lable at ScienceDirect

Soil Biology & Biochemistry 41 (2009) 1066–1074

Contents lists avai

Soil Biology & Biochemistry

journal homepage: www.elsevier .com/locate/soi lb io

Effective retention of litter-derived dissolved organic carbon in organic layers

Matthias Muller a, Christine Alewell a, Frank Hagedorn b,*

a Institute of Environmental Geosciences, University of Basel, Bernoullistrasse 30, CH-4056 Basel, Switzerlandb Swiss Federal Institute for Forest, Snow and Landscape Research (WSL), Zurcher Strasse 111, CH-8903 Birmensdorf, Switzerland

a r t i c l e i n f o

Article history:Received 13 October 2008Received in revised form3 February 2009Accepted 9 February 2009Available online 28 February 2009

Keywords:Carbon cyclingDecompositionDissolved organic matterLitterSoil organic matterPrimingStable isotopeTracer

* Corresponding author. Tel.: þ41 1 739 2463; fax:E-mail address: frank.hagedorn@wsl.ch (F. Hagedo

0038-0717/$ – see front matter � 2009 Elsevier Ltd.doi:10.1016/j.soilbio.2009.02.007

a b s t r a c t

This study aimed to gain insight into the generation and fate of dissolved organic carbon (DOC) in organiclayers. In a Free Air CO2 Enrichment Experiment at the alpine treeline, we estimated the contribution of13C-depleted recent plant C to DOC of mor-type organic layers. In an additional laboratory soil columnstudy with 40 leaching cycles, we traced the fate of 13C-labelled litter–DOC (22 and 45 mg l�1) in intactOa horizons at 2 and 15 �C. Results of the field study showed that DOC in the Oa horizon at 5 cm depthcontained only 20� 3% of less than six-year-old C, indicating minor contributions of throughfall, rootexudates, and fresh litter to leached DOC. In the soil column experiment, there was a sustained DOCleaching from native soil organic matter. Less than 10% of totally added litter–DOC was leached despitea rapid breakthrough of a bromide tracer (50� 7% within two days). Biodegradation contributed onlypartly to the DOC removal with 18–30% of added litter–DOC being mineralized in the Oa horizons at 2and 15 �C, respectively. This was substantially less than the potential 70%-biodegradability of the litter–DOC itself, which indicates a stabilization of litter–DOC in the Oa horizon. In summary, our results giveevidence on an apparent ‘exchange’ of DOC in thick organic layers with litter–DOC being retained and‘replaced’ by ‘older’ DOC leached from the large pool of indigenous soil organic matter.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Dissolved organic matter (DOM) plays an important role inecosystems: it links bio-, hydro- and pedosphere, represents themobile fraction of soil organic matter (Kalbitz et al., 2000), and actsas both an energy and C source for microorganisms (McDowell,2003). DOM is a key vehicle for the export of nutrients (e.g.Hagedorn et al., 2000; Michalzik et al., 2001), and determines thesolubility and mobility of metals and organic compounds (Tipping,2002). Furthermore, leaching of DOM from organic layers tomineral soils and sorption of DOM onto mineral surfaces are verylikely an important mechanism in stabilizing soil organic matter(Sollins et al., 1996; Kaiser and Guggenberger, 2000; Kalbitz andKaiser, 2008).

The generation and sources of DOC in soils are still uncertainand thus, impacts of environmental changes on the production andfate of DOC can hardly be predicted (Michalzik et al., 2003). Prin-cipally, DOC is a leaching product from plants, litter and humus andit is generated by microbial activity (McDowell and Likens, 1988;Kalbitz et al., 2000; Hagedorn et al., 2004). Root exudation and thus,

þ41 1 739 2215.rn).

All rights reserved.

recent photosynthates might also add to DOC in organic layers(Yano et al., 2000; Giesler et al., 2007). The relative contribution ofthese components is controversially discussed. Leaching experi-ments (e.g. Moore and Dalva, 2001; Park et al., 2002) have high-lighted that recent litter has a large potential to produce DOC, andhence it was suggested that fresh litter is a major DOC source(Currie and Aber, 1997). However, a substantial fraction of litter-derived DOC is easily decomposable and thus, might not passorganic layers (van Hees et al., 2005; Hagedorn et al., 2008). Littermanipulation experiments indicate that litter additions to forestfloors increase DOC leaching from the Oa horizons suggesting thatlitter contributes significantly to DOC (Froberg et al., 2005; Lajthaet al., 2005; Kalbitz et al., 2007). Exclusions of litter inputs,however, did not result in significant decreases in DOC leachingfrom lower forest floor layers, which implies that the major fractionof DOC is generated in Oe and Oa horizons and not in the litter layer.In a recent field study, Froberg et al. (2007) observed that onlya small fraction of added 13C-labelled spruce litter was leached asDOC from mor layers, but it remained unclear, if the litter-derivedDOC was removed by biodegradation or by a physico-chemicalinteraction with solid soil organic matter.

The aim of this study was to gain insight into DOC dynamics inundisturbed mor-type organic layers in particular to quantify howmuch of litter-derived DOC is mineralized, leached, or retained in

M. Muller et al. / Soil Biology & Biochemistry 41 (2009) 1066–1074 1067

Oa horizons. In a field study, we enriched alpine treeline ecosys-tems with 13C-depleted CO2 and traced the 13C-signal from therecent plant assimilates to soil-respired CO2 and leached DOC. Inaddition, we conducted a soil column experiment, where weapplied 13C-labelled litter–DOC to undisturbed soil cores of Oahorizons and followed its fate at different microbial activities at 2and 15 �C.

2. Material and methods

2.1. Study site description and field experiment

The Free Air CO2 Enrichment study (FACE) and the soil samplingfor the column experiment were carried out at 2180 m a.s.l. atStillberg in the Central Alps near Davos, Switzerland. Here, at thealpine treeline, seedlings of Larix decidua and Pinus uncinata wereplanted in 1975 in a previous large-scale afforestation experiment.Today, the trees have a height of 2 m. Dwarf shrubs such as Vacci-nium myrtillus, Vaccinium uliginosum, and Empetrum hermaphrodi-tum dominate in the understory under a sparse open canopy. Theterrain is relatively steep with north-east exposed slopes of 25–30�.Parent material is Paragneiss. Soil types are nutrient-poor sandydystric Leptosols and Podzols (Hagedorn et al., 2008). The organiclayers are Humimors dominated by Oa horizons with thicknessesbetween 5 and 15 cm (Bednorz et al., 2000). Topsoil characteristicsare given in Table 1.

In the FACE study, 20 plots each with one tree had been exposedto elevated CO2 since 2001. Pure CO2 (d13C¼�30&) is releasedthrough 24 vertically hanging laser-punched tubes, which are fixedon steel frames having areas of 1.1 m2 (Hattenschwiler et al., 2002).Growing season average of CO2 concentrations was 566� 75 ppmv

CO2 under elevated CO2 and 370� 3 ppmv under ambient CO2.

2.2. In situ C fluxes

Soil solution was collected in all plots by installing two ceramicsuction cups (SoilMoisture Equipment Corp., Santa Barbara, USA)per plot in Oa horizons at 3–7 cm depth. The suction cups wereevacuated at each sampling with a constant suction of 400 hPa forabout 16 h (overnight). In addition to the suction cups, we installedzero-tension lysimeters (8� 8 cm plexiglass plates with a poly-ethylene-net) at 5 cm soil depth which we connected to glassbottles. All soil water samples were stored in cooling boxes for thetransport to the laboratory.

Samples for soil-respired 13CO2 in the field were taken frompermanently installed PVC-collars (10-cm ID and a height of 5 cm)which were pressed to a depth of 2 cm into the organic layer inbetween dwarf shrubs. For the gas sampling, we closed thechambers with PVC-lids at least 4 h after stopping the CO2

enrichment to avoid contamination with 13C-depleted CO2. Thirtyminutes after closing the collars, we took gas samples by retrieving15 ml of air with 20 ml syringe through a septum and by injectingthe air in 12 ml pre-evacuated glass vials closed with an airtightrubber septum (volume of 12 ml, Exetainer gas testing vials, LabcoLimited, High Wycombe, UK).

Table 1Properties of surface soil horizons.

Horizon Depth Soil organic C(%)

C/N (Mass ratio)

Oa (5–0 cm) 40.8� 2.5 27.2� 0.7Aeh (0–5 cm) 10.3� 1.5 27.8� 0.7

Soil properties of the treeline ecosystems at 2200 m a.s.l., Stillberg, Switzerland. MeansCEC: cation exchange capacity; BS: base saturation.

2.3. Soil column study

For the soil column experiment we sampled 12 soil cores of Oahorizons at the Stillberg site with PVC-cylinders (Ø 11 cm). The soilcores had a thickness of approximately 5.5 cm and they wereplaced on glass filter plates with pore sizes of 10–16 mm. Littermaterial on top of the columns was removed by hand. The columnswere stored at 4 �C for three weeks before the column study star-ted. For the experiment, the soil columns were sprinkled 40 timesduring 80 days with (1) artificial rainwater as a control; or (2) 13C-labelled litter–DOC extracted with artificial rainwater. Treatmentswere conducted at 2 and 15 �C. The artificial rainfall was adjusted tonatural rain at the Stillberg site (0.5 mg Naþ l�1, 2.0 mg Kþ l�1,0.5 mg Mg2þ l�1, 1.4 mg Ca2þ l�1, 2.7 mg SO4

2� l�1, and 0.6 mgCl� l�1). During each leaching cycle 160 ml of solution was addedevery two days in drops within 2 h using a peristaltic pump. Thiscorresponded to a rainfall of 20 mm and approximately half thepore volume of the soil columns. Leachates were collected duringthe following two days by applying suctions of 40 hPa.

The experiment was conducted in four phases with 10 leachingcycles each. During the first phase, all soil columns were sprinkledfive times with artificial rainwater at 15 �C. Then, six out of the 12soil columns were incubated at 2 �C. In the second and third phases,litter–DOC was added to four of the six soil columns at eachtemperature while two control columns received rainwater only.The average DOC concentrations of the litter–DOC solutions were22 and 45 mg l�1 in the second and third phases, respectively. In thefourth phase, all soil columns were sprinkled again with artificialrainwater only. During the whole fourth phase, bromide (KBr) wasadded as a tracer to determine breakthrough curves from allcolumns.

The litter–DOC was extracted from air-dried 13C-labelled spruceneedle litter that was collected in a previous CO2 enrichment study(Hagedorn et al., 2005). For the extraction, we first added artificialrainwater to the litter in a weight ratio of 1:15. Then, after 24 h at20 �C, the solution was filtered at 0.45 mm and artificial rainwaterwas added again. This procedure was repeated eight times. Allextracts were combined to one litter–DOC solution except the firsttwo extracts, which had unnaturally high DOC concentrations.

2.4. Sampling and analysis

All solution samples were filtered through 0.45-mm cellulose-acetate filters (ME25, Whatman–Schleicher & Schuell) within twodays after sampling and then stored at 2 �C until analysis. The pH ofthe leachates (pH-meter, 691, Metrohm, Switzerland), electricalconductivity (conductometer 660, Metrohm, Switzerland), concen-trations of DOC and of total dissolved nitrogen (TN) (TOC/TN ana-lyser (TOC-V, Shimadzu Corp., Tokyo, Japan)) and nitrate andbromide concentrations (ion chromatography, 761 Compact IC,Metrohm, Switzerland) were measured in the leachates. In selectedsamples ammonium was measured colorimetrically by automatedflow injection analysis (RF-535 Fluorescence HPLC Monitor,Shimadzu).

The UV absorption at 285 and at 260 nm was determined witha Cary 50 UV spectrophotometer (Varian, Inc., Palo Alto, CA, USA).

pH (CaCl2) CEC eff(mmol C kg�1)

BS (%)

4.2� 0.1 169� 8 83� 33.8� 0.1 71� 5 37� 2

and standard errors of soils from 20 plots under ambient CO2.

M. Muller et al. / Soil Biology & Biochemistry 41 (2009) 1066–10741068

The specific UV absorbance (SUVA) as a measure of aromaticity (Chinet al., 1994) was then calculated by dividing measured UVabsorption at 285 nm by DOC concentrations in mg l�1.

Soil respiration rates were measured by closing the columns withPVC-caps, taking two gas samples per lysimeter within intervals of30–60 min and calculating the increase in CO2 concentration withtime. Gas samples were retrieved with 1 ml syringes throughrubber septa in the PVC-caps and analysed by gas chromatography(GC, SRI-8610C, SRI Instruments, Torrance, CA, USA). For themeasurements of d13C values of respired CO2, two additional gassamples were taken from the sealed lysimeters. Fifteen ml of airwas injected into pre-evacuated 12-ml glass vials (Exetainer gastesting vials, Labco Ltd., UK).

For soil carbon and its isotopes, soil samples were taken in theimmediate vicinity of the sampled soil columns. Roots wereremoved and soils were homogenised. Soils were dried at 40 �C,sieved at 2 mm and ground with a ball mill. Soil columns weredried at 40 �C and several samples per soil column down to 0.5 cmdepth were cut off with a scalpel at the end of the experiment tomeasure 13C-signatures in soil columns. Soil C contents of all soilsamples were determined by combustion analysis (Leco RC-412,LECO Corporation, USA).

Stable isotope analysis: The fraction of litter–DOC in DOC and CO2

was quantified by analysing d13C values of DOC in the leachates andd13C values in soil-respired CO2. In order to achieve greater andeasier-to-handle amounts of dried leachates, 1–2 ml of a K2SO4-solution containing 5 mg of salt was added to the leachates (usually100 ml) before freeze-drying (Hagedorn et al., 2004). The d13Cvalues of freeze-dried leachates and soil samples were determinedwith an automated continuous flow isotope ratio mass spectrom-eter (FEA-1112, Milan, Italy, interfaced with a DeltaPlus XL ThermoFinnigan, Bremen, Germany). The d13C values of soil-respired CO2

were measured with a gasbench II linked to a mass spectrometer(Thermo Finnigan DeltaPlus XL).

Results of C isotope analysis are expressed in d units (&) andreferenced back to the Vienna Pee Dee Belemnite (VPDB) standard.The long-term reproducibility for d13C data is generally better than0.1& (B. Seth, personal communication 2008).

2.5. DOC biodegradation

Biodegradability of litter–DOC and DOC leached from the soilcolumns was determined by measuring the decrease in DOCconcentrations with time at 15 �C (Hagedorn and Machwitz, 2007).We first adjusted solutions of 25 mg DOC l�1 to contain 2 mgNH4NO3–N l�1 and 0.2 mg K2HPO4–P l�1. Then, we added an inoc-ulum (extracts of fresh needle litter with 5 mM NaCl in 1:5 ratios)and glass fibre filters as surfaces for microbial growth. Duringincubation, all solutions were gently shaken by hand once a day.Glucose solutions (25 mg C l�1) served as controls.

2.6. Calculations

The d13C values of the soil-respired CO2 were calculated from thetwo 13CO2 measurements by a two end-member mixing calculation(Subke et al., 2004). The fractions of litter–DOC in C fluxes (CO2 andDOC) derived from the 13C-labelled litter–DOC during the soilcolumn study were calculated as follows:

flitter—DOC ¼d13Csample � d13Ccontrols

d13Clitter—DOC � d13Ccontrols

(1)

flitter–DOC¼ fraction of litter-derived C in respired CO2 or leachedDOC; d13Csample¼ d13C value of respired CO2 or leached DOC;d13Clitter–DOC¼ d13C value of the litter–DOC; and d13Ccontrols¼mean

d13C value of respired CO2 or leached DOC of the controls at thesame time.

For the C mass balance, the amount of leached DOC was calcu-lated by multiplying the average volume of the leachate withmeasured DOC concentrations. The carbon respired as CO2 wascalculated from the measured CO2 efflux by extrapolating to theoverall 48 h of each leaching cycle. Finally, the fluxes of litter-derived C were calculated by multiplying the average fractions oflitter–DOC per phase with the respective C flux.

3. Results

3.1. Solute transport

During each leaching cycle with 160 ml of rainwater input (withor without litter–DOC), 140 ml of solute was leached from thecolumns. Water transport through the columns was rather fast. TheBr� tracing showed that constant Br� concentrations were reachedafter five leaching cycles with 80–90% of the input concentrations(data not shown). At the first and second leaching cycle, Br�

concentrations were already 50� 7% and 75� 4% (Mean� Standarderror, n¼ 12) of the constant Br� concentrations.

The pH of the leachates ranged from 3.8 to 4.5 with a slightincrease in pH during the third and fourth phases. Adding litter–DOC did not affect pH-values. Electrical conductivity of the leach-ates was between 30 and 80 mS cm�1. Ammonium concentrationswere 10–80 mg N l�1 and thus, small.

3.2. DOC leaching and soil respiration

During the 80-day soil column experiment with 40 leachingcycles, DOC and CO2 fluxes remained almost constant over time inthe DOC-free rainwater control treatment at a given temperature(Fig. 1). Leaching of DOC accounted for 16–26% of the total C lossesfrom soil columns with Oa horizons. The mean concentrations ofDOC leached from the control soil columns without litter–DOCaddition were 20–30 mg DOC l�1 at 15 �C. They were similar tothose of the soil waters in the Oa horizons at 5 cm depth at theStillberg site: 28� 2 mg DOC l�1 in leachates of zero-tensionlysimeters and 40� 3 mg DOC l�1 in samples of suction cups.Average respiration rates from the soil column were 1.0� 0.2 mmolCO2 m�2 s�1 at 15 �C. This was about half of the mean in situ totalCO2 effluxes from soils, but approximately the same as heterotro-phic soil respiration at the study site (0.9� 0.2 mmol CO2 m�2 s�1;Hagedorn et al., 2008).

DOC leached from the Oa horizon had a high specific UVabsorbance (SUVA; Fig. 2), showing that it was highly aromatic anddominated by the so-called hydrophobic DOC (Chin et al., 1994;Dilling and Kaiser, 2002). In agreement, only a small fraction of DOCleached from Oa horizons was biodegradable with 0–14% of DOCbeing degraded during four weeks (Fig. 3). Again these DOC char-acteristics are very similar than those of DOC sampled in the Oahorizon at the field site (e.g. SUVA: 0.033� 0.002 l mg C�1 cm�1; 6%biodegradable DOC; Hagedorn et al., 2008).

DOC leaching and soil respiration strongly depended ontemperature as indicated by the immediate decline of DOC leachingand CO2 efflux after decreasing the incubation temperature from 15to 2 �C (Fig. 1). The temperature sensitivity was significantly greaterfor C mineralization than for DOC leaching (P< 0.05) with the soilrespiration rates and DOC concentrations decreasing by 80% and66%, respectively. The corresponding Q10 values were 3.4� 0.2 forsoil respiration but only 2.1�0.1 for leaching of DOC.

0 10 20 30 400

10

20

30

40

0.0

0.5

1.0

1.5

2.0

0

1

2

3

4

0.0

0.2

0.4

0.6

0.8

2°C

15°C

DO

C (m

g l-1)

Leaching cycle

2°C

15°C

controls

+ litter-DOC

CO

2-efflu

x (

mo

l m

-2s

-1)

Litter-DOC addition

0 22 45 0 ppm

CO

2-efflu

x (g

C m

-22d

-1)

DO

C-flu

x (g

C m

-22d

-1)

Fig. 1. Soil respiration and DOC leaching from Oa horizons in the soil column study.The 2 �C columns were incubated for the first five leaching cycles at 15 �C and then at2 �C. Means and standard errors of four replicates for the litter–DOC columns. Verticallines separate the four phases with additions of 20 mm of the respective solution perleaching cycle containing 0, 22, 45, and 0 mg l�1 litter–DOC. One leaching cycle isequivalent to two days. Note that the scale for C fluxes differs by a factor of 5 betweenCO2 efflux and DOC leaching.

0 10 20 30 400.01

0.02

0.03

0.04

0.05

0.01

0.02

0.03

0.04

0.05

2°C

Sp

ecific U

V ab

so

rb

an

ce at 285 n

m (l m

g C

-1cm

-1)

Leaching cycle

15°C

Litter-DOC addition

0 22 45 0 ppm

controls

+ litter-DOC

Fig. 2. Specific UV absorbance at 285 nm of leachates from Oa horizons in the soilcolumn study. Means and standard errors of four replicates for the litter–DOC columns.Vertical lines separate the four phases with additions of 20 mm of the respectivesolution per leaching cycle containing 0, 22, 45, and 0 mg l�1 litter–DOC. One leachingcycle is equivalent to two days.

0 5 10 15 20 25 300

20

40

60

80

100

DO

C b

io

de

gra

da

tio

n

(%

o

f in

tia

l D

OC

)

Days

15°C2°C

leached DOCOa horizon

litter-DOC

Fig. 3. DOC biodegradation at 15 �C under optimal nutrient supply. The lines are fittedwith a first-order exponential function [y¼ A(1� e�kt)]. For litter–DOC, A is 66% andk¼ 0.35 d�1; for leached DOC from Oa layers, A is 8.3% and k¼ 0.17 d�1. Means andstandard errors of four replicates in the case of leached DOC from individual soilcolumns.

M. Muller et al. / Soil Biology & Biochemistry 41 (2009) 1066–1074 1069

3.3. Adding litter-derived DOC

By comparison with DOC leached from the soil columns, thelitter–DOC was dominated by the so-called ‘hydrophilic’ fractionand had a low aromaticity as indicated by a low SUVA of 0.016 l mgC�1 cm�1. A significant fraction of the added litter–DOC was easilydegradable: 50�1% and 70� 0.5% of the litter–DOC was lostduring 5 and 28 days, respectively (Fig. 3).

The overall effect of the addition of litter–DOC on DOC leachedfrom the Oa horizon was rather small (Fig. 1). Adding 22 mg l�1

litter–DOC in the second phase did not lead to any changes in DOCleaching at both temperatures, but the addition of 45 mg litter–DOC l�1 almost doubled DOC concentrations at 2 �C. However, asthe absolute increase was only 5–9 mg DOC l�1, net DOC retentionwas still about 80–89% of litter–DOC inputs (Figs. 1 and 5). Theincrease in DOC leaching due to the litter–DOC was even smaller at15 �C with a four time greater respiratory activity. Here, DOCconcentrations in the leachates began to increase not before themiddle of the third phase, but this increase continued in the fourthphase after stopping the DOC addition. Concentrations of DONfollowed the DOC pattern (data not shown).

The specific UV absorbance at 285 nm generally increasedthroughout the experiment. It was not affected by the addition oflitter–DOC at 15 �C (Fig. 2). In contrast, at 2 �C, the SUVA decreased

M. Muller et al. / Soil Biology & Biochemistry 41 (2009) 1066–10741070

in the second and third phases evidencing the breakthrough oflitter–DOC with a much lower SUVA.

Soil respiration rates were not significantly affected by the inputof litter–DOC (Fig. 1). At 15 �C, total soil respiration was six timeshigher as the total amount of litter–DOC added, and thus, anypotential effect was not detectable. However, the integration acrossthe whole experimental period suggests that total soil respirationat 15 �C was increased by approximately 10%. At 2 �C and muchsmaller respiration rates, the C mineralized corresponded toapproximately 50% of the added litter–DOC. Nevertheless,compared to the control, a relevant increase did not occur.

3.4. Isotope tracing

The average d13C value of added litter–DOC was �38.8� 0.5&.Thus, the difference in 13C (D13C) between litter–DOC and SOM(�26.2� 0.2&) was about 13&. The average difference betweenadded litter–DOC and DOC leached from the control columns was11&. Therefore, the isotopic signal of litter-derived DOC was highenough to detect contributions of litter–DOC in soil-respired CO2

and in DOC leached from Oa horizons when they were higher than10%.

At 15 �C, the addition of 22 mg litter–DOC l�1 in the secondphase had no effect on d13C values of leached DOC, but in the thirdphase with higher litter–DOC additions, the D13C increased by 1.2–1.4& (Fig. 4). This relatively small change in the isotopic signal ofthe added litter–DOC corresponds to a fraction of added litter–DOCin leached DOC of 11�1%. At 2 �C with a smaller microbial activity,the D13C in leached DOC was greater with 1.0–1.9& and 4.4–5.5&

-34

-32

-30

-28

-26

-34

-32

-30

-28

-26

0 10 20 30 400.0

0.2

0.4

0.6

0.8

15°C

controls+ litter-DOC

2°C

controls+ litter-DOC

15°C2°C

flitter-D

OC in

D

OC

Leaching cycle

DOC

13C

o

f D

OC

(‰

)13C

o

f D

OC

(‰

)

Fig. 4. The d13C values of DOC in leachates and fractions of added litter–DOC in DOC at 15respired CO2 at 15 and 2 �C (right column). Means and standard errors of four replicates. Veper leaching cycle containing 0, 22, 45, and 0 mg l�1 litter–DOC. One leaching cycle is equi

in the second and third phases, which corresponded to 14� 3% and47� 5% of litter–DOC in leached DOC, respectively. However, due tothe low DOC concentrations in the Oa leachates at 2 �C, the absoluteamount of litter–DOC leached from Oa horizons was only 11–14% ofthe added 45 mg l�1 litter–DOC (Fig. 5).

The fraction of litter–DOC immediately declined after stoppingthe DOC addition in the fourth phase (Figs. 4 and 5). This impliesthat the increase in DOC leaching from the previous addition oflitter–DOC at 15 �C (Fig. 1) did not result from a retarded leaching oflitter–DOC but from an accelerated leaching of native C.

The addition of 13C-labelled litter–DOC was reflected ind13Crespired (Fig. 4). The highest difference in respired 13CO2 (D13C)was at the addition of 45 mg l�1 litter–DOC: 3.7& and 1.7& at 2 and15 �C, respectively. At 2 �C, the calculated fraction of added litter–DOC in respired CO2 reached up to 25� 4% during the third phase,and declined to about 10� 4% in the fourth phase (Fig. 4). At 15 �C,the fraction was at most 13� 3% in the third phase and it decreasedto only 5� 3% in the fourth phase.

The addition of litter–DOC did not change the d13C of solid soilorganic matter (data not shown), which is not surprising becausethe absolute amount of added C is small compared to the SOM pool.The total amount of added litter–DOC corresponds to only 3% of theSOM pool in the uppermost 0.5 cm of the Oa horizon.

3.5. Carbon mass balance

The 13C mass balance showed that 60–70% of the added litter–DOC (8.3–9.7 g C m�2) was retained in the organic layer, but theretention and the fate of litter-derived DOC depended on

-28

-26

-24

-22

-20

-28

-26

-24

-22

-20

0 10 20 30 400.0

0.2

0.4

0.6

0.8

15°C

controls+ litter-DOC

2°C

controls+ litter-DOC

15°C2°C

CO2

flitter-D

OC in

C

O2

Leaching cycle

13C

o

f C

O2 (‰

)13C

o

f C

O2 (‰

)

and 2 �C (left column). The d13C values of respired CO2 and fractions of litter–DOC inrtical lines separate the four phases with additions of 20 mm of the respective solutionvalent to two days.

0.0

0.2

0.4

0.6

0.8

1.0

0 10 20 30 400.0

0.2

0.4

0.6

0.8

1.0

Leaching cycle

input litter-DOCrespired CO2

leached DOC

15°C

2°C

Flu

xes o

f litter-D

OC

(g

C

m

-22d

-1)

input litter-DOCrespired CO2

leached DOC

Fig. 5. Leaching and mineralization of added litter–DOC from the Oa horizons in gC m�2 per leaching cycle in the soil column study at 15 and 2 �C. Means and standarderrors of four replicates. Vertical lines separate the four phases with additions of20 mm of the respective solution per leaching cycle containing 0, 22, 45, and 0 mg l�1

litter–DOC. One leaching cycle is equivalent to two days.

Fig. 6. Total C mass balance of added litter–DOC and native soil C at 15 and 2 �C in gC m�2. Dark colour represents C derived from added litter–DOC; the lighter colourrepresents native soil C.

In situ 13C tracing

M. Muller et al. / Soil Biology & Biochemistry 41 (2009) 1066–1074 1071

temperature (Figs. 5 and 6). About 18� 2% (2.5� 0.3 g C m�2) and30� 6% (4.3� 0.8 g C m�2) of added litter–DOC – was mineralizedat 2 and 15 �C, respectively (Fig. 6). Leaching was the least impor-tant pathway with less than 10% (1.4 g C m�2) of the totally addedlitter–DOC being leached from the soil columns, even at 2 �C witha very low biological activity (Fig. 6).

0.0

0.2

0.4

0.6

0.8

1.0

fractio

n recen

t C

Suctioncup5 cm

Zero-tension

lysimeter5 cm

RespiredCO2

LeachedDOC

LeachedDOC

Fig. 7. Fractions of recent plant-derived C (rhizosphere and litter) in soil-respired CO2,and leached DOC after six years of CO2 enrichment at the alpine treeline. The fractionof recent C was calculated from the difference in d13C between elevated and ambientCO2. Means and standard errors of 18–20 plots and four and two sampling campaignsfor soil-respired CO2 and leached DOC, respectively.

3.6. 13C tracing in the field

At the alpine treeline the addition of 13C-depleted CO2 for sixyears was clearly reflected in plant tissues with a decline in 13C by6.7� 0.4& (mean of tree needles and dwarf shrub leaves). Thetracing of this isotopic signal of recent plant-derived C in the plantand soil system clearly showed that the fraction of recent C wassignificantly greater in soil-respired CO2 than in leached DOC of theOa horizon at 5 cm soil depth (Fig. 7). The results also indicated thatDOC collected with zero-tension lysimeters representing therapidly leached DOC had a significant greater fraction of recent Cthan DOC collected with suction cups (P< 0.05). However, bothtypes of DOC contained relatively small fractions (20% and 30%) ofless than six-year-old C, implying that DOC was dominated byrelative old C.

4. Discussion

4.1. Sources of DOC in organic layers

Dissolved organic carbon in organic layers is generated bydifferent pathways: it could originate from root exudation, fromdecomposing litter and roots, or from older humified organicmaterial (Kalbitz et al., 2000; Neff and Asner, 2001; Hagedorn et al.,2004). The contribution of these DOC sources is, however, largelyunknown. Our six-year-long 13CO2 tracer study in the field showedthat fractions of recent, less than six-year-old, plant-derived C inDOC in the Oa horizon at 5 cm depth were small. This implies thatthroughfall, fresh litter or root exudates made minor contributionsto DOC leached from the thick Oa horizons (Fig. 7). Apparently, thegreatest fraction of leached DOC derives from older litter or fromthe Oa horizon itself. The soil column experiment supports this

M. Muller et al. / Soil Biology & Biochemistry 41 (2009) 1066–10741072

conclusion. The tracing of 13C-labelled litter–DOC clearly indicatedan effective retention of added litter–DOC in Oa horizons (60–70%;Figs. 5 and 6). Moreover, the repeated leaching of Oa horizonswithout litter–DOC addition (control columns) resulted in stableDOC leaching rates which demonstrates a sustained DOC genera-tion from the large pool of humified SOM. As a consequence, DOCleached from Oa horizons is dominated by older C.

4.2. Uncertainties

Our estimated fraction of litter–DOC in leached DOC could bebiased by a different DOC composition with a different d13C-signalin the Oa leachate than in the added litter–DOC. The natural 13Cabundance of the so-called hydrophobic DOC is 1–4& smaller thanof the hydrophilic one (Kaiser et al., 2001; Hagedorn, unpublisheddata). Using the linear relationship between SUVA at 260 nm andfraction of hydrophobic DOC (Dilling and Kaiser, 2002) indicatesa 40%-higher hydrophobic DOC fraction in Oa leachates than inlitter–DOC. Since the 13C-signal of litter–DOC was calculated fromthe difference in 13C between litter–DOC and Oa leachate of thecontrol columns (being more negative due to more hydrophobicDOC), we might have overestimated the denominator of the mixingequation and hence, underestimated the input signal. This, in turnsignifies that we might have overestimated the fractions of litter–DOC in Oa horizons, however, not by more than 8% as compared tothe values given in Figs. 4–6.

4.3. Effective retention of litter–DOC

In mineral soils, there is extensive evidence for DOC retentionthrough rapid sorption to mineral surfaces (e.g. Sollins et al., 1996;Kalbitz and Kaiser, 2008), but much less is known on the fate oflitter–DOC in organic layers (Qualls, 2000; Froberg et al., 2007). Oursoil column study indicates that the strong loss of litter–DOC withpassage through the Oa horizon can be partially related tobiodegradation, but other mechanism also contribute to the ratherrapid DOC removal (Figs. 5 and 6). The latter are most likelyphysico-chemical interaction processes with solid SOM, whichprolong the residence time of litter C and which are very likelyfollowed by microbially-driven incorporation into the solid phase.

Biodegradation seems the most obvious pathway of DOCremoval with passage through organic layers as evidenced for lowmolecular compounds such as amino acids and simple organicacids (Jones et al., 2004; van Hees et al., 2005). In a previous studyat this alpine treeline site, Hagedorn et al. (2008) found thatmineralization of 14C-labelled oxalate at 4 �C in organic layers wasrather fast with half-life times of a few hours. Our soil columnstudy, however, indicates that direct biodegradation of litter–DOCwas less important: more than 60% of added litter–DOC wasretained in the Oa horizon but only 30% was mineralized duringtwo months at 15 �C. Incorporation into microbial biomass mighthave additionally contributed to the biological DOC retention.Published microbial use efficiencies show that per unit respired CO2

from oxalate, lignin, phenols, and rygrass 0.05, 0.3, 0.5, and 0.7 unitsof soil microbial biomass are formed within the first days anddecline subsequently (Wu et al., 1993; Brant et al., 2006; Bahri et al.,2008). In turn, this suggests that the overall biodegradation(mineralization and incorporation into microbial biomass) wasmaximally 50% of added litter–DOC at the higher incubationtemperature. The direct biological removal of litter DOC was evenless important at 2 �C. Here, only 18% and 10% of added litter–DOCwas mineralized and leached, respectively (Figs. 5 and 6). We,therefore, conclude that retention of litter–DOC went beyond directbiodegradation. Our conclusion is supported by the field study ofFroberg et al. (2007), showing that a low recovery of 13C-labelled

coniferous litter in DOC leached from mor layers could not beexplained by biodegradation alone. The authors and Qualls (2000)hypothesized that most of the ‘litter-derived’ DOM is removed bysorption through electrostatic attraction or binding, hydrogenbonding and/or van der Waals forces (see e.g. Tipping and Woof,1991). Hydrophobic interactions of litter DOC with solid SOM mightalso be important for this initial DOC retention, in particular,because DOC becomes more enriched in aromatic moieties andhigher in molecular weight during biodegradation (Kalbitz et al.,2003; Hagedorn and Machwitz, 2007). An additional mechanismmight be the binding of DOM to polyvalent cations in the Ohhorizon being present in small amounts (

P(Al, Ca, Mg) ¼

150 mmolc kg�1).Although we cannot identify detailed retention mechanisms,

our results indicate that a substantial part of litter–DOC must havebeen stabilized in the Oa horizons.

While 70% of the litter–DOC itself was biodegradable within 28days, only 30% of added litter–DOC was mineralized in the Oahorizon (Figs. 3 and 6). The fourth phase of our column experiment –the addition of DOC-free rainwater – supports the very efficient andstrong stabilization of litter–DOC, because there was no release(or desorption) of retained litter–DOC and the mineralization oflitter–DOC declined rapidly (Fig. 5). Probably, a transformation oflitter–DOC into solid SOM contributes to this longer-term retention.The latter process might be microbially-driven, because a merechemical stabilization with decreasing concentrations is unlikely.Recent studies indicate that rather recalcitrant compounds aregenerated during biosynthesis into microbial biomass (Rillig et al.,2007).

4.4. Effects of DOC type, water flow and temperature

Obviously, the relative importance of biodegradation, physico-chemical retention, and leaching of DOC will depend on the type ofDOC. The litter–DOC, obtained by repeated leaching of spruce litterand discarding the initial extracts had the same SUVA as leachatesfrom Oi horizons in spruce and beech forests (Kalbitz et al., 2003).The 70%-biodegradability of the litter–DOC within 28 days wasslightly more than reported DOC mineralization rates of 20–60%per month observed for soil waters of Oi horizons (Qualls andHaines, 1992; Kalbitz et al., 2003; Kiikkila et al., 2006) and for DOCleached from pine and larch litter of the study site (Hagedorn andMachwitz, 2007). As a consequence, it seems likely that thecontribution of biodegradation to DOC retention in organic layers iseven smaller under field conditions than in our soil column study.

The 13C field data indicate that flow conditions controlling theresidence time have a major impact on the fate of litter-derivedDOC in organic layers. In the field, DOC sampled by zero-tensionplates comprised significantly greater fractions of recent C thansuction cups sampling soil waters at the same depth (Fig. 7). Thesetwo sampling methods collect different fractions of soils waters:while zero-tension lysimeters predominantly capture rapidlyflowing soil water from rainfalls and from macropores, suction cupsalso sample waters from finer pore sizes with longer residencetimes. Therefore, the greater fractions of recent C in DOC from zero-tension lysimeters indicate that some of the incoming DOC canbypass the retention in the Oa horizon by preferential flow, whichagrees with field studies in mineral soils (Hagedorn et al., 2000;Kaiser and Guggenberger, 2005). In the soil column study, therainfall rate was 20 mm within 2 h resulting in a 50� 7% leaching ofthe bromide tracer from the organic layer during the next two days.We consider this rainfall to be in the upper end of natural range,and we expect that less litter–DOC will be leached from organiclayers under average flow rates.

M. Muller et al. / Soil Biology & Biochemistry 41 (2009) 1066–1074 1073

Soil temperature had a different impact on leaching andmineralization of C. At lower temperatures, leaching of DOC wasrelatively more important than at higher temperatures (Figs. 1 and 6).This agrees with in situ measurements of C fluxes at the alpinetreeline but also with studies in other ecosystems, where DOCconcentrations showed a much smaller seasonal variation than soilrespiration (Michalzik et al., 2001; Hagedorn et al., 2008).

As a consequence, the leaching of litter–DOC will be particularlysignificant at snowmelt where large amounts of fresh litter fromprevious fall are available, soil temperature is low, and leachingrates are high. The occurrence of podzolic soils under cold and wetconditions (Driessen and Dudal, 1991) might be related to theincreasing significance of DOC leaching with decreasing tempera-ture and increasing flow rates.

4.5. Adding litter–DOC induced priming

Our results show priming by the litter–DOC. In the fourth phaseat 15 �C, the previous addition of litter–DOC promoted the leachingof native DOC (Figs.1 and 4). This supports an accelerated leaching ofold DOC under elevated CO2 at this field site (Hagedorn et al., 2008)and increased respiration from indigenous C following additions ofneedle litter (Sulzman et al., 2005). We suggest that DOC generationin thick organic layers is particularly prone to priming because (i)DOC production from largely decomposed organic matter is verylikely driven by fungal activity during lignin degradation (Kalbitzet al., 2006); (ii) altered C inputs primarily affects the fungalcommunity (Brant et al., 2006; Carney et al., 2007); and (iii)contributions of fungi to the soil microbial community are greatestin nutrient-poor organic layers (Pennanen et al., 1999).

4.6. Conclusions

Our 13C tracer experiments – in the field added as 13CO2 and inthe laboratory applied as litter–DOC on soil columns – show thatonly a small fraction of DOC leached from organic layers directlyoriginates from recent plant-derived C such as throughfall, freshlitter or root exudates. Less than 10% of added litter–DOC wasleached from the columns with Oa horizons and mineralizationaccounted only to 18–30% of the removal of litter–DOC. Theretention of litter–DOC was very rapid as indicated by smallleaching rates of litter–DOC despite a rapid breakthrough of addedbromide. We, therefore, suggest that physico-chemical processessuch as hydrophobic interactions and bridging to polyvalent cationsbut not biodegradation are primarily responsible for the initialretention of litter–DOC, very likely followed by a probably micro-bially-driven incorporation into solid SOM. The concomitant sus-tained leaching of ‘old’ DOC suggests an apparent ‘exchange’ of DOCin the organic layer with the largest fraction of litter–DOC beingretained and ‘replaced’ by DOC generated and leached from thelarge pool of indigenous SOM. Our results also show priming by theaddition of litter–DOC as it increased the leaching of native C.

Acknowledgements

We thank the following persons from the University of Basel:B. Seth for analysing stable isotopes; F. Conen, H. Strohm, M. Caroni,and H. Hurlimann for lab assistance; and C. Schneider for technicalsupport. We would also like to thank the following persons at theSwiss Federal Institute for Forest, Snow and Landscape Research(WSL): A. Zurcher for lab assistance, technical support, and con-ducting the biodegradation study; N. Hajjar for TOC analysis.

References

Bahri, H., Rasse, D.P., Rumpel, C., Dignac, M.-F., Bardoux, G., Mariotti, A., 2008. Lignindegradation during laboratory incubation followed by 13C isotope analysis. SoilBiology and Biochemistry 40, 1916–1922.

Bednorz, F., Reichstein, M., Broll, G., Holtmeier, F.-K., Urfer, W., 2000. Humus formsin the forest-alpine tundra ecotone at Stillberg (Dischmatal, Switzerland):spatial heterogeneity and classification. Arctic, Antarctic and Alpine Research32, 21–29.

Brant, J.B., Sulzman, E.W., Myrold, D.D., 2006. Microbial community utilization ofadded carbon substrates in response to long-term carbon input manipulation.Soil Biology and Biochemistry 38, 2219–2232.

Carney, K.M., Hungate, B.A., Drake, B.G., Megonigal, J.P., 2007. Altered soil microbialcommunity at elevated CO2 leads to loss of soil carbon. Proceedings of theNational Academy of Sciences of the United States of America 104, 4990–4995.

Chin, Y.-P., Alken, G., O’Loughlin, E., 1994. Molecular weight, polydispersity, andspectroscopic properties of aquatic humic substances. Environmental Scienceand Technology 28, 1853–1858.

Currie, W.S., Aber, J.D., 1997. Modeling leaching as a decomposition process inhumid montane forests. Ecology 78, 1844–1860.

Dilling, J., Kaiser, K., 2002. Estimation of the hydrophobic fraction of dissolvedorganic matter in water samples using UV photometry. Water Research 36,5037–5044.

Driessen, P.M., Dudal, R., 1991. The Major Soils of the World. Koninklijke WohrmannB.V. Zutphen, The Netherlands.

Froberg, M., Kleja, D.B., Bergkvist, B., Tipping, E., Mulder, J., 2005. Dissolved organiccarbon leaching from a coniferous forest floor – a field manipulation experi-ment. Biogeochemistry 75, 271–287.

Froberg, M., Kleja, D.B., Hagedorn, F., 2007. The contribution of fresh litter to dis-solved organic carbon leached from a coniferous forest floor. European Journalof Soil Science 58, 108–114.

Giesler, R., Hogberg, M.N., Strobel, B.W., Richter, A., Nordgren, A., Hogberg, P., 2007.Production of dissolved organic carbon and low-molecular weight organic acidsin soil solution driven by recent tree photosynthate. Biogeochemistry 84, 1–12.

Hagedorn, F., Schleppi, P., Waldner, P., Fluhler, H., 2000. Export of dissolved organiccarbon and nitrogen from Gleysol dominated catchments: the significance ofwater flow paths. Biogeochemistry 50, 137–161.

Hagedorn, F., Maurer, S., Blaser, P., 2004. A 13C tracer study to identify the origin ofdissolved organic carbon in forested mineral soils. European Journal of SoilScience 55, 91–100.

Hagedorn, F., Maurer, S., Bucher, J.B., Siegwolf, R.T.W., 2005. Immobilization, stabi-lization and remobilization of nitrogen in forest soils at elevated CO2: a 15N and13C tracer study. Global Change Biology 11, 1816–1827.

Hagedorn, F., Machwitz, M., 2007. Controls on dissolved organic matter leachingfrom forest litter grown under elevated atmospheric CO2. Soil Biology andBiochemistry 39, 1759–1769.

Hagedorn, F., van Hees, P.A.W., Handa, I.T., Hattenschwiler, S., 2008. Elevatedatmospheric CO2 fuels leaching of old dissolved organic matter at the alpinetree line. Global Biogeochemical Cycles 22, GB2004, doi:10.1029/2007GB003026.

Hattenschwiler, S., Handa, I.T., Egli, L., Asshoff, R., Ammann, W., Korner, C., 2002.Atmospheric CO2 enrichment of alpine treeline conifers. New Phytologist 156,363–375.

Jones, D.L., Farrar, J.F., Newsham, K.K., 2004. Rapid amino acid cycling in arctic andantarctic soils. Water, Air, and Soil Pollution: Focus 4, 169–175.

Kaiser, K., Guggenberger, G., 2000. The role of DOM sorption to mineral surfacesin the preservation of organic matter in soils. Organic Geochemistry 31,711–725.

Kaiser, K., Guggenberger, G., Zech, W., 2001. Isotopic fractionation of dissolvedorganic carbon in shallow forest soils as affected by sorption. European Journalof Soil Science 52, 585–597.

Kaiser, K., Guggenberger, G., 2005. Storm flow flushing in a structured soil changesthe composition of dissolved organic matter leached into the subsoil. Geoderma127, 177–187.

Kalbitz, K., Solinger, S., Park, J.-H., Michalzik, B., Matzner, E., 2000. Controls on thedynamics of dissolved organic matter in soils: a review. Soil Science 165, 277–304.

Kalbitz, K., Schwesig, D., Schmerwitz, J., Kaiser, K., Haumaier, L., Glaser, B.,Ellerbrock, R., Leinweber, P., 2003. Changes in properties of soil-derived dis-solved organic matter induced by biodegradation. Soil Biology and Biochem-istry 35, 1129–1142.

Kalbitz, K., Kaiser, K., Bargholz, J., Dardenne, P., 2006. Lignin degradation controlsthe production of dissolved organic matter in decomposing foliar litter. Euro-pean Journal Soil Science 57, 504–516.

Kalbitz, K., Meyer, A., Yang, R., Gerstberger, P., 2007. Response of dissolved organicmatter in the forest floor to long-term manipulation of litter and throughfallinputs. Biogeochemistry 86, 301–318.

Kalbitz, K., Kaiser, K., 2008. Contribution of dissolved organic matter to carbon storagein forest mineral soils. Journal of Plant Nutrition and Soil Science 171, 52–60.

Kiikkila, O., Kitunen, V., Smolander, A., 2006. Dissolved soil organic matter fromsurface organic horizons under birch and conifers: degradation in relation tochemical characteristics. Soil Biology and Biochemistry 38, 737–746.

Lajtha, K., Crow, S.E., Yano, Y., Kaushal, S.S., Sulzman, E., Sollins, P., Spears, J.D.H.,2005. Detrital controls on soil solution N and dissolved organic matter in soils:a field experiment. Biogeochemistry 76, 261–281.

M. Muller et al. / Soil Biology & Biochemistry 41 (2009) 1066–10741074

McDowell, W.H., Likens, G.E., 1988. Origin, composition, and flux of dissolvedorganic carbon in the Hubbard Brook Valley. Ecological Monographs 58,177–195.

McDowell, W.H., 2003. Dissolved organic matter in soils – future directions andunanswered questions. Geoderma 113, 179–186.

Michalzik, B., Kalbitz, K., Park, J.-H., Solinger, S., Matzner, E., 2001. Fluxes andconcentrations of dissolved organic carbon and nitrogen – a synthesis fortemperate forests. Biogeochemistry 52, 173–205.

Michalzik, B., Tipping, E., Mulder, J., Lancho, G.J.F., Matzner, E., Bryant, C., Clarke, N.,Lofts, S., Esteban, V.M.A., 2003. Modelling the production and transport ofdissolved organic carbon in forest soils. Biogeochemistry 66, 241–264.

Moore, T.R., Dalva, M., 2001. Some controls on the release of dissolved organiccarbon by plant tissues and soils. Soil Science 166, 38–47.

Neff, J.C., Asner, G.P., 2001. Dissolved organic carbon in terrestrial ecosystems:synthesis and a model. Ecosystems 4, 29–48.

Park, J.H., Kalbitz, K., Matzner, E., 2002. Resource control on the production ofdissolved organic carbon and nitrogen in a deciduous forest floor. Soil Biologyand Biochemistry 34, 813–822.

Pennanen, T., Liski, J., Bååth, E., Kitunen, V., Uotila, J., Westman, C.J., Fritze, H.,1999. Structure of the microbial communities in coniferous forest soils inrelation to site fertility and stand development stage. Microbial Ecology 38,168–179.

Qualls, R.G., Haines, B.L., 1992. Biodegradability of dissolved organic matter in forestthroughfall, soil solution, and stream water. Soil Science Society of America 56,578–586.

Qualls, R.G., 2000. Comparison of the behaviour of soluble organic and inorganicnutrients in forest soils. Forest Ecology and Management 138, 29–50.

Rillig, M.C., Caldwell, B.A., Wosten, H.A.B., Sollins, P., 2007. Role of proteins in soilcarbon and nitrogen storage: controls on persistence. Biogeochemistry 85,25–44.

Sulzman, E.W., Brant, J.B., Bowden, R.D., Lajtha, K., 2005. Contribution of above-ground litter, belowground litter, and rhizosphere respiration to total soil CO2efflux in an old growth coniferous forest. Biogeochemistry 73, 231–256.

Sollins, P., Homann, P., Caldwell, B.A., 1996. Stabilization and destabilization of soilorganic matter: mechanisms and controls. Geoderma 74, 65–105.

Subke, J.A., Hahn, V., Battipaglia, G., Linder, S., Buchmann, N., Cotrufo, M.F., 2004.Feedback interactions between needle litter decomposition and rhizosphereactivity. Oecologia 139, 551–559.

Tipping, E., Woof, C., 1991. The distribution of humic substances between the solidand the aqueous phases of acid organic soils – a description based on humicheterogeneity and charged-dependent sorption equilibria. Journal of SoilScience 42, 437–448.

Tipping, E., 2002. Cation Binding by Humic Substances. In: Cambridge Environ-mental Chemistry Series, vol. 12. Cambridge University Press.

van Hees, P.A.W., Jones, D.L., Finlay, R., Godbold, D.L., Lundstrom, U.S., 2005. Thecarbon we do not see: the impact of low molecular weight compounds oncarbon dynamics and respiration in forest soils – a review. Soil Biology andBiochemistry 37, 1–13.

Wu, J., Brookes, P.C., Jenkinson, D.S., 1993. Formation and destruction of microbialbiomass during the decomposition of glucose and ryegrass in soil. Soil Biologyand Biochemistry 25, 1435–1441.

Yano, W., McDowell, W.H., Aber, J.D., 2000. Biodegradable dissolved organic carbonin forest soil solution and effects of chronic nitrogen deposition. Soil Biologyand Biochemistry 32, 1743–1751.