Biogeochemistry (2007) 85:9–24 DOI 10.1007/s10533-007-9103-5

ORIGINAL PAPER

A conceptual model of organo-mineral interactions in soils: self-assembly of organic molecular fragments into zonal structures on mineral surfaces

M. Kleber · P. Sollins · R. Sutton

Received: 11 March 2006 / Accepted: 27 November 2006 / Published online: 15 June 2007 © Springer Science+Business Media B.V. 2007

Abstract In this paper, we propose a structure hydroxyls, forming stable inner-sphere com- for organo-mineral associations in soils based on plexes, or (ii) proteinaceous materials unfold recent insights concerning the molecular struc- upon adsorption, thus increasing adhesive ture of soil organic matter (SOM), and on exten- strength by adding hydrophobic interactions to sive published evidence from empirical studies of electrostatic binding. Entropic considerations organo-mineral interfaces. Our conceptual dictate that exposed hydrophobic portions of model assumes that SOM consists of a heteroge- amphiphilic molecules adsorbed directly to min- neous mixture of compounds that display a range eral surfaces be shielded from the polar aqueous of amphiphilic or surfactant-like properties, and phase through association with hydrophobic moi- are capable of self-organization in aqueous solu- eties of other amphiphilic molecules. This pro- tion. An extension of this self-organizational cess can create a membrane-like bilayer behavior in solution, we suggest that SOM sorbs containing a hydrophobic zone, whose compo- to mineral surfaces in a discrete zonal sequence. nents may exchange more easily with the sur- In the contact zone, the formation of particularly rounding soil solution than those in the contact strong organo-mineral associations appears to be zone, but which are still retained with consider- favored by situations where either (i) polar able force. Sorbed to the hydrophilic exterior of organic functional groups of amphiphiles interact hemimicellar coatings, or to adsorbed proteins, via ligand exchange with singly coordinated mineral are organic molecules forming an outer region, or kinetic zone, that is loosely retained by cation bridging, hydrogen bonding, and other interac- M. Kleber (&) tions. Organic material in the kinetic zone may Earth Sciences Division, Lawrence Berkeley National Laboratory, One Cyclotron Road, Berkeley, CA, experience high exchange rates with the sur- 94720, USA rounding soil solution, leading to short residence e-mail: [email protected] times for individual molecular fragments. The thickness of this outer region would depend more P. Sollins Department of Forest Science, Oregon State on input than on the availability of binding sites, University, Corvallis, OR, 97331, USA and would largely be controlled by exchange kinetics. Movement of organics into and out of R. Sutton this outer region can thus be viewed as similar to Department of Environmental Science, Policy and Management, University of California, a phase-partitioning process. The zonal concept #3114 Mulford Hall, Berkeley, CA, 94720-3114, USA of organo-mineral interactions presented here

123 10 Biogeochemistry (2007) 85:9–24 oVers a new basis for understanding and predicting Wandruszka 1997, 1998; von Wandruszka 1998; the retention of organic compounds, including von Wandruszka et al. 1999; Ferreria et al. 2001; contaminants, in soils and sediments. Martin-Neto et al. 2001; von Wandruszka and Engebretson 2001; Nanny and Kontas 2002) indi- Keywords Multilayer · Adsorption · Soil organic cates that soluble mixtures of organic molecules matter · Nitrogen · Protein · Amphiphilic · representing a signiWcant fraction of SOM can Organo-mineral · Micelle · Humic form organized structures called micelles within aqueous solution, structures that consist of hydro- philic exterior regions that shield hydrophobic Introduction interiors from contact with water molecules (von Wandruszka 1998). Because amphiphilic mole- Decades ago, earth scientists acknowledged the cules are requisite to the formation of micelles, ability of mineral particles to protect soil organic the ability of SOM components to form these matter (SOM) from biological attack (Jung 1943; structures suggests that many of these molecules Allison et al. 1949). In temperate, cultivated soils, are amphiphilic. SigniWcant to this insight, Wer- 50–75% of SOM exists within clay-sized organo- shaw (1993) previously developed a bilayer model mineral particles (Christensen 2001), and numer- of organo-mineral interactions (Fig. 1) that ous researchers have reported positive correla- sharply contrasted with the traditional view of tions between the contents of Wne mineral organo-mineral interactions (Stevenson 1985), particles and carbon in soils (Körschens 1980; which were visualized as associations of large, Nichols 1984; Burke et al. 1989; Mayer and Xing multifunctional polymers with mineral surfaces 2001). As a consequence, it is widely assumed that via a broad range of bonding mechanisms (Ste- the inhibiting eVect of soil minerals on decompo- venson 1985; Leinweber and Schulten 1998). Fur- sition can inXuence strongly the turnover of SOM ther, Wershaw and Pinckney (1980) postulated (Martin and Haider 1986; Theng et al. 1992; that decayed organic materials are often bound to Hsieh 1996; ParWtt et al. 1997). It follows that clay surfaces by amino acids or proteins, based on understanding SOM stabilization requires knowl- the observation that deamination of organo-min- edge of the processes operating at the organo- eral complexes with nitrous acid released organic mineral interface. Of particular importance but materials from the clay. great complexity are the questions of how It is our intention to develop this earlier work strongly SOM may bind to mineral surfaces, how into a generally applicable concept of the zonal such bonds form and eventually break, and how structure of organo-mineral associations (Fig. 2), the mechanisms of attachment of SOM to mineral based on the amphiphilicity of SOM fragments, surfaces aVect its residence time in soils. and the intimate involvement of proteinaceous While the characteristics of mineral surfaces compounds in stable organo-mineral associations. are reasonably well known, the chemical proper- As deWned here, a zonal structure is formed when ties of SOM continue to be the subject of much the organic matter attached to a mineral surface is debate. After all, while minerals crystallize segregated into more than one layer or zone of towards or dissolve from a deWned and compara- molecules, such that not all adsorbed molecules tively simple crystal structure, SOM encompasses are in contact with the mineral surface. Assuming organic molecules representing both compounds such a zonal structure, we are able to account released from living plant and microbial cells simultaneously for a number of phenomena (e.g., extracellular enzymes, surface-active pro- observed in soils and sediments, including: teins, chelating compounds, and many others) and complex plant, microbial and animal residues a) The decrease of C/N ratio with decreasing in various stages of alteration due to both biotic particle size (Hedges and Keil 1995; Aufdenk- and abiotic processes (Baldock and Skjemstad ampe et al. 2001); 2000). Considerable spectroscopic evidence b) The decrease of C/N ratio with increasing (Chien et al. 1997; Engebretson and von particle density (Sollins et al. 2006);

123 Biogeochemistry (2007) 85:9–24 11

O H

Al OH O Hydrophilic H H functional groups O O H

2+ CH Ca + 3 O Al O H 3 N

H 3 C - O H O H O O O H CH 3 2+ Hydroxylated Al O Ca H O mineral H H 3 C - surface O O O H

Al Hydrophobic 2+ structures Mg O O H

CH 3 O Al O + H Na O O 2+ H 3 C - Mg O O Al O P CH 3 O Electrostatic interaction H O O with soluble ions Direct bond with surface metal cation

Fig. 1 The “Wershaw bilayer model” of organo-mineral hydrophilic organic moieties and surface oxygens or hydrox- interactions (Wershaw et al. 1996a). Figure adapted from Es- ylated surface-metal cations. The hydrophobic portions of sington (2003). Accumulation of amphiphilic fragments on these surface molecules are shielded from the polar aqueous soil surfaces is initiated by electrostatic interactions between phase by a second layer of amphiphiles, forming a bilayer c) The decrease of 14C content with decreasing C content in heavy fractions (Sollins et al. To support the concept of zonal structure of 2006); organo-mineral complexes, we Wrst discuss the d) The preferential sorption of large, hydropho- idea of SOM as consisting of associations of bic organic compounds in sorption experi- organic molecules capable of micellar self-organi- ments with soils (Jardine et al. 1989; Guo and zation in aqueous solution. Given this important Chorover 2003) and in marine/estuarine sedi- concept, it is appropriate to view a signiWcant por- ments (Zhou et al. 1994); tion of SOM as consisting of a mixture of diverse e) The existence of at least two diVerent C and but typically amphiphilic molecules. We will pres- N pools in heavy fractions: an older, more sta- ent empirical evidence that supports the idea of a ble pool of C and N, and a more recent, fast zonal structure of organic accretions on mineral cycling pool of C and N (Trumbore et al. surfaces, demonstrate the special importance of 1989; Strickland et al. 1992; Swanston et al. proteinaceous compounds for the architecture of 2005); this structure, and show that the assumption of f) The observation that mineral-associated diVerent zones of sorption implies diVerential organic matter accumulates in A horizons exchange kinetics for organic fragments moving beyond levels consistent with monolayer cov- into and out of individual zones. erage (Eusterhues et al. 2005); g) The correlation of concentrations of poorly crystalline mineral phases with amounts of Amphiphilic molecules and the micellar oxidation-resistant SOM (Kleber et al. association model of soil organic matter 2005); h) Seasonal changes in water repellency in soils Critical insights regarding organo-mineral com- (Horne and McIntosh 2000). plexes arose from studies probing the behavior of

123 12 Biogeochemistry (2007) 85:9–24

contact zone of hydrophobic kinetic zone zone interactions (outer region) O R OH 2+ + - 2+ - M low charge H 3N R O M O

2:1 mineral O O O (smectite) O NH O - - + O O 2+ - with protein SH 2 M O O HN R - O O conditioning 2+ M - CH 3 H C O O NH 3

R O CH 3 O H HO P OH H 3C O O H 2:1 Fe O - OH O M2+ H mineral Fe H 3C - O O OH O O 2+ 2+ with O H M M Fe coating O Fe O of - - Fe OH N O O O 2+ hydrous OH O HO H M O H 3C O Fe - CH 3 iron - O OH O Fe oxide O M2+ O O - O O

O H3C H 3C - 2+ - O M OH O M2+ CH uncharged 3 - 1:1 OH O = octahedral charge OH siloxane O = tetrahedral charge CH (kaolinite) 3 M2+ = metal cation

Fig. 2 The zonal model of organo-mineral interactions. of adsorbed organic molecules from the polar aqueous The variety of surface types in soils is represented by a low- phase by a second layer of amphiphilic molecules. The charge smectite, a hydroxylated Fe-oxide coating, and a hydrophobic zone is thought to be discontinuous, as some hydrophobic kaolinite siloxane surface. This selection is proteins directly adsorbed to mineral surfaces may well ex- meant to illustrate major bonding mechanisms, and is not pose ionized or polar functional groups towards the soil intended to represent the full range of mineral surface solution. In the outer region or kinetic zone, further accu- types that may be encountered in soils. In the contact zone, mulation of organic molecular fragments is possible, prob- amphiphilic fragments accumulate on charged surfaces ably largely mediated by the presence of multivalent through electrostatic interactions, directing hydrophobic cations. Depending on stereochemical orientation, degree portions outwards toward the polar aqueous solution. Pro- of amphiphilicity, cation content of the soil solution, pH, teins serve as a surface conditioner that adds polar func- temperature, and perhaps several other controlling factors, tionality to low-charge siloxane surfaces. Some a variety of modes of attachment of organic molecular frag- hydrophobic organic compounds may also associate with ments to the intermediate region appears to be possible. noncharged mineral surfaces. The zone of hydrophobic The thickness of the outer zone is likely to depend on input interactions is equivalent to the hydrophobic region formed of organic materials and thus to be largely be controlled by as part of the Wershaw bilayer model, and results from the exchange kinetics entropically driven shielding of the hydrophobic portions natural organic fractions in aqueous solution. To signiWcant portion of this organic material, and it examine and manipulate the behavior of these contains many reactive polar or charged moieties complex organic materials within a controlled that respond to pH. In addition, behavior consis- laboratory environment, alkaline extraction is tent with micellar organization, observed using a often used to solubilize a large portion of SOM, diverse array of spectroscopic techniques includ- producing an operationally deWned humic frac- ing Xuorescence (Engebretson and von Wan- tion. That alkaline extraction eVectively removes druszka 1997, 1998; von Wandruszka 1998; von between 50% (Rice 2001) and 80% (ScheVer and Wandruszka et al. 1999; von Wandruszka and Schachtschabel 2002) of the organic material in Engebretson 2001; Nanny and Kontas 2002), elec- soil samples indicates that while the humic tron spin resonance (Ferreria et al. 2001; Martin- fraction represents only a portion of SOM, it is a Neto et al. 2001), and nuclear magnetic resonance

123 Biogeochemistry (2007) 85:9–24 13

(Chien et al. 1997), suggests that nonpolar moie- 1997, 1998; von Wandruszka 1998; von Wan- ties are also present within humic fractions. After druszka et al. 1999; Ferreria et al. 2001; Martin- all, it is the presence of hydrophobic functional Neto et al. 2001; von Wandruszka and Engebret- groups within amphiphilic molecules that triggers son 2001; Nanny and Kontas 2002) supports the entropically-driven self-organization to shield ability of complex natural organic substances to these nonpolar moieties from the polar aqueous self-assemble into associations featuring regions medium through formation of a micelle. Thus, the with diVerent chemical properties, similar behav- observation of micellar structure within humic ior is not typically ascribed to SOM adsorbed to extracts representing a major portion of SOM mineral surfaces. However, starting with the pre- indicates that a signiWcant fraction of the mole- viously mentioned work by Wershaw and col- cules making up this complex organic material are leagues (Wershaw and Pinckney 1980; Wershaw amphiphilic in nature. 1986; Wershaw 1993), information from a broad The components of decomposing SOM display range of scientiWc Welds has provided numerous a range of functional qualities based on chemical clues suggesting that SOM sorbs to minerals in and structural composition (Sutton and Sposito layers or zones that may have diVerent chemical 2005). We suggest that these chemical properties or functional properties. In the following sections represent a continuum of amphiphilicity that we use (i) the fate of organic pollutants in soils, ranges from molecules that are solely nonpolar sediments, and aquifers, (ii) the distribution of and hydrophobic, to those that are predomi- organic matter over mineral surfaces in natural nantly amphiphilic because they also contain systems, (iii) information from density fraction- hydrophilic, highly polar or charged functional ation experiments, (iv) evidence from the study of groups. Nonpolar, hydrophobic compounds are water repellency in soils, and Wnally, (v) data composed predominantly of alkyl and aromatic obtained through laboratory synthesis of layered functional groups, and may derive from sources organo-mineral structures, to draw inferences such as plant waxes and cutins. Occupying the suggesting that organic accretions on minerals mid-range of this chemical continuum are mildly consist of multiple zones of self-assembling mate- polar materials, such as many carbohydrates and rials. their derivatives, which feature functional groups like alcohols or ethers that do not ionize under Sorption of organic compounds to organic coat- typical soil and water pH conditions. In addition ings on mineral surfaces as a model of zonal org- to hydrophobic functional groups, the amphi- ano-mineral architecture philic compounds that populate the other extreme of this chemical spectrum contain more The idea that organic matter covers mineral sur- reactive functional groups like carboxyls and faces in a layered or zonal fashion (Fig. 2) is not amines, which can develop charge under typical new (Chassin 1979), with most early concepts of soil and water pH conditions. The dominant role organics sorbing to mineral surfaces in two or of amphiphilic molecules in the behavior of com- more layers resulting from a wealth of studies on plex organic mixtures within aqueous environ- the fate of organic pollutants in soils, sediments, ments cannot be ignored when considering the and aquifers. A review of this early work led mechanisms by which SOM adsorbs to mineral Voice and Weber (1983) to conclude that, surfaces. although hydrophobic bonding was a major sorp- tion mechanism for hydrophobic compounds, no single sorption mechanism adequately explained Experimental evidence in support of a zonal all experimental observations. Instead, Weber structure of organic accretions on mineral et al. (1983) state that “the organic carbon content surfaces of a solid appears to be the major factor in deter- mining its sorptive capacity.” While mounting experimental evidence (Chien A step forward in advancing mechanistic et al. 1997; Engebretson and von Wandruszka knowledge about zonal arrangements of organic

123 14 Biogeochemistry (2007) 85:9–24 molecules on mineral surfaces was taken by Mur- et al. 2002) and marine sediments (Ransom et al. phy et al. (1990), who, following up on the work 1998; Mayer 1999) is typically discontinuous. of Hunter and Liss (1982) among others, found While organic matter loadings on sedimentary that hydrophilic mineral particles exposed to mineral surfaces are often on the order of the so- humic fractions developed hydrophobic surfaces, called monolayer equivalent of 0.5–1.0 mg rendering them more capable of sorbing hydro- organic C m¡2 (Keil et al. 1994; Mayer 1994), the phobic organic compounds. Hydrophobic interac- fact that organic material is not distributed tions bind the outer layer hydrophobic evenly over the available mineral surface indi- compounds to the inner layer organics because cates that the material must instead be clustered the total hydrocarbon surface that is exposed to in small patches with some vertical extension. water is reduced, and entropy maximized, when Aluminosilicate sediments with low to moderate hydrophobic entities organize to reduce the non- loadings of organic matter (<3 mg polar surface area exposed to water molecules. organic C m¡2) generally have less than 15% of Jardine et al. (1989) provided additional support their surfaces coated, with organic materials for the importance of hydrophobic interactions to existing in discrete spots on the mineral surfaces adsorption of organic materials by comparing the (Arnarson and Keil 2001). sorption of dissolved organic carbon solutions Speculations about a layered architecture of that had been fractionated into operationally deW- these patches have been voiced by Kaiser and ned hydrophobic and hydrophilic organic por- Guggenberger (2003), who found that the tions. Hydrophobic organic solutes were reduction of SSA following sorption of increas- preferentially adsorbed by soils relative to hydro- ing amounts of DOC was not uniform: the philic organic solutes, leading the authors to con- decrease in SSA was larger at smaller organic clude that hydrophobic interactions driven by matter loadings than at larger ones. Among favorable entropy changes represent the primary other potential explanations, they hypothesized mechanism of adsorption for these organic sol- that “with increasing surface loading an increas- utes. Murphy et al. (1990) further showed that the ing portion of the sorbing molecules did not nature of the mineral surface, particularly the attach to mineral surfaces but formed organic concentration and spatial distribution of hydroxyl multilayers as a result of hydrophobic interac- sites, inXuenced the amount of hydrophobic tions or bridging by polyvalent cations between organic C adsorbed to organic coatings. The organic ligands of sorbed and soluted mole- chemical properties of the humic fraction used to cules.” This interpretation was subsequently coat the mineral particles inXuenced the amount supported by the work of Wang and Xing of hydrophobic compounds adsorbed, with the (2005), who found that, even under diVerent most aromatic humic material being the strongest organic matter loading levels, mineral surface sorbent for hydrophobic material. coverage remained nearly constant, indicating that “the coating must have increased in thick- Patchy organic matter distributions on mineral ness while retaining practically the same surface surfaces indicate zonal architecture of sorbed coverage at a higher loading. In such a multilayer materials arrangement, the Wrst several molecular layers close to the mineral surface may take a more Further evidence for the zonal or layered sorp- compacted form due to the attractive forces of the tion of organic matter to mineral surfaces comes mineral surface.” In addition, ocean sediment from the examination of natural organo-mineral studies show a direct relationship between complexes taken from soil and marine systems. organic matter content and mineral surface area Both speciWc surface area (SSA) determinations at organic loadings 2–5 times that of a mono- and microscopic analyses indicate that the layer equivalent (Hedges and Keil 1995), further distribution of organic materials on the mineral supporting the existence of minerals coated with surfaces of soils (Mayer and Xing 2001; Kahle multiple layers of organic molecules.

123 Biogeochemistry (2007) 85:9–24 15

Organo-mineral associations isolated through bound and more readily desorbed or decom- density fractionation may also suggest a zonal posed. That only part of the mineral-bound car- architecture of sorbed materials bon is protected from oxidation is corroborated by experiments that subject subsoils to chemical In addition to the ocean sediments described oxidation. The resulting C solubilization and min- above (Hedges and Keil 1995), soil particles iso- eral dissolution show that most of the organic lated through density fractionation have also pro- matter in subsoils is associated with mineral sur- vided evidence indicating that organic material is faces (Eusterhues et al. 2003; Mikutta et al. 2006). sorbed to minerals through both direct and indi- Thus mild chemical oxidation leaves an organic rect interactions with mineral surfaces. Euster- residue with a mass that correlates signiWcantly hues et al. (2005) found that organic matter with mineral surface F- reactivity, and a
14C loadings in heavy fractions (>2 g cm¡3) from a value lower than that of the original organic mat- series of topsoils were greater than could be ter pool (Kleber et al. 2005). explained solely by direct attachment to the avail- able mineral surfaces area in a monolayer fashion. Seasonal water repellency explained through They hypothesized that interactions between presence of organic material sorbed to mineral organic molecules leading to greater than mono- surfaces in a zonal fashion layer coverage would be necessary to explain the large amounts of sorbed organic material (Eus- Seasonal changes in soil water repellency may terhues et al. 2005). also be explained by alterations to the zonal Organic C in the heavy fraction of soils is com- architecture of sorbed organic matter induced by monly seen as being closely associated with min- seasonal changes in the soil environment. Non- erals and typically more depleted in 14C than seasonal water repellency in soils has often been light-fraction material, which is not intimately attributed to the presence of hydrophobic moie- bound to minerals (Trumbore and Zheng 1996). ties such as those found in long-chain fatty acids However, when Swanston et al. (2005) traced an (Ma’shum et al. 1988). The presence of hydropho- atmospheric 14C pulse into a forest ecosystem, bic compounds alone, however, does not explain they noticed that the elevated 14C signal appeared seasonal changes in water repellency. Using the not only in the light fraction, but also in the ‘molarity of ethanol droplet’ method to measure strongly mineral-associated dense fraction. They repellency, combined with a set of organic extrac- interpreted this observation as indicating the exis- tions, Horne and McIntosh (2000) attributed sea- tence of at least two diVerent C pools in the org- sonal changes in soil wettability to the existence ano-mineral fraction: an older, more stable pool of multiple, functionally diVerent layers of of C, and a younger, fast cycling C pool. Strick- organic matter on sandy grains. In their model, an land et al. (1992) found similarly that 15N-labeled inner layer of more hydrophobic compounds is inorganic N was incorporated rapidly into dense covered by a layer of amphiphilic compounds. (organo-mineral) fractions of Wve soils of greatly When the soil is wettable, the hydrophobic mate- diVering mineralogy, then released as both inor- rial is eVectively screened, especially if the outer ganic and organic N during a subsequent incuba- surface is well hydrated. Repellence occurs when tion. The existence of rapidly cycling C and N in the hydrophobic compounds making up the inner the dense or mineral-associated fraction can satis- layer are more exposed. This can be linked to sea- factorily be explained by a zonal structure of sonal moisture diVerences through a combination organic accretions on mineral surfaces. While the of factors: during the wet season, the outer layer organic materials directly adsorbed to the mineral will be well hydrated, amphiphilic compounds will surface are likely to be tightly bound and well be arranged in such a fashion as to have their protected from microbial degradation, the materi- polar portions oriented outwards, the carboxylate als sorbed in outer regions of the organo-mineral anion will predominate, and hydrophobic groups complex, and lacking direct interaction with the will be eVectively screened by more hydrophilic mineral surface, are presumably more loosely groups. With the onset of the dry season, a more

123 16 Biogeochemistry (2007) 85:9–24 hydrophobic outer surface develops as the amphi- after the beginning of the incubation. These mole- philic compounds change in orientation, the car- cules made up approximately 65% of the Wnal boxylate groups are protonated, and contraction coverage. They were irreversibly adsorbed and of the amphiphilic ‘surface screen’ exposes the did not measurably exchange with bulk solution interior hydrophobic layer. molecules. A second layer reached equilibrium after several hours of incubation and showed a Laboratory and industrial synthesis of multilayer pronounced and temperature-dependent organic assemblages exchange with bulk molecules. A third layer con- sisted of molecules that exchanged rapidly with Multilayered organo-mineral structures are rou- the bulk solution, but comprised only about 10% tinely manufactured and studied in the labora- of the total coverage. An important implication of tory, mainly for biomedical (Galeska et al. 2001) this work is that a protein-based multilayer struc- and biotechnical purposes (Halthur et al. 2004). ture allows occurrence of both hydrophobic Because these artiWcial organo-mineral struc- adsorption on a stable inner layer, as well as tures likely form as a result of some of the same phase-partitioning into a dynamic outer layer, adsorption mechanisms active in soils, they may thus explaining experimental results suggesting be viewed as potential analogs of natural organo- the coexistence of both kinds of sorption mecha- mineral complexes. In one such study of artiWcial nisms in the same system. organo-mineral assemblages, Spaeth et al. (1997) sorbed protein multilayers to hydrophobic silan- Reconciling the evidence for zonal structures on ized silicon wafers using alternating incubations mineral surfaces with poly-streptavidin and bovine serum albumin. They found that the thickness of the organic lay- The evidence compiled above suggests that ers increased 18.75 nm per incubation, yielding a SOM may attach to mineral surfaces in a zonal Wnal protein multilayer thickness of 350 nm. or layered fashion. In conjunction with the work Galeska et al. (2001) sorbed layers of the Ald- of Wershaw et al. (1995; 1996a; b) on adsorption rich commercial humic fraction onto silicon of compost leachates to aluminum oxide, studies wafers, noting that the expansion of the organic of the amounts or spatial distributions of organic Wlm could be signiWcantly altered by the incorpo- materials bound to mineral particles, and of vari- ration of salt, with the charge screening eVect con- ations in C turnover time and seasonal water tributing to thicker Wlm deposition irrespective of repellency, have provided a wealth of direct and pH. Further experimentation indicated that the indirect information supporting the concept of permeability of simple carbohydrates through the an organized, zonal architecture for SOM sorbed humic coating was a function of the number of to mineral surfaces. Experiments and observa- self-assembled layers of organic material. That tions in the laboratory prove that organic multi- the probe glucose penetrated thick Wlms com- layers on mineral surfaces can even be posed of many layers of organic matter more manufactured to serve speciWc purposes. Eco- readily than thin Wlms made up of few layers sug- logically speaking, the work of Schmidt et al. gests that organic molecules, including both SOM (1990) is especially signiWcant, as it assigns diVer- and xenobiotic substances, may not be tightly ent exchange kinetics to molecules making up bound to outer regions of organo-mineral com- diVerent parts of the organo-mineral complex, plexes. with molecules of the inner zone being the most Alkylated silicon oxide surfaces were used by stable, and molecules of the outermost zone hav- Schmidt et al. (1990) in studies of protein adsorp- ing the fastest exchange with the soil solution. tion on hydrophobic surfaces. They found that a Such diVerential, zone-dependent kinetics could model with at least three classes of adsorbed mol- explain why organo-mineral isolates from soils ecules was necessary to adequately describe their can contain C and N pools with diVerent turn- experimental results. A Wrst layer was formed by over times (Trumbore et al. 1989; Strickland the molecules that adsorbed within a short time et al. 1992; Swanston et al. 2005).

123 Biogeochemistry (2007) 85:9–24 17

Table 1 Relative Vascular plants Algae Bacteria Fungi composition of vascular plants, algae, bacteria, % Dry matter and fungi, compiled using data from Knicker Lignin 5–30 (2004) and White (1997) Cellulose 15–60 Hemicellulose 10–30 N-containing compounds 2–15 24–50 50–60 14–52 Lipids 2–10 10–35 1–42 Carbohydrates 40 4–32 8–60 Ratio

C:N ratio 20–50 (tree leaves) 6 5–8 t10 25–80 (herbaceous plants)

The role of proteins in organo-mineral (Almendros et al. 1991 Knicker et al. 1995; associations Knicker and Lüdemann 1995; Knicker 2000, 2002; Leinweber and Schulten 2000; Mahieu et al. 2000, Mineral-associated SOM exhibits C/N ratios of 7– 2002; Mathers et al. 2000; Zang et al. 2000), and 14, whereas water-extracted SOM has C/N ratios thus most likely in the form of proteins or protein of 26–55 (Aufdenkampe et al. 2001), indicating N residues. Tremblay and Brenner (2006) showed in enrichment of adsorbed organic materials. a 4-year decomposition experiment that on aver- Numerous observations, made in both soils and age 60–75% of the N in highly decomposed detri- marine/freshwater environments (Aufdenkampe tus was derived from heterotrophic bacteria, rather et al. 2001), similarly suggest that the adsorption than plant tissues. As the smallest organisms in the of nitrogenous compounds to mineral surfaces soil, bacteria and fungi are able to enter small, con- may be a broadly relevant ecological process. Wned pore spaces and die and decompose there, Thus, a model of organo-mineral associations and also contain far more N than vascular plant should account for the body of evidence suggest- materials or surface litter (Table 1). In fact, bacte- ing preferential adsorption of N-containing moie- ria are known to actively use proteins to attach to ties over other organic compounds to mineral surfaces (Bashan and Levanony 1988; Dufrene surfaces (Omoike and Chorover 2006; Hedges et al. 1999). Mycelial fungi also secrete a broad and Oades 1997). class of surface-active glycoproteins (Wright and Certainly, the relatively high strength of pro- Upadhaya 1996). Abundant evidence indicates tein adsorption to mineral surfaces (Theng 1979; that microbial products accumulate in Wne clay Chevallier et al. 2003; Wershaw 2004) is a long- fractions of soils (Leinweber and Schulten 1995; established (Ensminger and Gieseking 1942)— Kleber et al. 2004; Mertz et al. 2005), further cor- and often technologically exploited (Gougeon roborating the supposition that the more proteina- et al. 2003)—chemical phenomenon, and one that ceous microbial residues are likely to make initial has proved relevant to the laboratory synthesis of contact with mineral surfaces, rather than the less multilayer organo-mineral complexes, as dis- proteinaceous plant residues. cussed previously (Schmidt et al. 1990). We sug- Protein polyfunctionality and adsorption to gest that proteinaceous materials play a mineral surfaces—Proteins are a special class of prominent role in the formation and structure of amphiphilic molecules with a strong tendency to organo-mineral complexes due to both the rela- bind to surfaces and to resist desorption (Hlady tively high abundance of such compounds in soils, and Buijs 1996). They consist of chains of amino and the ability of these materials to adsorb irre- acids with both apolar and polar (either neutral or versibly to mineral surfaces. charged) side chains, folded into a conformation Protein availability at soil mineral surfaces—15N- in which as many apolar residues as possible are NMR analysis has demonstrated that a signiWcant packed into the interior, leaving mainly polar and amount of soil nitrogen is present in amide form charged residues at the surface.

123 18 Biogeochemistry (2007) 85:9–24

Given the diversity of functional groups mak- ability to bind to both hydrophilic and hydropho- ing up proteins, no single process dominates pro- bic mineral surfaces. Strong adsorption of deWned tein adsorption to mineral surfaces. Instead, three proteins on clean clays is well documented in the subprocesses drive adsorption behavior: (i) struc- laboratory, with the amount of adsorbed material tural rearrangement of the protein molecule; (ii) varying over nearly Wve orders of magnitude with dehydration of parts of the sorbent and protein the nature of the sorbate, sorbent, and experi- surfaces; and (iii) redistribution of charged groups mental conditions (Theng 1979; Burchill et al. at the organo-mineral interface (Haynes and 1981; Wang and Lee 1993; De Cristofaro and Vio- Norde 1994). Numerous electrostatic bonds can lante 2001; Ding and Henrichs 2002). form between reactive mineral sites and organic amide and other polar or charged functional N-containing biomolecules as contact materials groups during the adsorption process (Brash and within a zonal organo-mineral complex Horbett 1995). Due to their large size and coiled structure, proteins gain considerable conforma- The proposed special role of proteins in the org- tional energy by unfolding at the solid/liquid ano-mineral contact zone has implications for the interface, an entropically driven rearrangement attachment of additional organic compounds, or that produces numerous organo-mineral interac- the zonal architecture of organic materials tions and distinguishes protein adsorption from adsorbed to mineral surfaces. Organo-mineral that of other available biomolecules (Haynes and complexes featuring multiple layers of proteins Norde 1994). With adsorption to a mineral, pro- have been synthesized using a variety of experi- teins may increase the number of reactive sites on mental methods (Schmidt et al. 1990; Spaeth et al. the newly coated surface by exposing their abun- 1997; Halthur et al. 2004). The amounts of pro- dant polar functional groups to the soil solution. teins that may eventually sorb to mineral surfaces This eVect is known as support preconditioning can be large, approaching 1 g protein g¡1 clay for (Dufrene et al. 1996), and is considered a routine clean clays (e.g., Burchill et al. 1981; De Cristof- step preceding the adhesion of microorganisms to aro and Violante 2001) and buried ceramics mineral surfaces (Bos et al. 1999; Bakker et al. (Craig and Collins 2002), with record values of 2004). The surface charge densities of both pro- 2.4 g g¡1 (Greenland 1965) on montmorillonite, tein and surface can vary with pH, and adsorption implying a soil C concentration greater than that is typically greatest at the isoelectric point of the allowed for a mineral soil horizon according to protein (Ramsden 1995). Soil Taxonomy (Soil Survey StaV 1999). Proteinaceous compounds can bind especially However, observations from natural systems stably to mineral surfaces over long time periods, provide more compelling evidence regarding the as illustrated by the fruitless attempts of archaeol- critical role proteins play within soil organo-min- ogists to remove protein from archaeological arti- eral complexes. Physical fractionation data led facts for further study (Craig and Collins 2002). Sollins et al. (2006) to postulate that sorption of Amelung et al. (2006) used changes in chirality of proteins might create a basal layer to which other aspartic acid and lysine to show that some soil amphiphilic molecular fragments would adsorb proteins achieve residence times of centuries. more strongly than they would to clean clay sur- Sorption experiments (Hedges and Hare 1987; faces, creating a zonal eVect, with N-rich com- Wang and Lee 1993) as well as observational data pounds more abundant near mineral surfaces, and from marine sediments and riverine particles C-rich compounds more abundant farther from (Hedges and Hare 1987; Wang and Lee 1993; the surface. Physical fractionation reveals the ten- Mayer et al. 1995; Arnarson and Keil 2001; Auf- dency for the C/N ratio to decrease with increas- denkampe et al. 2001) also indicate strong and ing particle density (Turchenek and Oades 1979; stable binding of amino acids to clay minerals. Young and Spycher 1979). Lighter fractions Glycoproteins secreted by arbuscular mycorrhizal (<1.8 g cm¡3) have high C/N ratios because they fungi (Wright and Upadhaya 1996) stabilize soil are rich in plant structures high in lignin and cel- aggregates (Baveye 2006), likely due to their lulose. The continued downward trend in C/N

123 Biogeochemistry (2007) 85:9–24 19 ratios at densities beyond 2.2 and even 2.4 g cm¡3 properties depend mainly on the type, density, has previously been attributed to the accumula- and chemical reactivity of the mineral functional tion of proteinaceous material (Oades 1988), and groups, and on the pH of the soil solution. is consistent with our suggestion that N-rich com- Organic fragments directly attached to mineral pounds have a high probability of remaining surfaces are protected against decomposition and sorbed to mineral surfaces. do not participate in signiWcant exchange with Sollins et al. (2006) further suggested that organic compounds in the soil solution. This can sequential density fractionation combined with be explained in part by the strong, ligand- 14C analysis might oVer a way to assess the role of exchange bonding of charged or polar heads of N-rich compounds in the formation of organo- amphiphilic molecules to hydroxylated mineral mineral complexes. They pointed out that varia- surfaces (Tiberg et al. 1999). Proteins, a special tion in particle density correlated well with the class of amphiphilic molecules, can bind particu- percentage of C in the particles, and thus that C larly strongly to mineral surfaces because they loadings decreased with increasing particle den- reinforce the initial organo-mineral bonds that sity, suggesting thinner coatings of organics, and form with entropically favorable conformational in turn a more protein-rich inner layer. Assuming changes that produce further opportunities for these thin inner coatings were more stable, 14C more numerous adsorption interactions (Haynes age should increase with particle density. This and Norde 1994). proved to be true for an Andic forest soil they The hydrophobic zone—Amphiphilic mole- examined. However, large diVerences in mineral- cules adsorbed directly to mineral surfaces tend ogy of the density separates from this soil pre- to mask the physicochemical properties of these cluded ascribing the increase in 14C age solely to surfaces (Bos et al. 1999), to the extent that an coating thinness. outwardly oriented region of high hydrophobicity may be created, especially when the underlying mineral surface has high densities of reactive, sin- The zonal structure of organo-mineral complexes: gly coordinated hydroxyls (Murphy et al. 1990). a hypothesis The hydrophobic zone thus formed provides a suitable region for sorption of hydrophobic moie- Recent spectroscopic evidence suggests that the ties, including the hydrophobic portions of amphi- decomposing organic residues in soils can be philic molecules in the soil solution, leading to viewed as a mixture of largely amphiphilic frag- formation of a bilayer structure on the surface of ments (Chien et al. 1997; Engebretson and von the mineral. In contrast, the direct adsorption of Wandruszka 1997, 1998; von Wandruszka 1998; proteins to a mineral surface may lead to an von Wandruszka et al. 1999; Ferreria et al. 2001; increase in the number of reactive surface sites, Martin-Neto et al. 2001; von Wandruszka and rather than hydrophobic sites. Direct adsorption Engebretson 2001; Nanny and Kontas 2002). The of proteins may not result in formation of a amphiphilicity of many of these molecular frag- hydrophobic zone or a bilayer structure, but ments, and the polarity of the aqueous soil solu- instead enable the attachment of SOM compo- tion in which they are contained, cause them to nents through electrostatic interactions. Thus, the self-assemble into micellar associations when sus- hydrophobic zone must not be considered an pended at high concentrations, and into layered essential and continuous layer present within all or zonal structures when in contact with mineral organo-mineral associations, but rather a zone surfaces. Based on the evidence presented above, likely to be found to varying degrees in many of we propose that the self-assembly of SOM on these associations. mineral surfaces creates a layered or zonal struc- The kinetic zone—Depending on stereochemi- ture with regular features (Fig. 2). cal orientation, degree of amphiphilicity, cation The contact zone—When brought into contact content of the soil solution, pH, temperature, and with a mineral surface, amphiphilic SOM com- perhaps several other controlling factors, a vari- pounds form a contact zone whose structural ety of modes of attachment of further organic

123 20 Biogeochemistry (2007) 85:9–24 molecules appears to be possible. No common of amphiphiles arranged such that the polar por- principle governing these associations is apparent. tions point towards the aqueous phase, creating a However, there is reason to assume that the bilayer structure with a discrete hydrophobic organic compounds trapped in this outer region zone. Empirical evidence for organic surface are partitioned rather than sorbed, and that their loadings of 2–5 times the monolayer equivalent residence times within the outer region are (Hedges and Keil 1995) suggests that further shorter than the residence times of compounds in attachment of organics in a third region or kinetic the intermediate or inner regions. Because zone is possible, although for this outer zone or “Crowded conditions promote selfassembly” region, exchange rates of organic compounds with (Goodsell 2004), the thickness of the outer layer the soil solution are likely high, and residence is likely to depend more on input of organic mate- times brief. rials than on the availability of binding sites, and The arguments in support of the zonal hypoth- to be largely be controlled by exchange kinetics. esis of organo-mineral complexes are indirect in This suggests that more material will be sorbed to nature. We invite the scientiWc community to join kinetic zones in topsoil A horizons than in subsur- us in future attempts to challenge the zonal con- face B and C horizons. Movement of organics into cept and to help us further elucidate the structure and out of this outer region may thus resemble a and dynamics of organo-mineral associations. If phase-partitioning process, rather than classical experimentally and empirically validated, the adsorption. zonal model for organo-mineral associations in natural systems should not only open up a new road towards improved understanding of organic Conclusions C and N dynamics in soils and sediments, but also facilitate the modeling of the fate and transport of With the proposed zonal model, we attempt to contaminants. reconcile information from diVerent natural and artiWcial systems and from various scientiWc Welds Acknowledgements We thank M.S. Torn, T. Filley, C. into an integrated view of the organo-mineral Swanston, M. Kramer, G. Sposito and especially B. Caldwell for discussions and helpful comments on earlier drafts of the complex and its most important properties. Our manuscript. The comments of J. Chorover were particularly model encompasses interdependent hypotheses valuable and are gratefully appreciated. The contribution of regarding (i) the formation of organo-mineral MK results from previous support through the German Sci- associations, (ii) the speciWc architecture of the ence Foundation (DFG) priority program SPP 1090 and from current Wnancial support by the Climate Change Re- zones of adsorbed organic matter, and (iii) the search Division, OYce of Science, U.S. Department of En- exchange kinetics of these organic materials with ergy under Contract No. DE-AC03-76SF00098. Partial the surrounding soil solution. funding was provided by NSF SGER and USDA CSRI We suggest a zonal structure for organic matter grants to PS and MK and by the Andrews LTER program. accretions on mineral surfaces by recognizing that SOM consists largely of amphiphilic molecular References fragments with the thermodynamic impetus to self-assemble into micellar structures on mineral Allison FE, Sherman MS, Pinck LA (1949) Maintenance of particle surfaces suspended in a polar liquid soil organic matter: I. Inorganic soil colloid as a factor (Fig. 2). We further propose that microbial pro- in retention of carbon during formation of humus. Soil teins are better able to bind to mineral surfaces, Sci 68:463–478 Almendros G, Fründ R, Gonzalez-Vila FJ, Haider KM, as well more likely to arrive at these surfaces, Knicker H, Lüdemann HD (1991) Analysis of C-13 than are residues of vascular plants, thus explain- and N-15 CPMAS NMR-spectra of soil organic-matter ing the proposed existence of a particularly stably and composts. FEBS Lett 282:119–121 bound, N-rich inner layer of organic material. Amelung W, Zhang X, Flach KW (2006) Amino acids in grassland soils: Climatic eVects on concentrations and Hydrophobic portions of these surface-bound chirality. Geoderma 130:207–217 molecules can be protected from contact with Arnarson TS, Keil RG (2001) Organic-mineral interactions aqueous solution by the additional accumulation in marine sediments studied using density fraction-

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