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Geoderma 113 (2003) 211 – 235 www.elsevier.com/locate/geoderma Controls of bioavailability and biodegradability of dissolved organic matter in soils Bernd Marschner a,*, Karsten Kalbitz b a b Department Soil Science/Soil Ecology, Ruhr-University Bochum, D-44780 Bochum, Germany ă Department of Soil Ecology, Bayreuth Institute for Terrestrial Ecosystem Research (BITOK), University of Bayreuth, D-95440 Bayreuth, Germany Received 11 March 2002; accepted December 2002 Abstract In soils, dissolved organic matter (DOM) is probably the most bioavailable fraction of soil organic matter, since all microbial uptake mechanisms require a water environment Bioavailability describes the potential of microorganisms to interact with DOM It is a prerequisite for biodegradation and can be restricted, if DOM is present in small pores or within soil aggregates and therefore not accessible for microorganisms DOM biodegradation is defined as the utilisation of organic compounds by soil microorganisms quantified by the disappearance of DOM or O2 or by the evolution of CO2 The controlling factors for DOM biodegradability can be divided into three groups, namely, intrinsic DOM quality parameters, soil and solution parameters and external factors DOM characteristics that generally enhance its biodegradability are high contents of carbohydrates, organic acids and proteins for which the hydrophilic neutral fraction seems to be a good estimate In contrast, aromatic and hydrophobic structures that can also be assessed by UV absorbance decrease DOM biodegradability, either due to their recalcitrance or due to inhibiting effects on enzyme activity Effects of solution parameters such as Al, Fe, Ca and heavy metal concentrations on DOM biodegradability have been documented in various studies, however with different, sometimes conflicting results Inhibitory effects of metals are generally attributed to toxicity of the organic complexes or the free metal ions In contrast, the enhanced degradability observed in the presence of metal ions may be due to flocculation, as larger structures will provide better attachment for microbial colonies As degradation is dependent on microbial activity, the composition and density of the microbial population used in the degradation studies also influence biodegradation Site-specific factors, such as vegetation, land use and seasonality of meteorological parameters control DOM composition and soil and soil solution properties and therefore also affect its biodegradability * Corresponding author Tel.: +49-234-3222108; fax: +49-234-3214469 E-mail address: bernd.marschner@ruhr-uni-bochum.de (B Marschner) 0016-7061/02/$ - see front matter D 2002 Elsevier Science B.V All rights reserved doi:10.1016/S0016-7061(02)00362-2 212 B Marschner, K Kalbitz / Geoderma 113 (2003) 211–235 The major obstacle for a better understanding of the controls of DOM biodegradability is the lack of a standardised methodology or at least systematic comparisons between the large number of methods used to assess DOM biodegradability D 2002 Elsevier Science B.V All rights reserved Keywords: Bioavailability; Biodegradation; Dissolved organic matter; DOC Introduction In the past 10 years, much progress has been made in the understanding of dissolved organic matter (DOM) functions and dynamics in soils Today, it is commonly acknowledged that DOM can enhance the solubility and mobility of metals and organic compounds (Blaser, 1994; Piccolo, 1994; Zsolnay, 1996; Marschner, 1999) and thus contributes to pollutant transport or to micronutrient availability In the presence of DOM, weathering rates can be accelerated (Raulund-Rasmussen et al., 1998), and DOM plays a central role during podsolisation (Lundstrom et al., 1995) Furthermore, DOM contains ă organically bound nutrients such as N, P and S, and DOM dynamics will therefore also affect their mobility and availability (Kalbitz et al., 2000; Kaiser et al., 2001a) DOM is also a substrate for microorganisms In soils, DOM may be the most important C source since soil microorganisms are basically aquatic and all microbial uptake mechanisms require a water environment (Metting, 1993) Furthermore, the soluble state is presumably a prerequisite for the diffusion of substrates through microbial cell membranes so that the degradation of solid phase organic matter or large molecules can only occur after dissolution or hydrolysis by exoenzymes The initial phase of litter decomposition is also strongly related to the amount of soluble compounds in the litter (Williams and Gray, 1974) This was also shown by Marschner and Noble (2000), where CO2 release from a soil supplemented with different plant litters could largely be explained by the disappearance of DOC (Fig 1) Similar results were obtained with soils incubated at different temperatures (Marschner and Bredow, 2002) Cook and Allen (1992) also report positive relationships between initial DOC concentrations and CO2 release during the first weeks of a long-term incubation experiment However, at later stages, this relationship no longer existed which they attributed to the depletion of degradable DOM compounds Several other authors have found close correlations between DOM concentrations and denitrification potentials or rates (Bijay-Singh et al., 1988; Isermann and Henjes, 1990; Pu et al., 1999), thus indicating that the availability of biodegradable DOM may be a prerequisite for creating reducing condition in soils or in certain soil compartments (Zsolnay, 1996) On the other hand, Kalbitz et al (2003) found no evidence that DOM extracted from Oa, and A horizons is the most biodegradable fraction of soil organic matter DOM degradation is also an important process controlling DOM dynamics in soils DOM inputs into the mineral soil generally greatly exceed DOM outputs with seepage Until recently, this was mainly attributed to DOM retention through sorption (Guggenberger et al., 1998) However, some newer calculations indicate that total C pools should then be several orders of magnitude higher than generally observed in the field B Marschner, K Kalbitz / Geoderma 113 (2003) 211–235 213 Fig Relationship between the change in water-extractable soluble organic compounds (DOC) and cumulative CO2 evolution in an Australian pasture topsoil during a 21-day incubation with different plant litter materials (Marschner and Noble, 2000) (Guggenberger and Kaiser, 2003; Moore, 1997) Therefore, the mineralisation rate of DOM in subsoils must be much higher than thought previously (Qualls and Haines, 1992; Guggenberger et al., 1998), and thus, the biodegradation of DOM or of former DOM sorbed to mineral surfaces is the most likely explanation for the generally low DOM fluxes towards the groundwater reported by Michalzik et al (2001) In their extensive review on DOM dynamics in soils, Kalbitz et al (2000) also point out that the mechanisms and controls of DOM degradation in soils are still poorly understood If the earlier stated assumption is correct, that the soluble state is a prerequisite for the uptake and degradation of organic matter by microorganisms, then DOM should play a key role in the stabilisation and destabilisation of soil organic matter, and thus, in C dynamics and C pools of soils Sollins et al (1996) present a conceptual model of SOM stabilisation and destabilisation for which they differentiate between three general sets of characteristics affecting the stability of organic matter: recalcitrance, interactions and accessibility For the conceptual understanding of mechanisms and controls, this approach is very helpful However, one has to bear in mind that often, several mechanisms and processes interact to determine the stability or biodegradability of organic matter in soils In this paper, factors and mechanisms are reviewed that control the microbial degradation of DOM in soils The term ‘‘DOM’’ will be used for all organic substances smaller than 0.45 Am that are suspended in aqueous solutions Strictly speaking, the term DOM can only be applied to organic matter in soil solutions extracted with lysimeters In most studies where soluble organic matter is obtained after extraction from the soil with batch or percolation methods, this is not the truly dissolved phase, but the potentially soluble Zsolnay (1996) therefore suggests to clarify this by using the term ‘‘WEOM’’ (water-extractable organic matter) Since this review deals with the degradability of organic substances in the solution phase, a differentiation between the various methods whereby this solution phase was obtained is not regarded as essential for the problem 214 B Marschner, K Kalbitz / Geoderma 113 (2003) 211–235 Bioavailability versus biodegradability In pharmaceutical and toxicological studies with mammals, the term ‘‘bioavailability’’ is used to characterise the amount of a substance ingested and retained in the organism and thus becomes available for metabolic use It is therefore not a measure for the actual utilisation of this substance For organic molecules such as DOM compounds, this means that their uptake, i.e., bioavailability, must not necessarily result in their breakdown to smaller entities or to complete mineralisation On the other hand, microorganisms excrete exoenzymes that promote extracellular degradation of compounds that are otherwise not bioavailable according to the above definition Therefore, in the context of DOM, the term bioavailability describes the potential of microorganisms to interact with these substances As a measure for the actual utilisation of organic compounds by soil microorganisms, the term ‘‘biodegradability’’ is chosen In a strict sense, this still encompasses two alternative or sequential processes: (1) microbial uptake or breakdown of the original compounds which are then used for the biosynthesis of microbial cell materials (2) complete mineralisation to obtain energy and inorganic nutrients Depending on the analytical tools used to monitor the degradation process, these two processes are considered to different degrees If microbial utilization of DOM is determined by the increase in microbial biomass, then only the assimilated organic carbon (AOC) is considered (Escobar and Randall, 2001), while the mineralized fraction is neglected If DOC disappearance is used as a measure for biodegradation, it is not possible to differentiate between microbial incorporation and mineralisation Another aspect that points to the complexity of this issue is illustrated in the study by Amon and Benner (1996) where low-molecular DOM (< 1000 Da) was less degradable than high-molecular DOM However, bacterial growth efficiency was much higher with the low-molecular DOM fraction, thus indicating that this seemingly less-degradable fraction contained more compounds needed for bacterial biomass production AOC is a measure for the ability of water to support heterotrophic growth (Escobar and Randall, 2001) Therefore, AOC is mainly an important parameter for waterworks It represents only a small portion of the entire biodegradable DOM and will not be further discussed in this paper mainly dealing with soil DOM We will focus on biodegradability of terrestrial DOM, i.e., the utilisation of organic compounds by soil microorganisms quantified by the disappearance of DOM or O2 or by the evolution of CO2 Methods for the determination of DOM biodegradability As indicated in the previous section, no generally accepted standard method for the determination of DOM biodegradability exists so far A method published by the International Organization for Standardization determines the biochemical oxygen demand (BOD) of aqueous media, which is based on a 5-day incubation, using solid sewage sludge as an inoculum (ISO 10707, 1994) However, this method is mainly used to determine the B Marschner, K Kalbitz / Geoderma 113 (2003) 211–235 215 BOD in effluents from sewage treatment plants that have to meet certain BOD levels in many countries The methods used by soil scientists, groundwater hydrologists, limnologists or oceanographers to quantify the biodegradable organic carbon are quite diverse (Table 1) Most studies are conducted in cultures where any of the following parameters may vary: – – – – – – – – – type of incubation (batch culture, flow-through bioreactor) type and size of incubation vessel shaking duration of incubation initial DOC concentration nutrient additions type and amount of inoculum temperature measure for biodegradation (CO2 efflux, DDOC or DTOC) and frequency of measurements In addition, data analysis and documentation will either identify different pools of biodegradable DOM (labile, semi-labile, stable, fast, slow) based on degradation rates, or simply quantify the amounts of DOC mineralised or remaining after a certain time period As a matter of fact, in this review, no two studies performed in different laboratories used the same set of parameters for the determination of DOM biodegradability in their batch experiments This means that the reported results cannot be compared with each other, which is a major obstacle for scientific discussions and progress Of all the parameters listed above, two seem to be most crucial for the quantification of DOM biodegradability: duration of incubation and measure for biodegradation Many long-term incubations (>10 days) showed that DOM generally consists at least of a rapidly degradable fraction (fast BDOM or labile DOM), a fraction that is degraded more slowly, and the recalcitrant fraction that remains in solution even after very long incubation periods (up to 180 days) Little is known about the nature of the compounds in these different DOM pools, but it is generally assumed that the labile DOM consists mainly of simple carbohydrate monomers (i.e., glucose, fructose), low-molecular organic acids (i.e., citric, oxalic, succinic acid), amino acids, amino sugars and low-molecularweight proteins (Lynch, 1982; Qualls and Haines, 1992; Guggenberger et al., 1994; Kusel ă and Drake, 1999; Kaiser et al., 2001b; Koivula and Hanninen, 2001) These compounds ă can directly be utilised by a large number of different organisms and therefore not require a special set of enzymes (Lynch, 1982) The slowly degradable or relatively stable DOM fraction probably contains polysaccharides (i.e., breakdown products of cellulose, hemicellulose) and other plant or microbially derived compounds or degradation products that require special tools for degradation These enzymes are probably only produced when labile substrates are no longer available or they are limited to few organisms that therefore not need to compete for the labile substrates (k- vs R-strategists according to Paul and Clark, 1996) Even the so-called recalcitrant DOM fraction is not fully nondegradable as it would otherwise accumulate in subsoils to a much higher degree than observed (Moore, 1997) However, 216 Table Summary of methods used in different studies for the determination of the biodegradability of DOM or DOM fractions Biodegradation measure Additives – days – weeks H4 weeks Batch incubation in solution culture DOC or TOCa inoculum Pinney et al., 2000b Zsolnay and Steindl, 1991d; Qualls and Haines, 1992; Boissier and Fontvieille, 1993; Raymond and Bauer, 2001 inoculum + nutrients Zsolnay, 1996; Marschner and Bredow, 2002 Gilbert, 1988; Amon and Benner, 1994e, 1996e Benoit et al., 1968; Block et al., 1992c; Nelson et al., 1994; Boyer and Groffman, 1996; Volk et al., 2000b; Escobar et al., 2001b Andersson and Nilsson, 2001; Ogawa et al., 2001 CO2, O2 Duration of incubation V day DOC In soil DOC In soil CO2 a Jones and Edwards, 1998 inoculum + nutrients Flow-through reactor inoculum Lundquist et al., 1999 inoculum Volk et al., 1997; Yano et al., 1998, 2000; Søndergaard et al., 2000; Søndergaard and Worm, 2001 – ISO 10707 (1994); Amon et al., 2001d Brunner and Blaser, 1989 Jones and Edwards, 1998; Jones et al., 2001; Strom et al., 2001 ă TOC: Total organic carbon (analysis without filtration) Biologically active sand was used as an inoculum Biologically active sand was used as an inoculum besides suspended inocula d No direct addition of inoculum; use of unfiltered samples (Zsolany and Steindl, 1991 used 1-Am filters) e Besides O2 consumption, TOC concentration was measured f Quantification of DOM biodegradability by measuring the denitrification potential b c Møller et al., 1999f Hongve, 1999d; Hongve et al., 2000d; Søndergaard et al., 2000 Kalbitz et al., 2003 Jandl and Sletten, 1999; Jandl and Sollins, 1997; Moran et al., 2000 Merckx et al., 2001; Marschner and Noble, 2000; Marschner and Bredow, 2002 Boudot et al., 1989 B Marschner, K Kalbitz / Geoderma 113 (2003) 211–235 Type of incubation B Marschner, K Kalbitz / Geoderma 113 (2003) 211–235 217 this fraction must consist of structures that are not easily cleaved by enzymes, such as lignin degradation products or compounds strongly altered through preceding degradation steps (Joergensen, 1998) As pointed out by Qualls and Haines (1992) and Kalbitz et al (2003), soil solutions contain very different amounts of these fractions, and consequently, the kinetics of the degradation process will be very different Quantification of the contribution of DOM to the stable C pool in the mineral subsoil requires a quantification of rapidly and slowly degradable DOM fractions and their mean residence times Knowledge about the size of the biodegradable DOM fraction is not sufficient Kalbitz et al (2003) reported two DOM solutions with a similar portion of biodegradable DOC, whereas the decomposition constants of the rapidly and slowly degradable fraction differed to a great extent The other aspect that can strongly influence the result of a biodegradation assay is how DOM degradation is quantified If the change in DOC concentration is used, then samples taken from the incubation solution will need to be filtered at 0.45 Am Any DOC transformed into microbial biomass or other particulate carbon resulting from coagulation and precipitation will be largely retained on the filter and its disappearance will therefore falsely be interpreted as degradation On the other hand, if samples are left unfiltered and TOC is used as a measure, microbially incorporated DOC will be regarded as not degraded According to Søndergaard et al (2000), microbial biomass may be in the range of 10% of TOC, so that the error made with TOC or DOC measurements may be small However, the error could be much higher if all particulate C is considered (C which does not pass through a 0.45-Am filter) Thus, Kalbitz et al (2003) reported a DOC mineralisation after 90 days of only 9% from CO2 data, although the DOC content of this sample declined by 50% However, a problem with TOC analysis is that an adequate and reproducible sampling of the suspension and a complete combustion of the particles cannot be guaranteed The other measure for DOM mineralisation is CO2 efflux from the samples (Table 1) However, even with this method, errors can occur due to CO2 dissolution in water (3.4 g CO2/l under atmospheric pressure in distilled water) if CO2 is not trapped and depleted from the atmosphere of the incubation vessel and when the solution was not in equilibrium with atmospheric CO2 initially Another approach for the determination of DOM degradability is using ‘‘bioreactors’’ filled with glass beads that are colonized by microorganisms to form so-called biofilms on their surfaces (Yano et al., 1998; Søndergaard et al., 2000) DOM solutions are passed through such flow-through reactors and DOM degradation is determined from the difference in DOC concentrations between in- and out-flow, usually with residence times of 10 – 24 h In a comparative study, Søndergaard et al (2000) showed that the degradability of DOM determined with such a system is closely related to DOM degradability in batch cultures after 135 –151 days (r2= 0.73) and reaches about 90% of the batch values This high efficiency of the bioreactor can be explained by the relatively high microbial density compared to batch cultures which allows more intensive microbial interactions with DOM and its degradation products within the biofilm Pinney et al (2000) describe another type of bioreactor where they used biologically active sand in a batch vessel 218 B Marschner, K Kalbitz / Geoderma 113 (2003) 211–235 However, these flow-through bioreactors initially require long equilibration times (up to months) and continuous maintenance to achieve reproducible degradation rates (McDowell, personal communication) Other problems of these reactors are the release of DOM into the solution and the adsorption of DOM onto the biofilms Furthermore, it seems impossible to determine the pool sizes and the residence times of a rapidly and a slowly degradable fraction The other listed parameters will also affect the result of DOM biodegradation measurements because these are mainly discussed under soil and solution properties (Section 5.2) Here are some examples Shaking of DOM solutions during the incubation could hinder the development of hyphae which could result in an underestimation of the biodegradation by fungi Addition of nutrients will at least accelerate DOM biodegradation resulting in higher degradation rates in comparison to incubation without addition of nutrients (Schmerwitz, 2001) Furthermore, an enhanced coagulation and precipitation due to increased ionic strengths (Kalbitz et al., 2000) is imaginable Finally, data about the density of added microorganisms are scarce in published studies on DOM biodegradation, ranging from 1000– 2000 (Miettinen et al., 1999) to 104 CFU (Volk et al., 2000), and 0.48 Â 105 cells mlÀ (Buffam et al., 2001) in the inoculated sample Mostly, it is reported that 1% (v/v) of inoculum was added Controls of DOM bioavailability The bioavailability of DOM is reduced if the possibilities of microorganisms to interact with DOM are restricted These may be physical restrictions, such as inaccesibility of DOM in very small pores or chemical restrictions, such as DOM sorption to solid surfaces 4.1 Pore size DOM in small pores is not accessible for microorganisms, i.e., in pores with diameters below 0.2 Am (Zsolnay, 1997) This pore size class contains water that is not plant available and hardly participates in transport processes Consequently, DOM in these pores will only become bioavailable through diffusion into larger water-filled pores Although enzymes excreted by microorganism may enter these pores, this is also limited to diffusion, as well as the movement of the breakdown products out of the pores In some clayey soils, up to the 50% (v/v) of the total pore volume is in this size class and DOM could be preserved there from microbial breakdown However, to date, little is known about the amounts and quality of DOM in different pore size classes, because the soil water in the smallest pore sizes cannot be directly extracted for analysis Zsolnay and Steinweg (2000) have attempted to overcome this problem by using a stepwise extraction technique to obtain DOM fractions from different pore size classes In a first step, the undisturbed samples are percolated to obtain the so-called mobile DOM Soil solution from the mesopores (0.2 –6 Am) is then extracted by centrifugation, and the remaining DOM is extracted in a batch-shake procedure, with a mild salt solution For the three soils examined by Steinweg (2002), DOM in the percolates was always the least biodegradable, thus supporting the assumption that this DOM pool should be depleted first B Marschner, K Kalbitz / Geoderma 113 (2003) 211–235 219 due to its high bioavailability In the other two fractions, DOM degradability was similar and much higher, thus indicating some physical protection in less-accessible pores Since 71 –82% of DOM was obtained in the batch extracts, DOM conservation in the smallest pores may be of major importance in soils 4.2 Soil aggregation A similar mechanism of restricted bioavailability of DOM in small pores may occur within aggregates since various studies have shown that the disruption of aggregates stimulates microbial activity (i.e., Elliott, 1986; Ladd et al., 1993) and that aggregates contain more young and less-altered plant-derived organic matter than the bulk soil (Skjemstad et al., 1990; Puget et al., 1995; Six et al., 2000) However, no studies have been encountered where DOM from within aggregates was compared to bulk soil DOM in terms of its biodegradability 4.3 Sorption In the presence of mineral solid phases, the mineralisation of plant-derived carbohydrates or simple organic compounds such as glucose and citrate can be greatly reduced, especially if charged molecules like citrate or oxalate interact with charged minerals such as clay minerals or goethite (Jones and Edwards, 1998; Miltner and Zech, 1998; Strom ă et al., 2001) The observed reduced biodegradability of soil organic matter through sorption to mineral surfaces is considered to be one or the most important stabilisation processes, and it is extensively reviewed by Sollins et al (1996) and Kaiser and Guggenberger (2000) However, the mechanisms of this sorption process are still as poorly understood, as the reasons why sorbed materials may be less degradable In contrast, Guggenberger and Kaiser (2003) estimated a mean residence time of the sorbed organic carbon of about –30 years and thus challenged the commonly assumed sorptive stabilization of DOM They hypothesised that natural soil surfaces are covered by biofilms with a high affinity for DOM, so that the observed ‘‘sorption’’ may indeed enhance bioavailability and subsequent biodegradation Only sorption onto purely mineral surfaces would thus result in an effective stabilization of DOM (Guggenberger and Kaiser, 2003) On the other hand, sorption is generally not irreversible, so that sorbed materials may return to the solution phase and thus become bioavailable again Desorption will depend on the nature of the sorbate and sorbent and is affected by solution composition Kaiser and Guggenberger (2000) have shown that the hydrophilic DOM fraction is much more easily desorbed than the hydrophobic DOM fraction, which is also less biodegradable (see below) 4.4 Drought DOM availability for microorganisms is reduced when soils become dry, since this greatly limits microbial activity and decreases diffusive transport processes towards the remaining moist sites of activity The strong stimulation of microbial activity after rewetting dry soils is therefore often attributed to the accumulation of easily degradable 220 B Marschner, K Kalbitz / Geoderma 113 (2003) 211–235 substances such as cellular materials from the desiccated organisms in the dry soil (Lundquist et al., 1999; Zsolnay et al., 1999; Merckx et al., 2001) Factors controlling DOM biodegradability The biodegradability of DOM is controlled by numerous factors that can be divided into three categories The first set of factors are intrinsic DOM characteristics that are determined by molecular structure, functional group content or size of the molecules The second set of factors consists of soil properties that can influence the degradation process, such as nutrient availability, microbial community structure and the presence of toxic substances or other soil solution constituents At the third level, external factors such as the temperature and rainfall regime and the associated vegetational cycles will induce a seasonal variability of both DOM inputs and microbial activity which can affect intrinsic DOM quality parameters and soil and soil solution properties 5.1 Intrinsic DOM quality parameters 5.1.1 Molecular size Considering the uptake mechanisms of microorganisms, one could expect that smaller DOM molecules or units should be ingested and degraded preferentially Evidence for this was found in one of our studies where the biodegradability of DOM in ultrafiltrates of the size class < 1000 Da was three- to fourfold higher than in the size class < 10,000 Da or in the bulk DOM solution (Table 2) However, this was only true for DOM extracted from soil samples that were collected in early spring In summer, biodegradability of DOM was much lower, with no differentiation between size classes The reason for this is probably the depletion of degradable compounds by the activated microorganisms during late spring and summer On the other hand, the preferential degradation of small compounds in the spring sample may not be a size effect but due to chemical characteristics Kaiser et al (2001b) showed for a forest soil that easily degradable carbohydrates, amino sugars and proteins accumulate during winter and these compounds would largely appear in the small size class For aquatic DOM, Amon and Benner (1994, 1996) found opposite results In their samples obtained from the Gulf of Mexico and the Amazon River and nearby coastal ocean waters, they determined a much higher C mineralisation from larger DOM size faction (>1000 Da) compared to smaller DOM Since most DOM in the Amazon was in the larger size fraction and marine DOM consisted mainly of the small size fraction, they Table Effect of sampling date on the biodegradability of total DOM and DOM in two size fractions (ultrafiltration) from solutions obtained from percolating undisturbed soil samples from an arable field with 0.01 M CaCl2 (DOC after 5-day incubation at 20 jC in % of initial DOC) Sampling date Total DOM DOM < 10 kDa DOM < kDa March July 12.1 a 3.3 a 16.2 a 3.5 a 51.3 b 4.4 a Values in rows followed by the same letter are not significantly different ( p < 0.05, Duncan test) B Marschner, K Kalbitz / Geoderma 113 (2003) 211–235 221 concluded that the larger molecules are more bioreactive not due to their size, but because they are fresher, i.e., less degraded than the smaller compounds Therefore, again size is only a secondary attribute and the primary factor controlling DOM biodegradability would be structural characteristics 5.1.2 Chemical structure and spectroscopic properties Carbohydrates and amino acids are highly decomposable in soils (Haider, 1992) and are utilized preferentially by microorganisms during degradation of different compounds in DOM solutions (Volk et al., 1997; Amon et al., 2001; Kalbitz et al., 2003) However, Volk et al (1997) stated that the often used classification of carbohydrates as labile DOM components should be seen with caution, as carbohydrates can also be bound to stable DOM compounds Compounds with alkyl or aromatic structural units generally accumulate during the decomposition of soil organic matter (Baldock et al., 1992; Kogel-Knabner et al., 1992; ¨ Baldock and Preston, 1995; Huang et al., 1999) and have thus been associated with a low biodegradability Boissier and Fontvieille (1993) found that phenols and polyphenols were closely related to the amount of nondegradable DOM in incubation experiments Similarly, Wershaw and Kennedy (1998) observed a relative increase in aromatic structures during litter decomposition Kalbitz et al (2003) showed that the biodegradability of DOM extracted from forest litter layers was negatively correlated to its content in aromatic structures determined with 1H-NMR Spectroscopic properties are commonly determined for DOM characterisation Traina et al (1990) and Chin et al (1994) have shown that the specific UV absorbance of humic and fulvic acids between 250 and 280 nm is closely correlated to their content in aromatic structures Since aromatic structures are generally quite recalcitrant, one would expect a negative relationship between the specific UV absorbance and the biodegradability of DOM This indeed was observed in one of our studies (Fig 2a, Jodemann unpublished results) with a ă linear correlation coefficient of r = 0.69 for 28 solutions obtained from an arable soil that had been stored fresh, air-dried or frozen and then extracted with mM CaCl2 solution with a percolation procedure with either undisturbed samples or after homogenization In the same samples, the decrease in DOC concentration after biodegradation was highly significantly correlated (r = 0.85) with an increase in specific UV absorbance (Fig 2b), thus indicating that UV-inactive substances were degraded preferentially Other authors also reported close correlations between DOM degradability and specific UV absorbance (Gilbert, 1988; Zoungrana et al., 1998; Pinney et al., 2000; Kalbitz et al., 2003) and some even found nonlinear relationships, where biodegradability increases exponentially with decreasing UV absorbance However, specific UV absorbance of DOM is not always a reliable predictor for biodegradability Marschner and Bredow (2002) show that the biodegradability of DOM from soil samples incubated at different temperatures varied greatly from 8% to 61% but was not related to specific UV absorbance of either total DOM or of its size fractions If one accepts the assumption that UV absorbance is a measure for the recalcitrant aromatic structures, then these results clearly show that the non-aromatic compounds also greatly differ in biodegradability A low biodegradability of aliphatic compounds may be due to 222 B Marschner, K Kalbitz / Geoderma 113 (2003) 211–235 Fig Relationship between DOC degradation and specific UV absorbance of DOC extracted with 0.01 M CaCl2 solution from differently treated top soil samples from an arable field Sample treatments included drying and freezing prior to extraction (a) Relationship between initial UV absorbance and degradation of DOC after days of incubation (b) Relationship between the change in UV absorbance during incubation and the degradation of DOC binding to aromatic structures (i.e., lignocellulose) or to a high degree of polymerisation or oxidation (Guggenberger et al., 1994), but this cannot be assessed with simple spectroscopic methods More recently, fluorescence spectroscopy has been used successfully to obtain information about the biodegradability of DOM (Glatzel et al., 2003; Kalbitz et al., 2003) using the assumption that more condensed aromatic structures with a red-shifted fluorescence are less biodegradable than structures with a low degree of condensation and conjugation Zsolnay et al (1999) showed that a humification index calculated from fluorescence data can help to differentiate between microbial cell lysis products and more humified DOM Parlanti et al (2000) stressed the usefulness of fluorescence spectroscopy as an indicator for biological activity and humification in coastal waters B Marschner, K Kalbitz / Geoderma 113 (2003) 211–235 223 5.1.3 Fractionation according to polarity and acidity A DOM fractionation scheme used frequently is the one developed by Leenheer (1981), where DOM is separated according to its polarity and acidity Jandl and Sollins (1997) performed incubation studies with such different DOM fractions obtained from water extracts of forest soil samples For the acidic fractions (hydrophilic and hydrophobic), degradation was extremely low ( < 5%) during 120 days of incubation In contrast, the hydrophilic neutral fraction was mineralised to almost 15%, mostly during the first days Since this fraction is enriched with carbohydrates from cellulose and hemicellulose breakdown and from microbial origin (Guggenberger et al., 1994), its high biodegradability is probably due to these easily utilisable substances As a consequence, this fraction generally amounts to less than 20% of total DOC in soil solutions (Vance and David, 1991; Qualls and Haines, 1992; Guggenberger and Zech, 1993; Guggenberger et al., 1994; Andersson and Nilsson, 2001; Kaiser et al., 2001b) In consequence, the hydrophilic and hydrophobic acid fractions generally dominate, which is attributed to their content of recalcitrant compounds and their higher degree of biodegradation (Guggenberger et al., 1994) Kalbitz et al (2003) found a close negative correlation between DOM degradability and hydrophobic DOM portions The hydrophobic acid fraction is preferentially sorbed to mineral surfaces, especially Fe and Al oxyhydroxides (Dai et al., 1996; Kaiser and Guggenberger, 2000) As a consequence, the hydrophilic fraction becomes more dominant with increasing depth and decreasing DOC concentrations, so that biodegradability of DOM may increase, as observed by Qualls and Haines (1992) 5.2 Soil and solution properties Soil properties that affect the physical and chemical environment of the microbial degrader community are expected to affect their activity, and therefore, the degradation of DOM in situ Furthermore, intrinsic DOM properties are affected by soil and solution properties However, most studies of DOM biodegradability are conducted in vitro, i.e., in solution cultures without the soil solid phase Therefore, only effects of varying solution parameters on DOM biodegradability can be assessed 5.2.1 Nutrients, salts, pH, O2 A question that is addressed repeatedly in these studies concerns the addition of inorganic nutrients to the bioassays If the potential biodegradability of DOM is to be assessed, all other limits to microbial activity should be eliminated, i.e., macro- and micronutrient supplies optimized This is generally restricted to addition of N, P and K, since it is assumed that other nutrients are generally present in adequate amounts in the soil solutions or soil extracts In a study with five selected DOM solutions extracted from forest floors, peat and agricultural soils, Schmerwitz (2001) found either no effects of NPK additions on DOM biodegradability or only minor stimulating or depressing effects (Fig 3) Enhancement occurred with DOM of low degradability, while slightly negative nutrient effects were observed for the highly degradable DOM samples Nelson et al (1994) determined the effects of N additions on the mineralisation of DOM obtained from water extracts of soil samples taken at different depths of a pasture profile In the subsoil (80 – 224 B Marschner, K Kalbitz / Geoderma 113 (2003) 211–235 Fig Effects of nutrient addition (N + P + K) on the degradation of DOC from different forest floor solutions during a 10-day incubation (adapted from Schmerwitz, 2001) 100 cm), DOM degradability was not N limited, but in the topsoil, N additions increased CO2 release from the DOM solutions by roughly 25% Nelson et al (1994) attribute this to a relatively high supply of fresh root exudates and soluble root compounds that are easily degradable, so that biodegradability becomes N limited This appears to be an exception, since microbial growth in soils is generally limited by the availability of C substrates (Lynch, 1982), and therefore mainly stimulated by inputs of fresh C sources However, at this point, it may be helpful to consider an important aspect of substrate utilisation by microorganisms All heterotrophic organisms need C sources for two purposes One is as an energy substrate where organic carbon is largely mineralised to CO2 This form of C utilisation mainly depends on the size of the microbial population, and therefore microbial biomass can be estimated from the CO2 release after glucose addition with the substrate-induced respiration (SIR) method of Anderson and Domsch (1978) With these pure energy substrates, the second purpose, microbial growth, will not occur unless important nutrients like N and P are present that are needed for the synthesis of new biomass Therefore, nutrient limitations for the degradation of DOM will only arise when the ratio of biodegradable DOM to microbial biomass is too wide, and when DOM is poor in N or P In soils, this is probably rarely the case However, in solution bioassays that need inoculation, microbial growth may be a prerequisite for DOM degradation and therefore may be stimulated by nutrient additions In their incubation experiments with different DOM fractions, Jandl and Sletten (1999) also tested the effects of Ca on DOM degradation At 1:1 molar ratios of Ca/DOC, the degradation of hydrophobic acids from litter extracts and soil solutions was increased 1.5to 6-fold compared to a Ca-free control For the hydrophilic fractions, Ca had a slightly inhibitory effect and even glucose was degraded less in solutions containing Ca Since glucose cannot form stable complexes with Ca, the authors suggest that Ca forms complexes with metabolites and thus stabilises them against further biodegradation Similar mechanisms may be active with the hydrophilic fractions The stimulatory effects B Marschner, K Kalbitz / Geoderma 113 (2003) 211–235 225 of Ca on the degradation of the hydrophobic acid fraction are assumed to be due to flocculation, as larger structures will provide attachment for microbial colonies (Jandl and Sletten, 1999) However, such precipitation of DOM could also result in a decreased bioavailability because DOM is removed from the aqueous phase In soils, Ca additions generally reduce DOM solubility, affecting mainly the large size fraction (Romkens and ă Dolfing, 1998) It has not yet been established if this has positive or negative effects on the degradation of the sorbed or the remaining soluble DOM Other solution parameters such as O2, pH and soluble salts will also influence microbial activity and the solubility and configuration of DOM molecules Under anoxic conditions, DOC concentrations are commonly elevated (Hunchak-Kariouk et al., 1997; Hagedorn et al., 2000), which is either attributed to the absence of oxide sorption sites or to the greatly reduced microbial activity so that easily degradable organic compounds like acetate may accumulate in the soil solution (Kusel and Drake, 1999) At low pH, DOM solubility is ă generally lower and molecules are more condensed than at higher pH, while Na+ or K+ can increase DOM solubility and cause an expansion of DOM molecules in contrast to Ca effects (Ghosh and Schnitzer, 1980; Murphy et al., 1994) Since most of these solution parameters will also directly affect the composition and activity of the microbial community (Metting, 1993), it should be difficult to separate these effects from those of configurational changes on biodegradability 5.2.2 Metal concentration While nutrient availability in the soil solution can affect DOM degradability through its effects on microbial activity, other solution components can directly interact with DOM and thus may alter its biodegradability In acid forest soils, Al and Fe can form relatively stable complexes with DOM and may thus be mobilized and transported in the soil profile, as it is observed during podsolisation (Blaser, 1994) When these complexes form, DOM is altered structurally, as evidenced by changes in fluorescence (Blaser et al., 1999) and molecular size (Ritchie and Posner, 1982; Jandl and Sletten, 1999) Several authors have assumed that DOM in these complexes is stabilised against biodegradation due to toxic effects from the metals, especially Al (Brunner and Blaser, 1989; Jones et al., 2001) However, experimental data for this are scarce and conflicting Jandl and Sletten (1999) observed inhibiting, enhancing and no effects of Al additions on the mineralisation of different DOM fractions from forest floor solutions Since the strongest inhibiting effects were observed for the carbohydrate-rich hydrophilic neutral fraction and for glucose, direct Al toxicity is more likely since these uncharged molecules are poor metal complexers Jones et al (2001) found no Al effects on the mineralisation of organic acids unless Al concentrations were increased to mM, where they were equally effective in reducing the mineralisation of 0.5 mM citrate or oxalate Boudot et al (1989) found inhibiting effects of Al and Fe on citrate mineralisation only at molar ratios of at least 0.97 and suspect that this is likely due to precipitation of metal oxides and subsequent sorption of their organic model substance On the other hand, Lundstrom et al (1995) showed that the mineralisaă tion of organic metal complexes may be a prerequisite for podsolisation They percolated litter extracts and organic acids through columns filled with C material and observed the formation of an eluvial horizon depleted in Al and Fe after 10 months However, an Aland Fe-enriched illuvial horizon only was formed in the biologically active columns where 226 B Marschner, K Kalbitz / Geoderma 113 (2003) 211–235 the mineralisation of the complexes caused the precipitation of the metals In the sterile treatments, no Al or Fe deposition was observed within the columns These reports indicate that the complexation of potentially toxic metals by DOM will not necessarily reduce its biodegradability but instead may even enhance microbial activity by reducing the free metal ion concentrations and thus their toxicity This would be similar to the beneficial effects of DOM on Al toxicity to roots and microalgae (Marschner, 1995; Parent et al., 1996) Certain heavy metals such as Cu, Pb and Hg can also form very stable complexes with DOM which can alter metal toxicity to aquatic organisms (Oikari et al., 1992; Alberts et al., 2001) and the structural composition of DOM (Blaser et al., 1999) However, no studies were encountered where the degradability of metal complexes was compared to that of metal-free DOM Inhibitory effects of heavy metals on microbial activity in soils have been studied extensively and this has been reviewed recently by Giller et al (1998) In a very original experimental setup, Merckx et al (2001) showed that Zn additions to soil ranging from 50 or 500 mg/kg inhibited the degradation of DOM that had been released after rewetting the dried soil However, the authors interpret this as a direct toxic effect rather than a stabilization of DOM by the complexed metals 5.2.3 Organic compounds As mentioned above, aromatic DOM compounds are generally more stable than molecules with aliphatic structures In addition to this, soluble polyphenols, phenolic acids and plant-derived tannins have been shown to inhibit the activity of various enzymes (Benoit et al., 1968; Williams and Gray, 1974; Gianfreda et al., 1995; Wetzel, 2000) The inhibitory effects of tannins on enzyme activity are less pronounced in the presence of polyvalent cations such as Al3 +, Fe3 + or Mn2 + (Gianfreda et al., 1995), which may also explain the stimulatory effects of Al additions on the degradation of the forest soil-derived hydrophobic acid fraction mentioned above (Jandl and Sletten, 1999) Other natural organic compounds that may even be toxic to soil microorganisms and can thus affect degradation processes include terpenoids (Bremner and McCarty, 1993) and certain amino acids like mimosine (Soedarjo and Borthakur, 1998) Fritze et al (1998) report that DOM extracted from burned forest floor greatly reduced CO2 release when added to soil samples They found the highest concentration of toxic DOM in the hydrophilic base fraction but were not able to identify the responsible compounds 5.2.4 Composition of the microbial community From a general ecological and evolutionary understanding, one would expect the autochthonous microbial population to be best adapted to the optimal utilisation of the organic compounds present in a certain soil This hypothesis was proven by Block et al (1992), who tested the effect of different inocula on the biodegradation of aquatic DOM They concluded that indigenous mixed bacterial populations should be used to determine the biodegradability of DOM However, land use changes and introduction of different plant species alters the quality and quantity of organic matter entering the soil system (Sanger et al., 1997; Bauhus et al., 1998; Coudron and Newman, 1998; Chen and Stark, 2000), which should also alter the quality of DOM In order to assess the influence of different microbial communities on DOM degradation, Schmerwitz (2001) inoculated four B Marschner, K Kalbitz / Geoderma 113 (2003) 211–235 227 DOM solutions with soil extracts obtained from these four different sites (beech forest, spruce forest, peat and agricultural soil) and with a mixed inoculum containing the microorganisms from all sites The degradability of the DOM solutions differed by up to a factor of two depending on the origin of the inoculum However, the highest DOM degradation was not observed with the native inoculum, and noninoculated samples also showed high DOM degradations Although the reasons for the different inoculum efficiencies are not known, these results clearly show that the assessment of DOM biodegradability depends on the type and origin of the microorganisms used for the test Møller et al (1999) shed some light on the complexity of microbial interactions that may occur during these degradation processes They incubated sterilized beech litter with different cultures of bacteria, fungi and combinations of both and determined the biodegradability of extractable DOM by determining its denitrification potential (DNP) As expected, the DNP of DOM from the sterile leaves was highest, because the fresh plant components had not been utilised by microorganisms yet After incubation of the leaves with bacteria alone or with bacteria + fungi, DNP of extractable DOM was lower, but the lowest DNP was determined for DOM extracted after incubation with a cellulytic fungus (Humicola sp.), especially in the presence of bacteria which apparently very effectively removed a large proportion of the biodegradable DOM from the solution through mineralisation and incorporation into the microbial biomass This is a nice example for the important interactions of different organisms during the degradation of DOM 5.3 External factors The DOM characteristics and soil properties controlling DOM biodegradability are not only site-specific characteristics, but they will also vary in time due to seasonal changes in OM inputs, temperature and moisture regime DOC concentrations in soil solutions are generally highest during summer (Hongve, 1999; Kalbitz and Popp, 1999; Kaiser et al., 2001b; Yano et al., 2000) which is attributed to the combined effects of increased release of root exudates and microbial metabolites Concentration effects due to reduced water content can also occur, but are of minor magnitude Wet – dry cycles during summer can also contribute to elevated DOM concentrations, due to aggregate disruption, microbial cell lysis and stimulated microbial activity (Zsolnay and Gorlitz, 1994; Borken et al., ă 1999; Lundquist et al., 1999) Only few data are available on the seasonal variability of DOM quality or biodegradability Kaiser et al (2001b) showed from 13C-NMR analyses that DOM in forest floor leachates contained more low-molecular organic acids and less aromatic, O-alkyl structures and COOH groups during winter than in summer From this, they conclude that winter DOM should be more degradable than summer DOM and that this is due to the accumulation of easily degradable compounds during the dormant season These findings agree well with other studies which generally observe higher DOM degradabilities in winter/spring than in summer/fall (Qualls and Haines, 1992; Nelson et al., 1994; Hongve, 1999; Lundquist et al., 1999) However, Yano et al (2000) determined DOM biodegradabilities as high as 40% in summer soil solutions while during winter, degradability was only 10 –20% Since the nondegradable DOM concentrations remained fairly stable during the year, they attributed the observed seasonality of easily degradable DOM primarily to 228 B Marschner, K Kalbitz / Geoderma 113 (2003) 211–235 Table Effects of soil sample incubation temperature on DOM extractability (percolation with 0.01 M CaCl2) and on DOM degradability (DOC after 5-day incubation at 20 jC in % of initial DOC) Before incubation After incubation at jC À1 DOC [mmol kg ] DOC degradation [%] 26 a 12 a 20 jC 35 jC 18 b 8a 7c 48 b 3d 61 b Values in rows followed by the same letter are not significantly different ( p < 0.05, Duncan test) Data from Marschner and Bredow (2002) the release of organic compounds from roots Another source of easily degradable DOM during the growing season could be canopy-derived plant leachates Within the physiological range of – 35 jC, temperature is known to stimulate microbial activity (Paul and Clark, 1996) As a consequence, DOM production may increase at higher temperatures due to enhanced microbial breakdown of larger insoluble compounds to soluble entities, as observed by Christ and David (1996) On the other hand, increased microbial activity also results in enhanced biodegradation and mineralisation of DOM If this process dominates over the enhanced production, the net effect of a temperature-stimulated microbial activity will be DOM depletion This was observed by Marschner and Bredow (2002), where extractable DOM decreased with increasing temperature, while at the same time DOM became more biodegradable (Table 3) This stands in contrast to the concept that easily degradable DOM should be depleted preferentially in soils One possible explanation is that the high microbial activity caused a rapid depletion of substrates and nutrients, resulting in the die-back of part of the microbial population, releasing easily degradable cell constituents into the soil solution In any case, these studies show that the external controls on DOM production and DOM quality, especially in terms of biodegradability, are only poorly understood Conclusions This review shows that the biodegradability of DOM in soil solutions or in aqueous soil extracts varies greatly with soil depth, sampling date or site characteristics Some of the differences can be attributed to the presence of varying amounts of specific chemical compounds like sugars, proteins, phenols or tannins that are known for their different biodegradability However, as with other soil organic matter, DOM contains only a small fraction of these identifiable compounds, while the major part are structures that have been altered by microbial and biochemical degradation processes The degradability of these compounds will be mainly determined by the presence of structural components for which enzymatic tools exist within the microbial community Little is known about the diversity and efficiency of these enzymes, so that it seems unlikely that an exact characterisation of structural components will allow the prediction of DOM degradability However, the correlations between biodegradability and certain DOM characteristics, such as specific UV absorbance, aromaticity, hydrophilic neutral or hydrophobic acid content show that certain analytical tools can help to explain differences in DOM degradability However, B Marschner, K Kalbitz / Geoderma 113 (2003) 211–235 229 conclusions about causal relationships cannot be easily drawn from these results For example, the generally negative correlation between UV absorbance as a measure for aromaticity and degradability can be due to the recalcitrance of aromatic structures or due to inhibitory effects of these compounds on enzyme activity Among the studies investigating other factors that may influence DOM degradability, results are often conflicting or ambiguous so that not even concepts about causalities can be developed yet This concerns the effects of nutrients or metals on biodegradability, while other potential controlling factors such as pH or salts have not been assessed at all A summary of the discussed controlling factors is presented in Fig Here also, some parameters have been included, which may be of relevance although no experimental evidence for this has been encountered The major obstacle for a better understanding of the controls of DOM biodegradability is the lack of a standardised methodology or at least systematic comparisons between the various methods used to assess DOM biodegradability The high variability in incubation durations, inoculum or nutrient additions and the different measures for the quantification of DOM degradation greatly hinder comparisons between the studies Therefore, efforts should be put in the development of a standardised protocol for DOM degradation studies For this, interlaboratory comparisons have to be made that assess the variability of degradabilities determined with the different methods on the same samples A first such study is currently under way among six laboratories in Europe and North America Methodological research should also include the specific features of soils Almost nothing is known about the effects of natural soil surfaces with a high density and diversity of microorganisms on DOM biodegradation Finally, the question of bioavailability has been addressed in only very few studies In naturally structured soils, certain amounts of DOM may not be accessible for micro- Fig Summary of the three groups of parameters that have been identified as controlling factors for DOM degradability Bold: verified in 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microorganisms, then DOM should play a key role in the stabilisation and destabilisation of soil organic matter, and thus, in C dynamics and C pools of soils. .. reserved Keywords: Bioavailability; Biodegradation; Dissolved organic matter; DOC Introduction In the past 10 years, much progress has been made in the understanding of dissolved organic matter (DOM)... potential and fractions of organic carbon in air-dried and field-moist soils Soil Biol Biochem 20, 737 – 741 Blaser, P., 1994 The role of natural organic matter in the dynamics of metals in forest soils