the C and N cycle in soils.

H. Knicker / Soil Biology & Biochemistry 43 (2011) 1118e11291126

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 arerecalcitrant towards high temperatures. As constituent of the socalled “Black Carbon” (BC) fraction, they are expected to contributemainly to the storage of recalcitrant compounds (Knicker et al.,2008). Whereas, BC is frequently included in global C-cyclingmodels (González-Pérez et al., 2004), the concept of this “BlackNitrogen” (BN) as an important fraction of charcoal in soils was onlyrecently 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 derivedfrom plant litter and humic material (Knicker et al., 1996a;Almendros et al., 2003), the formation and stabilization of BN hascertainly the same potential importance for C sequestration as thatattributed to BC. Products formed during heating of proteins andpeptides are cyclic di- and oligopeptides (Reeves and Francis, 1998;Meetani et al., 2003). At lower temperatures (200 �Ce300 �C), lowmolecular weight heterocyclic compounds and above 500 �C,polynuclear N-containing aromatic structures have been observed(Sharma et al., 2003). A further reaction, occurring during charringof biomaterial is the Maillard reaction (Ikan et al., 1996). During thisprocess, ammonia or free amino groups of amino acids and aminosugars react with carbonyl groups in sugar compounds to formSchiff bases, which subsequently undergo rearrangements toproduce dark-colored melanoidins. Such heterocyclic-Ncompounds were detected in fire-affected SOM (Knicker andSkjemstad, 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 forthe quality of SOM, the contribution of BN structures to the bulkchar was estimated to account for more than 50% of the organic C ina recently examined grass char (Knicker et al., 2008) and at least12% to the total BC pool of charred peat. Therefore, burning maylead to a relative enrichment of organic N by transforming biomassinto pyrogenic material that is incorporated into the soil and altersthe long-term N availability for biomass production. Thus, apartfrom a direct impact on the quality and quantity of the refractorysoil organic C pool, the formation and addition of BN to soils hasalso 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 inits organic and inorganic forms, determines the amount of Cimmobilized in living biomass and litter which are the sources forthe SOM pool. Moreover, recent studies indicate that the N

accessibility for microorganisms affects the degradation patternand the turnover rates of the different soil organic C pools. Thoseinteractions between the C and N cycles are well recognized inecological literature but often remained neglected in modelsdescribing the cycling of organic matter in soil. Most of the modelsdescribing C and N dynamics in soils distinguish between the N andC cycle, admitting the interactive character of the two cycles butstill consider them to act independently. However, such a viewunderrates the important chemical premise which is thatcompounds forming organic N are also contributing to the organicC fraction. This literally direct connection between the C and N poolimplies that mechanisms affecting the N stabilization must havea considerable impact on size and quality of the different organic Cpools, too (Fig. 6). Bearing in mind that in humified SOM withnarrow atomic C/N ratios < 23 N-containing organic compoundsare important constituents, their chemical properties must be ofhigh relevance for the mechanisms involved in SOM stabilizationand the respective degradation patterns.

With respect to the chemical nature of SON, there is accumu-lating evidence suggesting that peptideous material makes up thedominating organic N form in soils unaffected by fire and that its

H. Knicker / Soil Biology & Biochemistry 43 (2011) 1118e1129 1127

contribution to the SOC pool is increasing with increasing age ofthis pool. Thus, in spite of being frequently classified as labile andfast turning SOM components, peptides must represent moreimportant players within the SOM-sequestration process thancommonly assumed. With their amphiphilic character and the factthat approximately two functional groups per amino acid unit canbe charged they are excellent candidates for sorptive interactionswith the soil mineral phase. Such interactions would even beenforced if the peptide moiety occurs in association with carbo-hydrates as is the case for microbial cell wall residues. Theirincreasing contribution to the organic matter of particle fractionswith decreasing size may thus be related to their preferentialadsorption to surfaces of mineral oxides. In calcareous soils, Ca2þ

ions are likely to be involved as bridging elements. On the otherhand, the fact that peptides also make up the dominant N form inpeat deposits indicates that apart from physical protection viamineral interactions other or additional mechanisms must aggra-vate their degradation potential. Most likely steric hindrance(Knicker and Lüdemann, 1995), encapsulation (Knicker andHatcher, 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 inthe present work, one can conclude that not only SON butin particular peptide-like materials are indeed under-rated playersin the C sequestration in soils. The information of this review clearlypoints to the conclusion that they are an important soil organicmatter fraction, but how they affect SOM or even soil functionalityis still in the dark. Therefore, directing the research effort towardsan understanding on the relationship between peptide chemistryand SOM functionality may be a future important step forimproving our knowledge of SOM formation and its role of SOM asa C sink.

Acknowledgment

Dr. F.J. González-Vila is gratefully acknowledged for helpfuldiscussion and suggestions. Dr. S.I. Nilsson, Dr. G. Almendros,Dr. D.W. Hopkins and Dr. J.P. Shimel are thanked for critical proofreading and their constructive comments. I also want to thank theanonymous reviewers for their helpful suggestions. Financialsupport was obtained from the Ministerio de Ciencia e Innovación(Spain) and the Deutsche Forschungsgesellschaft (DFG) (Germany).

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