biodegradation of soil-derived dissolved organic matter as related to its properties

Biodegradation of soil-derived dissolved organic

matter as related to its properties

K. Kalbitz*, J. Schmerwitz, D. Schwesig, E. Matzner

Department of Soil Ecology, Bayreuth Institute for Terrestrial Ecosystem Research (BITOK),

University of Bayreuth, D-95440 Bayreuth, Germany

Received 18 December 2001; accepted 9 December 2002

Abstract

Quantifying the contribution of dissolved organic matter (DOM) to C sequestration in soils

requires knowledge about extent and rate of its biodegradation. Since degradation experiments are

time consuming, estimating the biodegradability of DOM by more easily measurable properties

seems valuable. Our goal was therefore to investigate the biodegradation of DOM of different

origin and to relate its extent and rate to properties such as UV absorbance, synchronous and

emission scan fluorescence, XAD-8 sorption chromatography and 1H-NMR spectroscopy. We

extracted DOM from 13 different samples (maize straw, forest floors, peats, agricultural soils) and

carried out a 90-day liquid incubation experiment. DOM biodegradation was quantified by CO2

evolution. Rapidly and slowly mineralizable portions of dissolved organic carbon (DOC) as a

measure of labile and stable DOC and the respective mineralization rate constants and half-lives

were calculated by a double exponential model. The extent and rate of DOM biodegradation from

less humified organic material (straw, litter and fermentation layers of forest floors) were high

resulting in 61–93% of DOC being mineralized. The labile fraction comprised 59–88% of total

DOC. DOM extracted from agricultural soils was of an intermediate biodegradability with a CO2

evolution comprising 17–32% of total DOC. Labile DOC represented 14–25% of total DOC.

DOM extracted from peats and Oa forest floor layers was relatively stable (mineralization of 4–9%

of total DOC, labile DOC: 3–6%). The half-life of the labile DOC pool was short (2–5 days),

whereas that of the stable DOC pool ranged from 0.2 years (DOM from less humified material) to

8.6 years (DOM from the Oa layer under spruce). Extent and rate of DOM biodegradation were

closely but nonlinearly related to DOM properties. Solutions exceeding a threshold value of UV

absorbance, aromaticity, XAD-8 adsorbable C or of humification indices derived from fluorescence

spectra or having carbohydrate contents below a certain level were stable against biodegradation.

0016-7061/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

doi:10.1016/S0016-7061(02)00365-8

* Corresponding author. Tel.: +49-921-55-5624; fax: +49-921-55-5799.

E-mail address: [email protected] (K. Kalbitz).

www.elsevier.com/locate/geoderma

Geoderma 113 (2003) 273–291

Relatively simple methods like UV spectroscopy, XAD-8 fractionation and fluorescence emission

spectroscopy were suitable to estimate the biodegradation of DOM.

D 2002 Elsevier Science B.V. All rights reserved.

Keywords: C sequestration; Dissolved organic matter; Fluorescence spectroscopy; 1H-NMR spectroscopy; UV/

VIS spectroscopy; XAD-8 fractionation

1. Introduction

The importance of dissolved organic matter (DOM) for nutrient cycles in terrestrial

ecosystems is well recognized (Qualls et al., 1991; Smith et al., 1998). However, the

contribution of DOM to C sequestration in the mineral subsoil is largely unknown.

Besides C inputs from plant roots, the flux of dissolved organic carbon (DOC) from the

forest floor to the mineral subsoil has been estimated to be about 115–500 kg C ha� 1

year� 1 in forest ecosystems (Guggenberger and Zech, 1993; Currie et al., 1996;

Michalzik and Matzner, 1999; Solinger et al., 2001; Kaiser et al., 2001). However,

DOC concentrations in deep soil horizons are typically low and the C output from the

mineral subsoil in leachate is comparably small (5–66 kg C ha� 1 year� 1) (Guggenberger

and Zech, 1993; Michalzik and Matzner, 1999; Solinger et al., 2001). Therefore, a

considerable portion of DOC is retained in the mineral subsoil and could be sequestered.

Currie et al. (1996) estimated a DOC retention in forest mineral soils of about 217–366

kg C ha� 1 year� 1.

According to recent knowledge, sorption of DOC on mineral surfaces rather than

DOC mineralization to CO2 is responsible for maintaining low DOC concentrations in

the mineral subsoil (summarized by Kalbitz et al., 2000; Kaiser and Guggenberger,

2000). Guggenberger and Kaiser (2003) related the retained amount of DOC in the

mineral subsoil (input minus output) to the DOC sorption capacity of the soil

measured in the laboratory. On the basis of measured DOC fluxes, they estimated

the DOC sorption capacity in the field to be exhausted within a few years. However,

field data did not indicate a net increase in DOC concentrations in the mineral subsoil

indicating no decrease in DOC retention. Therefore, this model calculation has two

interpretations: (i) DOC mineralization occurs to a larger extent than previously

assumed and/or (ii) a continuous replenishment of sorption sites takes place probably

by degradation of sorbed organic matter. Degradation of dissolved and sorbed organic

matter diminishes the potential of DOM to become stabilized in soil. Hence, DOM

degradation needs to be quantified in order to estimate the impact of DOM on C

sequestration.

Numerous incubation studies in the laboratory have shown that 10–44% of DOM in

soil solutions were microbially degradable (Jandl and Sletten, 1999; Kalbitz et al., 2000;

Yano et al., 2000; Sachse et al., 2001). DOM released from fresh leaf litter was degradable

up to 75% (Hongve et al., 2000). The biodegradable fraction of DOM varies with soil

depth, land use and soil contamination (Boyer and Groffman, 1996; Lundquist et al., 1999;

Merckx et al., 2001). It decreases with increasing soil depth and is lower in forest than in

agricultural soils (Boyer and Groffman, 1996).

K. Kalbitz et al. / Geoderma 113 (2003) 273–291274

Qualls and Haines (1992) recognized rapidly and slowly degradable DOM in a long-

term incubation experiment. Besides information on the pool size of rapidly and slowly

degradable DOM, information is needed about the degradation rates of these two fractions

to estimate the contribution of DOM to C storage in soils.

It seems reasonable to assume that the observed differences in biodegradation of DOM

are caused by differences in its composition. Hydrophobic acids are less accessible to

microbial degradation than hydrophilic compounds (Qualls and Haines, 1992; Jandl and

Sollins, 1997; Jandl and Sletten, 1999). Correlations have been found between the

degradable portion of DOM and (i) the elemental composition (Sun et al., 1997) as well

as (ii) the specific absorbance in UV light of DOM (Gilbert, 1988; Zoungrana et al., 1998;

Pinney et al., 2000). However, there are no systematic studies relating properties of DOM to

its biodegradability.

Therefore, the objectives of this study were:� to quantify the extent of biodegradation of DOM of different origin and to

determine degradation rate constants for the rapidly and slowly degradable DOM

pools and� to identify DOM properties suitable to predict extent and rate of DOM biodegra-

dation.

2. Material and methods

2.1. Samples

In May 2000, we sampled forest floors, peat layers and A horizons in two forests, a fen

area and three arable fields, respectively (Table 1). At a Norway spruce [Picea abies (L.)

Karst.] site in the Fichtelgebirge (Germany, Michalzik and Matzner, 1999), we sampled

litter (Oi-spruce), fermentation (Oe-spruce) and humified layers (Oa-spruce). We also

sampled litter (Oi-beech) and the Oe/Oa layer (Oa-beech) of a deciduous stand in

Northern Bavaria (Germany) with European beech (Fagus sylvatica L.) as the

dominant tree species (Solinger et al., 2001). In northeastern Germany, we collected

samples of the uppermost 20 cm at plots of a fen area (‘‘Droemling’’; Table 1). The

selected plots (fen-1 to fen-4) are described in detail by Kalbitz et al. (1999).

The A horizons of three different plots of the long-term agricultural field experiment

Bad Lauchstadt (Sachsen-Anhalt, Germany) were sampled in order to include samples

with varying DOM properties (Korschens et al., 1994; Weigel et al., 1998). We chose a

plot not fertilized since 1902 (BL-0), a plot with mineral fertilization (BL-NPK) and a plot

with manure addition every other year since 1902 (30 t ha� 1; BL-manure). In addition, we

sampled maize straw immediately after harvesting from a field near Bayreuth in October

2000.

The sampled materials were sieved (5 mm), visible roots and animals were removed

and finally well mixed. The maize straw and the beech leaves were cut to pieces of a size

of about 1–2 cm2 to facilitate a successful mixing. Finally, the material was packed into

polyethylene bags (portions of 300 g) and stored at � 20 jC. Subsamples were air dried

and stored as sources of inoculum.

K. Kalbitz et al. / Geoderma 113 (2003) 273–291 275

2.2. Preparation of DOM solutions

Our sample treatment was intended to ensure a maximum homogeneity among replicate

extracts from a single soil with respect to amount and composition of the extracted DOM.

We also intended to obtain DOM as similar as possible to in situ DOM.

Before extraction, the frozen samples were thawed overnight. Ultrapure water was

added to the soil (soil/water ratio: Table 1), and the suspensions were stirred manually

three times within 24 h. In this time, an equilibrium between the solid and the liquid phase

should be established (Zsolnay, 1996; Qualls, 2000). The solutions were then passed

through a ceramic plate (pore size 1 Am) and filtered through 0.2-Am membrane filters

(cellulose acetate; OE 676, Schleicher & Schuell) to remove microorganisms. Prior to the

first use, each ceramic plate had been cleaned (NaOH, HCl, ultrapure water) and

conditioned with the corresponding DOM solution up to a constant UV absorbance at

254 nm. Because of low DOC concentrations, the arable soil samples (fen-1, BL-0, BL-

NPK, BL-manure) were extracted at narrower soil-to-solution ratios (Table 1), an end-

over-end shaking of the soil suspension (2 h) followed by centrifugation (2100� g).

Table 1

Total organic carbon (TOC) and total organic nitrogen (TON) content of the solid material used for extraction of

DOM solutions, the soil/solution ratio for extraction and DOC content of the DOM solutions

Sample/site TOC

(mg g� 1)

TON

(mg g� 1)

DOCa

(mg l� 1)

DOCb

(mg g�1)

Soil/solution

ratio (w/w)c

Beech forest (Dystric Cambisol)

Litter layer (Oi-beech) 456 15.7 604 7.385 0.1

Fermentation/humified layer (Oa-beech) 295 16.2 67.4 1.702 0.1

Spruce forest (Haplic Podzol)

Litter layer (Oi-spruce) 474 17.4 99.3 1.642 0.1

Fermented layer (Oe-spruce) 435 21.2 149 2.496 0.1

Humified layer (Oa-spruce) 312 13.9 38.1 1.175 0.1

Fen area (Gleyic Mollisols and Terric Histosols)

Arable soil (fen-1) 43.3 3.0 19.8 0.015 1.5

Grassland soil (fen-2) 48.3 3.6 18.4 0.215 0.2

Grassland in succession (fen-3) 96.1 7.2 40.5 0.642 0.1

Almost natural forest (fen-4) 383 26.8 75.7 2.239 0.1

Field sites of a long-term agricultural trial (Haplic Chernozem)

Without any fertilization (BL-0) 12.8 1.3 7.2 0.006 1.5

Mineral fertilization (BL-NPK) 12.5 1.1 12.6 0.012 1.5

Organic fertilization (BL-manure) 14.7 1.4 13.9 0.012 1.5

Maize straw

Maize straw 420 8.6 819 33.0 0.1

a Measured DOC concentration in the extract.b Calculated DOC content of the solid phase using DOC concentration in the extract, amount of solid material

(dry weight) and water volume.c Applied ratio to extract DOM.


Otherwise, the procedure was identical to that of the other samples. Extraction and

filtration was carried out at 5 jC.

2.3. Inoculation

We used the samples of Oa-spruce, Oi-beech, fen-4 and BL-manure to obtain a mixed

inoculum which contained microorganisms from all sites and ensured a broad diversity.

Before extraction, air-dried subsamples were rewetted to field capacity and incubated for 2

weeks at 20 jC to reactivate the microorganisms. The soils were shaken separately for 30

min with a 4 mM CaCl2 solution and filtered through 5-Am filters (Millipore SMWP

4700). In the solutions, the total cell number was counted (Schramm et al., 2000) and the

four solutions were combined to give an inoculum with a similar number of cells from

each solution. We added 7 ml of the inoculum to each incubation flask which was equal to

about 2.5� 105 cells ml� 1 inoculum.

2.4. Incubation experiments

Before incubation, DOM solutions with more than 20 mg C l� 1 were diluted to avoid

excessive growth of microorganisms (Hongve et al., 2000). We added in triplicate 700 ml

of each of the DOM solutions into 1-l incubation flasks. No nutrients were added. After

inoculation, we added five glass-fibre filters to provide surface area for the establishment of

biofilms (Qualls and Haines, 1992). The flasks were sealed, incubated in the dark at 20 jCfor 90 days, and gently shaken by hand every day. Biodegradation of DOC was quantified

using CO2 measurements to avoid an overestimation of biodegradation due to formation of

colloidal, particulate and precipitated C that might be excluded from DOC measurements.

Indeed, we observed clouding of solutions during the experiment. The incubation flasks

were opened after 6, 14, 29 and 54 days to measure pH and to aerate the DOM solutions for

10 min using filtered compressed air. To test the activity of the microorganisms, we used a

glucose solution (20 mg C l� 1, with additions of N, P, K as NH4NO3, K2HPO4) as a

control. A second control with ultrapure water and inoculum was used to measure the C

evolution from the added inoculum.

2.5. Analytical methods

We measured the CO2 concentration in the headspace of each incubation flask at 3-day

intervals at the beginning of the experiment and 14 days at the end (gas chromatograph HP

6890, Hewlett Packard, thermal conductivity detector). We applied a slight pressure of

about 200 hPa in each flask to ensure a proper sampling of the headspace and measured

the pressure before each CO2 analysis. The CO2 concentration in the gas phase was

calculated using the general gas equation, and by using solubility constants and the

measured pH, we calculated the CO2 in the liquid phase.

Each DOM solution was analyzed before incubation for:� DOC concentration (High TOC, Elementar)� UV absorbance at 280 nm (UVIKON 930, Bio-Tek Instruments) as a parameter to

estimate the aromaticity of DOM (Chin et al., 1994; McKnight et al., 1997)


� Synchronous and emission scan fluorescence spectra followed by calculations of two

humification indices (SFM 25, Bio-Tek Instruments; Kalbitz and Geyer, 2001; Zsolnay

et al., 1999) as an expression of the complexity and condensation of the molecules

(HIXem: humification index deduced from emission spectra; HIXsyn: humification

index deduced from synchronous spectra)� Liquid state 1H-NMR spectra (Avance DRX 500 spectrometer, Bruker Analytik) to

measure the portions of H associated with O-containing functional groups and with

aromatic compounds (Kaiser et al., 2002)� XAD-8 adsorbable carbon as a measure of hydrophobic DOM portions (Raastad and

Mulder, 1999; Kaiser et al., 2001).

For the UV and fluorescence measurements, the DOC concentration was adjusted to 10

mg C l� 1, the pH to 7.7 and the ionic strength to 1000 AS cm� 1 to ensure a comparability

of all DOM solutions. For NMR spectroscopy (see details in Kaiser et al., 2002), the DOM

solutions from the fen and field sites were treated with a strong cation exchanger (AG MP

50, BioRad) to reduce the salt content before the samples were freeze dried. The most

prominent signals were those of H associated with O-containing functional groups (3.0–

4.8 ppm). A large portion of the signals in this region should be due to carbohydrate H

(Kaiser et al., 2002), and we used the assumption that this part of the spectrum is

representative for carbohydrates. Less abundant were signals at 5.5–10.0 ppm which are

caused by aromatic H. The C and N content of the solid material was measured using a

CHN-O-RAPID (Foss Heraeus).

2.6. Calculations and terminology

Based on the assumption that DOM is a mixture of components with different

degradation rates and different extents of degradability, we fitted a double exponential

model with two distinct pools with different rates to the measured mineralization:

mineralized DOCð% of total DOCÞ ¼ ð100� aÞð1� e�k1tÞ þ að1� e�k2tÞ

where: t: time [days]; 100� a: the part of DOC that is rapidly mineralizable = labile DOC

[%]; a: the part of DOC that is slowly mineralizable = stable DOC [%]; k1: mineralization

rate constant of labile DOC [day� 1]; k2: mineralization rate constant of stable DOC

[day� 1]

We also calculated:

half � life of the labile DOC ¼ ln2=k1

half � life of the stable DOC ¼ ln2=k2

mean residence time of the labile DOC ¼ 1=k1

mean residence time of the stable DOC ¼ 1=k2


The curves were fitted using a least-square optimization method (Quasi-Newton).

We calculated linear and logarithmic regressions to relate the C mineralization to

DOM properties. Cluster analysis (k-means clustering) was run for classifying DOM

solutions. Input data comprised the measured and calculated DOM properties (11

variables) listed in Table 2 (without half-life and r2) and Table 3. Principal components

analysis and hierarchical cluster analysis were applied to highlight relations between

DOM properties and its biodegradation.

In a strict sense, DOM biodegradation encompasses two alternative or sequential

processes: (1) the breakdown and transformation of DOM to products that can be used as

precursors for the biosynthesis of microbial cell materials and (2) a complete mineraliza-

tion to obtain energy and inorganic nutrients (Marschner and Kalbitz, 2003). In our study

we quantified process (2). The extent of biodegradation is expressed as the percentage of

DOC mineralized after 90-day incubation. The rate of biodegradation is quantified by the

Table 2

Quantitative measures of the biodegradation of DOM after 90 days incubation: percentage of mineralized DOC,

sizes of the labile and stable DOC pools, mineralization rate constants and half-life for the labile (k1) and the

stable (k2) DOC pools (samples represent the means of three replicates)

DOM

solution

Mineralized

DOC

Labile

DOCa

Stable

DOCb

k1c

(day� 1)

k2d

(day� 1)

Half-life 1e

(day)

Half-life 2f

(year)

r2

(% of total DOC)

First group with a high biodegradation

Oi-beech 65.0 58.9 41.1 0.2661 0.00166 2.6 1.1 0.98

Oi-spruce 61.4 56.9 43.1 0.2585 0.00122 2.7 1.6 0.97

Oe-spruce 93.4 87.5 12.5 0.2635 0.00556 2.6 0.3 0.97

Maize straw 88.6 68.5 31.5 0.1564 0.00849 4.4 0.2 0.97

Second group with an intermediate biodegradation

BL-0 30.0 25.1 74.9 0.2970 0.00081 2.3 2.3 0.99

BL-NPK 17.2 13.5 86.5 0.2236 0.00053 3.1 3.6 0.95

BL-manure 31.8 21.5 78.5 0.2737 0.00172 2.5 1.1 0.99

Third group with a low biodegradation

Oa-spruce 7.2 5.6 94.4 0.1382 0.00022 5.0 8.6 0.98

Oa-beech 9.1 5.3 94.7 0.1126 0.00047 6.2 4.0 0.98

fen-1 7.8 5.0 95.0 0.1247 0.00033 5.6 5.8 0.98

fen-2 9.4 5.7 94.3 0.1635 0.00050 4.2 3.8 0.96

fen-3 8.5 4.7 95.3 0.3074 0.00055 2.3 3.5 0.95

fen-4 5.3 2.7 97.3 0.1486 0.00028 4.7 6.8 0.92

r2: r-square (coefficient of determination) of the double exponential model.a Rapidly mineralizable DOC; calculated using a double exponential model.b Slowly mineralizable DOC; calculated using a double exponential model.c Mineralization rate constant of the labile DOC pool (double exponential model).d Mineralization rate constant of the stable DOC pool (double exponential model).e Half-life of the labile DOC pool.f Half-life of the stable DOC pool.


mineralization constants of the rapidly mineralizable (k1) and slowly mineralizable (k2)

DOC.

3. Results

3.1. DOM biodegradation—extent and dynamic

After 90 days at 20jC, 5–93% of the DOC was mineralized (Fig. 1, Table 2). In all

samples, we found a high mineralization rate during the first days and later on a

mineralization rate that was 2–3 orders of magnitude lower. The CO2 evolution from

any of the tested samples exhibited no temporal course typical for a lag phase of DOM

degradation. Consequently, the density and activity of microorganisms were sufficient in

all incubations. This was confirmed by the fast mineralization of glucose (almost complete

mineralization within 3 weeks). The DOC mineralization could be adequately described

using the double exponential model (Table 2). Due to large differences, the 13 samples

were classified by cluster analysis into three distinct groups using mineralization

Table 3

Properties of the DOM solutions before incubation

DOM

solution

A 280a

(l mg C� 1 cm� 1)

HIXsynb

(– )

HIXemc

(– )

XAD-8d

(%)

Aromatic

He (%)

Carbohydrate

Hf (%)

First group with a high biodegradation

Oi-beech 0.0162 0.58 4.7 24 6 37

Oi-spruce 0.0156 0.35 2.4 29 6 40

Oe-spruce 0.0065 0.66 1.4 21 6 44

Maize straw 0.0064 0.31 0.7 22 5 34

Second group with an intermediate biodegradation

BL-0 0.0235 0.43 12.5 45 9 21

BL-NPK 0.0291 0.47 19.1 55 n.d. 21

BL-manure 0.0370 0.64 20.9 46 12 24

Third group with a low biodegradation

Oa-beech 0.0309 0.75 14.0 77 11 24

Oa-spruce 0.0413 2.39 16.3 73 13 31

fen-1 0.0280 0.63 11.4 48 9 29

fen-2 0.0416 1.50 25.5 65 10 14

fen-3 0.0417 1.24 23.7 67 12 17

fen-4 0.0436 1.32 27.4 75 13 18

n.d.: Spectrum was not interpretable in this region.a Specific absorbance at 280 nm.b Humification index using synchronous fluorescence spectra (band ratio of intensities: 460/345 nm) (Kalbitz

and Geyer, 2001).c Humification index using emission fluorescence spectra (ratio of areas: 435–480 nm/300–345 nm)

(Zsolnay et al., 1999).d % of DOC sorbed onto XAD-8 resin.e 1H-NMR, aromatic H (5.5–10.0 ppm; % of H).f 1H-NMR, H associated with O-containing functionalities (3.0–4.8 ppm; % of H).


Fig. 1. Dynamics of DOC mineralization of different DOM extracts (group 1; high biodegradation, group 2;

intermediate biodegradation, group 3; low biodegradation; samples, see Table 1; means and standard deviations of

three replicates).


parameters (Table 2) and chemical properties (Table 3). This numerical classification

coincides with a classification using the different extent of mineralization.

The first group in which the extent and rates of mineralization were the highest

comprised DOM extracted from maize straw, from the Oi layers of the spruce and beech

forests and from the Oe layer of the spruce forest. In these samples, 61–93% of total DOC

was mineralized within 90 days (Table 2, Fig. 1). The portion of rapidly mineralizable

(labile) DOC in these solutions ranged from 57% to 88%. The k1 values of labile DOC

were high (Table 2) with a half-life of about 3 days. In contrast, the k2 values of the

portion of slowly mineralizable (stable) DOC were 2 orders of magnitude lower (half-life:

0.2–1.6 years).

The second group with an intermediate extent of mineralization and lower k values

consisted of DOM extracted from the three plots of the long-term agricultural experiment

(BL-0, BL-NPK, BL-manure). Within this group, 17–32% of the total DOC was

mineralized after 90 days (Fig. 1, Table 2). The portion of labile DOC ranged from

14% to 25% of total DOC. The k1 values were similar to those in the first group. However,

the k2 values of the stable DOC (mean half-life, 2.3 years) were almost 1 order of

magnitude lower than those of DOM solutions with high biodegradation. Only DOM

extracted from the manured plot showed a relatively high k2 value, which was similar to

that of the spruce-Oi sample (half-life: 1.1 years).

The third group of DOM solutions with a low extent and rate of mineralization

comprised extracts from Oa layers of forest floors and from fen soils with an extent of

mineralization ranging between 5% and 9%. The labile portion was small (3–6%) with a

relatively short half-life of f 5.1 days. One exception was the DOM extracted from fen-3

(Table 2). The k2 values were only slightly lower (mean half-life of these samples: 5.4

years) than those of group 2 with an intermediate extent of mineralization. DOM from the

Oa layer of the spruce forest was the most stable one with a half-life of 8.6 years for the

stable DOC pool.

3.2. DOM biodegradation in relation to DOM properties

We obtained significant logarithmic correlations between DOM properties and the

percentage of DOC mineralized (Fig. 2). The three DOM solutions in the first group with

high mineralization differed strongly from the other samples having lower specific UV

absorbances, smaller portions of XAD-8 adsorbable carbon, lower humification indices

(emission mode; HIXem), and less aromatic H. Furthermore, these highly biodegradable

DOM samples had high contents of carbohydrates. Comparing the mean values for the

three specified groups (circles in Fig. 2), those of group 2 (intermediate biodegradation)

had a lower specific UVabsorbance, a lower portion of XAD-8 adsorbable carbon, a lower

HIXem, and a less aromatic H content than those of group 3 (low biodegradation). The

mean values of the percentage of mineralized DOC correlated linearly with the UV

absorbance and the content of aromatic H.

DOM properties were not correlated to portions of labile and stable DOC because these

portions were highly intercorrelated with the percentage of DOC mineralized (r = 0.999).

The k1 values did not correlate with any of the DOM properties, whereas the k2 values

correlated logarithmically with the specific UV absorbance (r2 = 0.77), the HIXem


(r2 = 0.73), the proportion of XAD-8 adsorbable C (r2 = 0.57) and the proportion of

aromatic H (r2 = 0.50).

The following DOM properties were related to the microbial stability of DOM: (i) UV

absorbance, (ii) aromaticity, (iii) XAD-8 adsorbable carbon as an expression of hydro-

phobicity, (iv) degree of complexity and condensation of the molecules (expressed by the

HIXem), (v) content of carbohydrates. Humification indices deduced from synchronous

fluorescence spectra (HIXsyn ) were not useful to predict the biodegradation of DOM.

The UV absorbance correlated very well with the content of aromatic compounds

(r2 = 0.91) (Fig. 3). Furthermore, relationships existed between the content of aromatic H

bonds and the XAD-8 adsorbable C as well as the HIXem (Fig. 3). These properties were

negatively related to the content of carbohydrates.

Fig. 2. Logarithmic regressions between DOM properties and DOC mineralization after 90 days (n and solid line,

r2). Indicated are the linear regressions using average values for each of the three groups (o and dotted line;

groups and samples, see Fig. 1).


Fig. 3. Linear regressions between different DOM properties (x-axis: parameters derived from 1H-NMR spectra;

y-axis: UVabsorbance at 280 nm, XAD-8 adsorbable C; humification indices derived from emission (HIXem) and

synchronous (HIXsyn) fluorescence spectra).


4. Discussion

4.1. DOM extraction

DOC concentrations obtained by extraction of previously frozen solid material (Table

1) were in the same range as those found in soil solution obtained in situ by lysimeters

(except for the Oi-beech and the Oe-spruce). This is somewhat contradictory to laboratory

studies showing an additional DOM release after freeze/thaw cycles (reviewed by Zsolnay,

1996), most likely due to disruption of microbial tissues (DeLuca et al., 1992). Another

unique feature of our sample treatment was the cutting of maize straw and beech leaves to

ensure homogenization. The cut leaves released much more DOC as expected from DOC

concentrations beneath the Oi layer at the sampling site (36 mg C l� 1; Solinger et al.,

2001), but the humification indices were similar. Hongve et al. (2000) reported a higher

biodegradation of beech than of spruce litter DOM. The similar biodegradation of DOM

from spruce and beech litter in our study implied that cutting of the organic material did

not result in an overestimation of DOM biodegradation. Furthermore, the high DOC

concentration and the high biodegradation of the Oe-spruce DOM are not generally

representative for Oe material. The wide range of DOC concentrations (20–160 mg l� 1

for different extractions) indicates an inhomogeneity of this material.

4.2. Extent and dynamics of DOM biodegradation

Besides the portion of DOC that was mineralized, the size and mineralization constants

of the labile and stable DOC pools also depended on the origin of DOM. DOM extracted

from maize straw and litter layers showed the highest percentage of C mineralized and the

highest portion of labile DOC. Both the labile and stable DOC pools from these materials

had higher mineralization rates than those of the other DOM solutions. It seems reasonable

that the extent and rate of DOM degradation decreased with increasing degree of

decomposition of the solid organic matter, because of a preferential decomposition of

labile material (Haider, 1992; Dai et al., 2002).

The percentage of C mineralized (61–93% of total DOC) of samples extracted from

maize straw, from spruce and beech litter and from the fermentation layer of a spruce forest

was high as compared with the results of Qualls and Haines (1992). They found that 33%

of litter DOM (collected by zero-tension lysimeters) was degraded after 134 days.

Consequently, their degradation rate constants k1 were only about half of the k1 values

we determined. Hongve et al. (2000) reported a refractory behaviour of DOM from spruce

litter (0% biodegradation), whereas DOM from deciduous litter was highly degradable

(75% after 6 weeks). The reasons for these large differences in the literature remain an

open question and cannot be explained simply by the different methods used including

extraction of DOM, incubation and quantification.

Like Boyer and Groffman (1996), we found a higher percentage of C mineralized and

higher k values for DOM extracted from agricultural soils than from forest floors.

Probably, this reflects a different litter composition and a faster turnover of organic matter

in agricultural as compared with forest soils. The accordance of our data on DOM from the

agricultural soils (group 2, Table 2) with the results of Nelson et al. (1994), Boyer and


Groffman (1996) and Zsolnay and Steindl (1991) could also be caused by a similar

procedure to extract DOM.

The agricultural DOM samples BL-0 and BL-manure showed similar percentages of

DOC mineralization (f 30%). However, the mineralization rate constant of the stable

DOC portion of the BL-manure sample was twice that of the BL-0. In this case, DOM

originating from the manured plot was less stable than DOM from the plot without

fertilization. Gregorich et al. (2003) also found increased biodegradation of water-

extractable organic C after manure addition. The lower stability thus could be caused

by DOM released from the fresh, largely undecomposed organic material added (k value

for manure H k value for soil C). Furthermore, DOM derived from the Oa layer of the

spruce forest was more stable and had lower k values than DOM from the Oa layer of the

beech forest, although the mineralized portion of DOC was similar in both cases (Table 2).

These examples highlight the importance to study the dynamics of DOM biodegradation

to assess the contribution of DOC to C sequestration. Besides information on total

amounts of DOC mineralized, the pool sizes of labile and stable DOC and their mean

residence times are of interest.

The low biodegradation of DOM extracted from the Oa layer of a spruce and a

beech forest and from fen topsoils (5–9%) may also refer to a methodological problem.

Higher levels of degradation and higher degradation constants in other studies (e.g.,

Qualls and Haines, 1992; Boyer and Groffman, 1996) could be caused by the widely

used quantification of biodegradation by DOC measurements. Especially in long-term

experiments (e.g., Qualls and Haines, 1992), C can be removed from the liquid phase

by the formation of particulate organic matter through coagulation and precipitation. It

is very difficult to sample such suspensions adequately for DOC analysis. Often,

samples are filtered before DOC analysis which removes particulate organic matter.

Furthermore, harvest of particulate organic C during the incubation like those done by

Qualls and Haines (1992) contributes to an overestimation of the biodegradability of

DOM.

The k values of the stable DOC pool are lower than the corresponding k value for lignin

(0.003 day� 1; Paul and Clark, 1996) and in the same range as those used for the stable C

pool in the Century model (0.0005 day� 1; Paul and Clark, 1996). This is in contradiction

to the widespread assumption that DOM represents the most biodegradable pool of soil

organic matter.

4.3. Prediction of DOM biodegradation using DOM properties

Recalcitrance of organic matter should be one factor affecting its stabilization in soils

(Sollins et al., 1996). Examples of recalcitrant compounds are alkyls and aromatics which

can accumulate during the decomposition of organic matter (Baldock et al., 1992; Kogel-

Knabner et al., 1992; Huang et al., 1999). Our results showed that the aromaticity, the

degree of complexity and condensation of the molecules, the hydrophobicity and the

content of carbohydrates affect the microbial stability of DOM, confirming results for soil

organic matter (Haider, 1992; Capriel, 1997; Piccolo et al., 1999). There was no linear

relationship between chemical and structural properties of DOM and its biodegradation.

However, it seems that DOM solutions exceeding (UV absorbance, XAD-8 adsorbable C,


aromaticity, humification index) or falling below (carbohydrates) certain threshold values

are relatively stable with a mineralization of less than 10–20% of total DOC. The

corresponding values of the mentioned DOM properties can be easily obtained from Fig.

2. It was noteworthy that the simplest measurements such as UV absorbance and XAD-8

fractionation agreed best with the extent of DOC mineralization (Fig. 2).

Our study confirms previous results that carbohydrates are preferentially utilized by

microorganisms during degradation of the different DOM compounds (Volk et al., 1997;

Amon et al., 2001). Qualls and Haines (1992) related the extent of DOM degradation to

the initial content of hydrophilic neutrals (mainly carbohydrates). However, Volk et al.

(1997) stated that the often-used classification of carbohydrates as labile DOM compo-

nents should be treated with caution as carbohydrates can also be bound to stable DOM

compounds. Binding of carbohydrates to aromatic compounds is typical for hydrophobic

DOM fractions (Guggenberger et al., 1994) which are refractory (Jandl and Sollins,

1997).

In several studies, the extent of DOM degradation was inversely related to the UV

absorbance and to the hydrophobic fractions (Gilbert, 1988; Qualls and Haines, 1992;

Jandl and Sollins, 1997; Zoungrana et al., 1998; Jandl and Sletten, 1999; Pinney et al.,

2000), which is confirmed by our results. Until this study, fluorescence data were not

directly related to the extent of DOM degradation. Parlanti et al. (2000) stressed the

usefulness of fluorescence spectroscopy to gain information on the biological activity in

coastal waters. Our results suggested (Fig. 2) that the condensation status of aromatics,

revealed by fluorescence spectroscopy, is one of the DOM properties affecting its

microbial stability.

The UV absorbance and the proportion of XAD-8 adsorbable C were more strongly

correlated to the biodegradability than the amount of aromatic H. This indicates that

Fig. 4. Dendrogram for the hierarchical distribution of all measured parameters (see Tables 2 and 3 for

abbreviations and the values of these parameters).


besides aromatics, alkyl compounds with double bonds could be responsible for the

microbial stability of DOM. Almendros and Dorado (1999) pointed out that the aromatic

content of humic acids is not necessarily decisive for their biodegradability.

Principal components analysis did not improve the interpretation of our results.

However, clustering of DOM properties revealed a close relation between the XAD-8

adsorbable carbon and the stable DOC pool (Fig 4). This relation indicates the importance

of hydrophobic compounds, probably derived from lignin, to form stable organic matter.

Furthermore, the labile DOC, the content of carbohydrates and the percentage of

mineralized DOC form a second group. This second group emphasizes the function of

carbohydrates to be the main substrate for microorganisms in the initial state of DOM

mineralization. The aromatic content, the mineralization constants k1 and k2 and the UV

and fluorescence parameter belong to a third group indicating possible predictability of

DOM biodegradation by the most easily measurable property–the UV absorbance. The

results of the cluster analysis illustrate in a qualitative way the close relationships between

chemical properties of DOM and its biodegradation.

5. Conclusions

The extent and rate of DOM biodegradation decreases with progressing decomposition

and humification of the parent solid material where DOM is derived from. DOM

biodegradation is closely related to chemical DOM properties. It seems that especially

aromatic structures are highly stable, whereas carbohydrates serve as the main substrate for

microorganisms. Simple measurements like UV absorbance, XAD-8 fractionation and

fluorescence emission spectroscopy can be used to estimate the potential extent and rate of

DOM biodegradation. Although the rather expensive NMR spectroscopy did not improve

the prediction of DOM biodegradation, it was helpful to elucidate the complex interactions

between DOM properties and biodegradation.

We did not find evidence that DOM is necessarily the most biodegradable fraction of

soil organic matter. Like the solid phase, DOM comprises both labile and relatively stable

portions. Considering the high DOM fluxes in forest soils and a mean residence time up to

12.5 years, DOM percolating into the mineral soil can contribute to stable C in forest

mineral soils. However, it is unknown how interactions between DOM and the solid soil

phase will affect DOM biodegradation. Both inhibition caused by sorption and enhanced

biodegradation due to a higher density and probably better source-adaptation of micro-

organisms at the sorption sites seem possible.

Acknowledgements

Financial support came from the Deutsche Forschungsgemeinschaft and the German

Ministry of Education and Research (BMBF; project No. BEO 51-0339476). We thank Dr.

Ludwig Haumaier for recording the NMR spectra and Dr. Klaus Kaiser for support in their

interpretation. Special thanks goes to Dr. Bjorn Berg for helpful discussions and to the two

referees for helpful comments.


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