fractionation of dissolved organic carbon from soil...

Fractionation of dissolved organic carbon from soilsolution with immobilized metal ion affinitychromatography

I. PAUNOVICa , R. SCHULIN

a & B. NOWACKa,b

aInstitute of Terrestrial Ecosystems, ETH Zurich, Universitaetstrasse 16, 8092 Zurich, and bEMPA- Materials Science and Technology,

Lerchenfeldstrasse 5, 9014 St. Gallen, Switzerland

Summary

The presence and identity of Cu-complexing ligands in soil solution strongly affects biogeochemistry,

bioavailability and the fate of Cu in soils. In this study, we compared the influence of heavy metal

pollution, vegetation and soil type on the amount and characterization of ligands able to form ternary

complexes with Cu in soil solution. For separation and characterization, we applied immobilized metal ion

affinity chromatography (IMAC) combined with fluorescence spectroscopy. All separated IMAC-frac-

tions exhibited excitation-emission wavelengths of humic-like fluorescence (240–285/365–434 nm). Pro-

tein-type fluorescence (270–280/295–365 nm) and fluorescence at 330–340/375–385 nm were detected only

in the retained fraction whereas carboxylate-type fluorescence (300–310/420–430 nm) was observed only in

the non-retained fraction. These findings are in agreement with the behaviour of model ligands. The

IMAC-retained ligands represented between 5 and 30% of dissolved organic carbon. The soil type and

the vegetation had the largest influence on the quality and quantity of Cu ligands able to form ternary

complexes. In the topsoil, the IMAC retained fraction was greater in soil without vegetation (16%)

compared with soil with vegetation (12%). A larger amount (75%) of the protein-type ligands able to

form ternary complexes with Cu was found in soil with vegetation compared with plant-free soil (69%).

Metal pollution also affected the composition of the extracted ligands; the fraction with protein-type

ligands decreased from 75% in unpolluted to 65% in the polluted topsoil. The results show that

IMAC-retained ligands are related to the biological activity in soils.

Introduction

In waters and soils, copper is generally strongly complexed by

dissolved organic carbon (DOC), and this complexation influ-

ences the biogeochemical cycling, bioavailability and toxicity of

Cu (Temminghoff, 1997; Kalbitz &Wennrich, 1998). A number

of factors control the characteristics, functions and dynamics of

soil DOC, such as vegetation, soil type and microbial activity

(Kalbitz et al., 2000, 2003; Chantigny, 2003; Marschner & Kal-

bitz, 2003). Variation in DOC composition and concentration

can have a diverse effect on Cu speciation in soil. The chemical

nature of the ligands in DOC that bind Cu has resisted thor-

ough characterization because of the complex nature, the

small concentrations of Cu complexes, and the presence of

a mixture of ligands.

Immobilized metal affinity chromatography (IMAC) is gain-

ing widespread popularity as an effective tool for the separation

and characterization of a variety of biotic macromolecules

(Chaga, 2001;Gaberc-Porekar&Menart, 2001). IMAC is based

on the ability of ligands in solution to form coordination com-

plexes with metal ions that are fixed to the immobilized phase of

a column, through partial complexation by a surface bound

multi-dentate ligand. IMAC is most commonly used to isolate

ligands binding soft metal ions (e.g. Cu, Zn, Ni).

IMAC has been employed to isolate natural organic ligands

from aquatic environments and to separate humic and fulvic

substances (Burba et al., 2000; Wu et al., 2002). In combination

with other techniques, isolated fractions have been character-

ized by complexation capacity, molecular size distribution,

and amino acid characterization (Gordon et al., 1996; Donat

et al., 1997; Midorikawa & Tanoue, 1998).

Several studies have used fluorescence spectroscopy to char-

acterize aquatic Cu ligands isolated with IMAC (Wu&Tanoue,Correspondence: B. Nowack. E-mail: [email protected]

Received 11 January 2007; revised version accepted 13 September 2007

198# 2007 The Authors

Journal compilation # 2007 British Society of Soil Science

European Journal of Soil Science, April 2008, 59, 198–207 doi: 10.1111/j.1365-2389.2007.00975.x

2001; Wu et al., 2001). Such spectroscopy is a sensitive method

for characterizing natural organic matter in soils (Senesi et al.,

1991; Luster et al., 1996) and natural waters (Coble, 1996).

The fluorescence signals originate mainly from aromatic DOC

compounds. Differences in the aromatic DOC fraction can

thus be determined by comparison of the fluorescence spectra,

so enabling different types of sources to be distinguished

(Senesi et al., 1991). Attempts have been made to identify indi-

vidual fluorescent components and to relate this to specific sig-

nals in DOC fluorescence spectra (Senesi, 1990). However, due

to the complex nature of DOC, effects such as spectral over-

lapping, peak broadening and shifting have limited the success

of a florescence spectroscopy approach.

In a previous study, we developed and validated an IMAC

method for Cu ligand separation from soil solution and investi-

gated the retention behaviour of simple model ligands in the

IMAC column (Paunovic et al., 2005). We found that the

ability to form ternary complexes of the structure ligand-

Cu-ligand was the dominant factor governing the retention

of a ligand. The value of the stability constant of the Cu-

complexes was found to have only a minor influence on the

retention. The not-retained fraction was composed of ligands

unable to form ternary complexes in the column (mainly

ligands with carboxylic functional groups) and of all com-

pounds that are not able to bind to Cu in solution.

In the present study, we used the IMAC method to separate

and isolate ligands able to form ternary complexes with Cu from

soil solution.Model ecosystem soil solutions were used to exam-

ine complex effects of vegetation, subsoil type and heavy metal

pollution on DOC characteristics. The objective of this study

was to investigate the effects of the key variables of the ecosys-

tem on the amounts and characteristics of Cu-binding ligand

able to form ternary complexes fractions isolated by IMAC.

Fluorescence spectroscopy was used to characterize the struc-

ture and functional properties of the IMAC-separated fractions.

Materials and methods

Sample site and sample collection

The model ecosystem experiment from which the soil solution

samples were obtained has been described in more detail by

Nowack et al. (2006) and will only be described briefly here.

The experiment had a factorial design in order to investigate

effects of metal pollution and plant processes and their inter-

actions in young forest stands under controlled, quasi-natural

conditions. The soil profile consisted of a 50-cm deep quartz

sand layer that functions as drainage packing, an 80 cm layer

of subsoil, and a 15-cm layer of topsoil. The lysimeters had

a surface area of 3 m2. The subsoil was acidic loamy sand from

a Haplic Alisol in one half of the treatments or a calcareous

sandy loam from a Calcaric Fluvisol in the other half of the

treatments. The topsoil was a weakly acidic loam (Luvisol)

from an unpolluted agricultural field. In half of the lysimeters

with each subsoil type, the topsoil had been contaminated arti-

ficially with filter dust from a non-ferrous metal smelter, con-

taining Zn, Cu, Cd, and Pb, 4 years before the sampling. Some

physical and chemical characteristics of the three soils are

given in Table 1.

In the polluted topsoil, the final metal concentrations were

3000 Zn, 640 Cu, 90 Pb, and 10 Cd mg kg�1. Each model eco-

system was planted with 14 trees (six Picea abies (L.) Karst.,

four Populus tremula L., two Salix viminalis L. and two Betula

pendula) and understory plants (Carex sylvatica Hudson, Allium

ursinum L., Tanacetum vulgare L.) (Menon et al., 2005).

Four soil columns, one per treatment, were installed in the

immediate vicinity of open-top chambers as plant-free referen-

ces (Rais, 2005). The columns were 78 cm in diameter and

packed with topsoil, subsoil and quartz sand in the same way

as the model ecosystem lysimeters.

Soil solutions were sampled at 10, 40 and 70 cm depth with

10-cm long porous suction cups made of nylon (EcoTech

GmbH, Bonn, Germany) (Rais et al., 2006). The samplers were

installed vertically with the centre of the cup at the specified

depth. There were two samplers in each lysimeter connected

with Teflon tubing to the same sampling bottle. Nylon suction

cups were found to have the smallest influence on metal con-

centration of a range of materials tested and DOC from the

soil solution was not significantly adsorbed at neutral pH

(Rais et al., 2006). The sampling bottles were made of poly-

propylene (EcoTech GmbH). One week before each sampling

event, the collection bottles were emptied. Thus, each sample

represents a sampling period of 1 week. The sampling bottles

were kept in boxes buried in soil pits in order to keep them

cool and dark to minimize microbial and algal activity. How-

ever, we cannot exclude the possibility that more labile organic

material had already been degraded within 1 week. After they

had been collected, the samples were filtered through a 0.45

Table 1 Physical and chemical characteristics of the soil materials used

in the model ecosystem experiment (from Nowack et al., 2006)

Topsoil

Acidic

subsoil

Calcareous

subsoil

Sand/% 36 87 74

Silt/% 49 8 16

Clay/% 15 5 10

pH (1:2 soil: 0.01 mol CaCl2) 6.6 4.2 7.4

Cinorga/g kg�1 < 1 < 1 21

Corgb/g kg�1 15.1 3.2 11.2

Cutotc (control; polluted)/mg kg�1 28; 640 7.4 14

Zntotc (control; polluted)/mg kg�1 97; 3000 39 58

Cdtotc (control; polluted)/mg kg�1 0.1; 10 < 0.2 0.2

Pbtotc (control; polluted)/mg kg�1 37; 90 13 19

aCinorg, inorganic carbon.bCorg, organic carbon.cTotal metal concentration.

Fractionation of DOC from soil solution with IMAC 199

# 2007 The Authors

Journal compilation # 2007 British Society of Soil Science, European Journal of Soil Science, 59, 198–207

mm pore-size filter and stored at 4°C in the dark. The samples

that were used in this study were collected in October 2004.

IMAC set-up

We used HiTrap Chelating HP 5 ml columns (Amersham Phar-

macia Biotec, Wadenswil, Switzerland) throughout this study.

Chelating Sepharose� High Performance, the chelating gel in

the column, consists of cross-linked agarose beads to which imi-

nodiacetate (IDA)moieties are covalently bound by hydrophilic

spacer arms. After loading with Cu, this type of column matrix

provides low specificity, excessive capacity and great adsorption

affinity for all copper ligands. The IMACcolumnwas connected

to a HPLC pump (Jasco PU-980; Omnilab, Mettmenstetten,

Switzerland), an ultraviolet detector (Jasco UV-970; Omnilab)

and a fluorescence detector (Jasco FP-2020 plus; Omnilab).

The column was washed with 25 ml of distilled water to

remove any trace of storage solution (200 g litre�1 ethanol)

and then loaded with copper by adding 1 ml of 0.1 mol litre�1

CuSO4 solution. Loosely bound copper ions were removed by

flushing the column at first with 20 ml of distilled water and

then with 30 ml of binding buffer (2 mmol litre�1 3-morpho-

lino-2-hydroxypropanesulphonic acid (MOPSO), 0.5 mol

litre�1 NaCl, pH 6.9), until a stable UV adsorption baseline

was obtained.

IMAC fluorescence measurements were determined at excita-

tion-emission wavelength pairs of 240/420, 280/320, 330/380

and 330/420 nm and UV absorption was measured at 254 nm.

IMAC isolation procedure

The IMAC isolation procedure used in this study was as

described by Paunovic et al. (2005). Briefly, the procedure con-

sists of the following five steps: column equilibration, sample

loading, washing with buffer, sample elution, and re-equilibra-

tion of the column. All experiments were performed at a volu-

metric flow rate of 2 ml minute�1. For equilibration, the

column was washed with the binding buffer (above). The sam-

ple was loaded by switching from binding buffer to sample for

a specified time and acquisition of data was started. Sample

volumes applied to the column were 1–3 ml. After sample

loading the column was washed with 20 ml of binding buffer

to remove the non-retained compounds. The ligands retained

on the column were then removed from the column with 20 ml

of elution buffer (2 mmol litre�1 phosphate buffer, pH 3.5, 0.5

mol litre�1 NaCl). Prior to running the next experiment, the

column was thoroughly washed and re-equilibrated with bind-

ing buffer. After each experimental run Cu ions were stripped

from the column matrix by applying 25 ml of 0.25 mol litre�1

EDTA in order to regenerate the column.

IMAC chromatograms were recorded in duplicate with

closely correlated sample volumes. The peak areas of duplicates

differed by not more than 5%. All soil solution DOC was com-

pletely recovered at reproducible elution times.

Fluorescence measurements

Three fractions of the IMAC-separated soil solution were col-

lected: from 6 to 9 minutes for non-retained ligands, from 17 to

19 minutes for the first retained peak and from 19 to 21 minutes

for the second retained peak. The fractions were analysed using

a LS 50 B fluorescence spectrophotometer (Perkin Elmer, Bea-

consfield, UK). A series of emission spectra were recorded for

each sample from 250 to 650 nm in 10-nm steps. The excitation

wavelength ranged from 240 to 500 nm and increased in 10-nm

steps. Three-dimensional excitation-emissionmatrixes (3DEEM)

were obtained by combining sample emission spectra for differ-

ent excitationwavelengths. To remove possible column contam-

ination, 3DEEM of IMAC mobile solutions were recorded and

used as blanks. The 3DEEM of the binding buffer (MOPSO)

was subtracted from the 3DEEM of non-retained IMAC frac-

tions, whereas the eluting buffer (2 mmol litre�1 phosphate

buffer at pH 3.5) was used as blank for the retained fraction

measurements. Replicate scans did not differ by more than 5%

in peak location and intensity.

Results

IMAC procedure

All soil solution IMAC chromatograms revealed three peaks.

Figure 1(a) shows representative IMAC chromatograms of

soil solutions collected at depths of 10, 40, and 70 cm below

the vegetation. The first peak corresponds to the non-retained

fraction (peak I) with an elution time of 7.0 � 0.5 minutes.

The retained fraction was always divided into two peaks. The

first retained peak (peak II) occurred at 18.0 � 0.5 minu-

tes and the second at an elution time of 19.3 � 0.5 minutes

(peak III).

The effect of using the stated detection excitation/emission

wavelength on the quantity of IMAC-isolated ligands recorded

was examined by analysing a soil solution sample at four differ-

ent wavelength pairs. As Figure 2 shows, the total peak area of

the sum of peaks I, II and III depended on the selection of the

excitation/emission (ex/em) pair and thus on the intensity of

the signal. However, the chosen ex/em pair affected neither the

shape of the retained IMAC peak nor the respective elution

times. The relative size of the retained peak area (sum of peaks

II and III), given as percentage of total peak area (sumof peaks I,

II, and III), was 13% for the three different ex/em pairs. The

exception was the detection at 280/320 nm, where the retained

fraction increased to 39% of the total peak area, which suggests

that the retained fraction had a considerable protein content, as

the wavelength of 280/320 nm is typical for protein fluorescence.

Based on the results of these measurements, that is the great

signal stability, good peak abundance and excellent reproduc-

ibility, the quantity of retained IMAC ligands throughout this

study will be presented as the percentage of total peak area

measured at 330/420 nm.

200 I. Paunovic et al.

# 2007 The Authors


Analysis of soil solution samples from the model ecosystem

The IMAC chromatograms of all samples showed broad peaks

that confirm the broad diversity of copper complexing ligands in

soil solutions. Depending on the sample and depth, the retained

fraction accounted for 6.8–23.9%of total peak area. In contrast

to Gordon et al. (1996), and Midorikawa & Tanoue (1996), we

found no effect of the soil solution pH on the percentage of

the retained fraction (R2 ¼ 0.0784, P > 0.15), within the pH

range of our samples (5.6–8.6).

The percentage of the retained fraction was greater in the

acidic than the calcareous subsoil (Figure 3). In the acidic sub-

soil, the retained fraction was 14%, the same as in the topsoil,

whereas only half of that was found in the calcareous subsoil. In

both subsoils the retained fraction increased from 40- to 70-cm

depth.

In the presence of vegetation, the percentage of retained ligands

was less in topsoil than in plant-free topsoil (Figure 4). While the

percentage of the retained fraction increased with the depth in the

presence of plants, it decreased in the plant-free soils.

Metal pollution did not affect the size of the retained fraction.

However, the shape of peaks II and III in the IMAC chromato-

grams differed between polluted and non-polluted soil, thus

indicating differences in composition (refer to Figure 1b). Fig-

ure 5(a) shows that in the presence of vegetation,metal pollution

decreased the percentage of peak II, more than in the absence of

plants.Metal pollutionof the topsoil also influenced the percent-

age of peak II in the unpolluted subsoil. As can be seen in Fig-

ure 5(b), the calcareous subsoil had a greater percentage of peak

II under unpolluted topsoil than under polluted topsoil. In the

acidic subsoil, no effect of pollution on the percentage of peak II

was observed. This effect did not differ between 40- and 70-cm

sampling depths (data not shown).

Fluorescence spectroscopy of IMAC fractions

The ratios between fluorescence intensity at 330/420 nm andUV

absorption at 254 nm differed between the non-retained (peak I)

and the retained fractions (peaks II and III) (Figure 6). For the

non-retained peak the ratio was 0.5� 0.02, for the two retained

peaks it was 1.46 � 0.3 (SD, n ¼ 44).

Figure 7 shows the 3DEEM plots of IMAC-fractionated

ligands fractions (peaks I, II and III) and fluorescence ex/em

maxima of separated soil solutions are shown in Table 2. The

contour plots of unfractionated soil solutions exhibit broad areas

characteristic of humic substances (Coble, 1996). After IMAC

separation, the three isolated factions (peaks I, II and III) were

less complex. Table 3 shows the ex/emmaxima of unfractionated

soil solutionand the three fractions for the soil solution samples at

different depths, and in the presence or absence of vegetation.

Comparing the contour plots of the ex/em matrices of the

fluorescence intensities revealed that the ex/em maximum of

240–258/365–434 nm was present in all samples, independent

of their origin, and in all separated fractions (peak A, Figure 7).

A second peak (peak A¢, Figure 7), with an ex/em maximum at

300–322/415–425 nm, was observed in all samples of the non-

retained fraction. This peakwas not found in any of the retained

fraction samples. Two other peaks were observed in some of the

retained fractions. The first (peak B) at 330–340/375–385 nm

was detected in IMAC peak III fractions of soil solution from

the topsoil with vegetation and in the calcareous subsoil under

vegetation. The second (peak C) with an excitation/emission

maximum at 270–280/295–365 nm was observed in the IMAC

peak II of unpolluted topsoil samples and in samples from both

subsoil types under vegetation.

A comparison of the location of the peaks in the ex/emmatrix

with fluorophore peaks found in matrices of natural samples

suggests that the peaks A, A¢, B and C correspond to peaks a¢,a, b and g/d described by Parlanti et al. (2000) and Sierra et al.

(2005), to peaks C, A, M and T described by Coble (1996) and

a)

0 5 10 15 20

0

1x104

2x104

III

II

I

Fluo

resc

ence

at 3

30/4

20 /n

m

Time/minutes

10 cm 40 cm 70 cm

b)

10 15 20

0

1x104

2x104

3x104

III

II

Fluo

resc

ence

at 3

30/4

20/n

m

Time/minutes

unpolluted topsoil polluted topsoil

Figure 1 (a) Immobilized metal ion affinity chromatography (IMAC)

chromatograms (fluorescence signal at 330/420 nm) of soils solutions

collected in the unpolluted topsoil (10 cm depth) with vegetation and

in the calcareous subsoil at 40 and 70 cm depth (with vegetation); (b)

IMAC chromatograms of retained fractions of polluted and unpol-

luted topsoil (with vegetation).


# 2007 The Authors


to peak classes II, I and III described by Wu & Tanoue (2001),

respectively.

The emission wavelengths of peak A were shorter in samples

of the non-retained fractions than of the retained fractions. Such

a blue-shift in fluorescence emission results from an increased

energy difference between the ground state and the first excited

state of a molecule (Coble, 1996).

Discussion

Characterization of IMAC-retained ligands

In previous work, we have shown that not all Cu-complexing

ligands interact with Cu in the IMAC column and that not all

are retained (Paunovic et al., 2005). The retention of copper-

binding ligands on the IMAC column depends primarily on

the ability of the ligands to form metal, ternary complexes,

that is surface complexes with the structure ligand-Cu-ligand,

and to a much lesser degree on the binding strength.

The respective elution times of peaks I, II and III were the

same as the elution times of the model ligands from the previous

study (Paunovic et al., 2005). Model ligands containing only

carboxylic groups were not retained on the column (peak I),

proteins and peptides were eluted at the same time as ligands

in peak II, while N-heterocyclic ligands had an elution time

corresponding to peak III. Thus, the elution times can be used

as an indicator of the chemical characteristics of the separated

ligands (Paunovic et al., 2005).

330/420 240/420 330/380 280/3200

5.0x105

1.0x106

1.5x106

2.0x106

2.5x106

a)

excitation/ emission wavelengths/nm

Tota

l pea

k ar

ea /A

rbitr

ary

units

330/420 240/420 330/380 280/320

b)

excitation/ emission wavelengths/nm

0

5

10

15

20

25

30

35

40

Ret

aine

d pe

ak o

f tot

al p

eak

area

/%

Figure 2 Detection of IMAC chromato-

grams at different excitation/emission wave-

lengths: (a) Value of total peak area and (b)

percentage of retained peak area at the ex/em

pairs of 330/420, 240/420, 330/380 and

280/320 nm.

80

60

40

20

00 10 15 20 25

Dep

th /c

m

Retained peak of total peak area /%5

Figure 3 Effect of soil type on the percentage of the retained IMAC

peak. Topsoil (D), acidic subsoil (u), calcareous subsoil (j). Bars are

standard errors (n ¼ 5).

80

60

40

20

00 10 15 20 25

Dep

th /c

m

Retained peak of total peak area /% 5

Figure 4 Effect of vegetation on the percentage of the IMAC peak;

percentage of retained peak of the plant-free system solution (s) and

soil with vegetation (d). Bars are standard errors (n ¼ 5).


# 2007 The Authors


The excitation/emission peak maxima observed in this study

have been consistent with existing studies of fluorescence prop-

erties of DOC (Coble, 1996; Parlanti et al., 2000; Wu et al.,

2001; Sierra et al., 2005). In previous studies, the wavelength

of the ex/em maximum of peak A has been attributed to

humic-like substances (Senesi et al., 1991; Coble, 1996). There-

fore, the presence of this peak in all IMAC fractions indicates

that in all fractions humic acids are present with different

functional groups. The ex/em wavelength of the A¢ peak (300–

310/420–430 nm) has been attributed to carboxylic acid

groups (Midorikawa & Tanoue, 1998; Wu & Tanoue, 2001).

Fluorescence of peak B (ex/em of 330–340/375–385 nm) has

been detected in a study of marine DOC as well as in leaf litter

extracts in terrestrial systems (Luster et al., 1996). Fluores-

cence of peak C (ex/em 270–290/300–360 nm) is characteristic

of proteins or aromatic amino acids and their metabolites

(Determann et al., 1998; Mayer et al., 1999).

The chemical identity deduced from the retention times of

the IMAC chromatograms and the results from fluorescence

spectroscopy support each other very well. In the non-retained

fraction only two types of fluorescence have been detected,

humic-like and fluorescence characteristic of ligands with car-

boxylic acid groups. Based on the model ligand study, we

expected to find carboxylic acids mainly in the non-retained

fraction (Paunovic et al., 2005).

In the retained fraction, protein-like fluorescence and fluores-

cence at 330–340/375–385 nm in the ex/em matrix, was detected

(peak B). The presence of protein-like fluorescence in IMAC

fractions agrees well with the model-ligand study (Paunovic

et al., 2005) and with findings of Hsu & Hatcher (2005) and

Knicker et al. (2000) that some proteins, despite their bio-

chemical biodegradability, can be protected in soil solution.

The origin of the fluorescence of peak B was related to ele-

vated biological activity (Parlanti, 2000), but to our know-

ledge, the chemical structures responsible for fluorescence at

the ex/em wavelengths of peak B have not been identified.

Further, the maximum of fluorescence emission of the humic-

like fluorescence of the retained fraction was shifted towards

longer wavelengths in comparison with the non-retained frac-

tion. A blue shift of the emission can be caused by a decrease in

the number of aromatic rings, by a reduction of conjugated

bonds in a chain structure, by the conversion of a linear ring

system to a non-linear system, or by elimination of carbonyl

and hydroxyl functional groups (Senesi et al., 1990; Coble,

1996). The occurrence of a blue shift in the fluorescence of the

humic peak and the presence of two specific fluorescence

peaks in combination with a large variation in the ratio

between fluorescence intensity and UV absorption further

characterize the retained IMAC fraction. The retained IMAC

fractions seem to contain a large variety of fluorophores, that

is ligands with a large content of proteinous structure, a small

content of aromatic moieties, and a lack of carboxylic func-

tional groups.

Figure 5 Effect of heavy metal pollution on the size of IMAC peak

II as percentage of the total retained fraction (peaks II and III) (a) in

solution samples from topsoil with and without vegetation, (b) of

acidic and calcareous subsoil (40 cm) with and without pollution.

Bars are standard errors (n ¼ 5).

80

60

40

20

00 0.5 1.0 1.5 2.0

Dep

th/c

m

Fluorescence intensity/UV absorbance

Figure 6 Ratio between fluorescence at 330/420 nm and UV absor-

bance at 254 nm for the non-retained IMAC fraction (d) and the

retained fraction (s). Bars are standard errors (n ¼ 30).


# 2007 The Authors


The consistence between the results of the fluorescence char-

acterization and the study ofmodel-ligand behaviour (Paunovic

et al., 2005) demonstrates that IMAC elution times can be

used as indicator of the chemical characteristics of ligands.

Quantity and quality of IMAC-retained ligands

Large differences between the amounts of extracted Cu ligands

were observed between vegetated and non-vegetated soils. In the

subsoil, the presence of plants increased the amount of Cu

ligands extracted, thus indicating the contribution of vegetation

to organic ligands able to form ternary complexes with Cu. The

extracted Cu ligands could have originated either directly from

plants (e.g. root exudates or from decaying plant material) or

from microbial decomposition of soil organic matter, in

response to a stimulation of microbial activity in the rhizo-

sphere. Soil column experiments with the same soil have shown

that plants can locally have a large influence on DOC concen-

tration in soil solution (Zhao et al., 2007).

The presence of vegetation also affected the quality of the

Cu ligands isolated. The relative retained-peak area of peak II

measured at the wavelength typical for fluorescence of proteins,

280/320 nm, was much larger than at the other wavelengths,

which indicates that the peak II contained large amounts of

proteinous Cu ligands. As root and microbial exudates are one

of the sources of peptides and proteinous compounds in soil

solution (Jones et al., 1994; Neumann & Romheld, 1999), the

quantity and quality of the IMAC-retained protein fraction

could be used as an indicator of the changes in DOC due to

the biological activity in soils.

Root growth and soil enzyme activities were reduced in metal

contaminated topsoil of the model ecosystems from which the

soil solution samples were obtained (Menon et al., 2005; Now-

ack et al., 2006). A decrease in the protein fraction was found

in the polluted topsoil compared with the metal-free soil,

showing that elevated concentrations of metals, in particular

of Cu and Zn, did not affect the quantity, but the quality of

the IMAC retained ligands. An additional link between the

quantity of IMAC protein fraction and soil biological activity

was found by comparing unpolluted topsoil with and without

vegetation. A greater percentage of the IMAC protein fraction

was found in the unpolluted topsoil with vegetation than in

the bare soil, which is in line with elevated root zone biological

activity.

A further verification that the amount of the IMAC protein

fraction is related to rhizosphere activity was found in the sub-

soils. In the calcareous subsoil, the growth conditions weremore

favourable for deeper rooting, while in the acid subsoil root

growth was limited (Menon et al., 2005). Metal pollution in

the topsoil affected the root density in the calcareous subsoil

at 40 cm depth but not in the acidic subsoil (Menon et al.,

2005). Accordingly, we found that in the calcareous subsoil

Figure 7 3DEEM contour plots of IMAC fractions from topsoil solution without vegetation (upper row) and topsoil with vegetation (lower row).

A, A¢, B, and C represent ex/em maxima (see text).


# 2007 The Authors


below the polluted topsoil, a greater quantity of the protein

fraction was bound in ternary complexes, while in the acid

subsoil no influence of heavy metal on the IMAC protein frac-

tion was observed.

Parlanti (2000) attributed the emission excitation wavelength

of peak B (ex/em of 330–340/375–385 nm) to biological activity.

Here, we observed peak B in the topsoil and the calcareous sub-

soil under vegetation and did not detect it in the acidic subsoil,

which agrees with a biological origin of this fluorescence.

Environmental role of IMAC-retained ligands

It has been shown that very stable binding sites for Cu are asso-

ciated with N-containing functional groups (Alberts & Takacs,

1999). Such ligands couldbe foundmainly in peak III. The ex/em

wavelength of strong complexing ligands in leaf litter extracts as

determined by Luster et al. (1996), was identical to that of

the maximum fluorescence of ligands found in the retained

fraction. In order to examine the environmental role of

the IMAC-isolated fraction, we compared the amount of

Table 3 Excitation/emission maxima of unfractionated soil solution and three immobilized metal ion affinity chromatography (IMAC) soil solution

fractions at different depths and in the presence or absence of vegetation. Designation of identified peaks relates to Figure 7

Sample Bulk Non-retained fraction (Peak I) 1st retained fraction (Peak II) 2nd retained fraction (Peak III)

Soil Treatment Depth/cm ex/nma em/nmb ex/nma em/nmb Peak ex/nma em/nmb Peak ex/nma em/nmb Peak

Topsoil No plants 10 240–260 380–460 240 432 A 240 365 A 240 351 A

300 418 A¢ 270 336 C — — —

Topsoil Plants 10 240–400 350–450 257.5 434 A 245 432 A 250 426 A

322 423 A¢ — — — 330 375 B

Calc. subsoil No plants 40 240–400 350–460 252.5 428 A 245 429 A 240 405 A

310 424 A¢ — — — — — —

Calc. subsoil Plants 40 240–400 350–460 240 427 A 240 378 A 240 412 A

300 422 A¢ 275 365 C 340 385 B

Acidic subsoil Plants 40 240–250 410–440 240 417 A 250 320 A 250 317 A

300 412 A¢ 270 289 C — — —

aex, excitation wavelength.bem, emission wavelength.

Table 2 Immobilized metal ion affinity chromatography (IMAC) retained peak area (as % of total area) for soil solutions sampled at different depths,

in the absence and presence of plants, and from polluted and unpolluted soil. Values of dissolved organic carbon (DOC), pH total and free

copper concentrations of soil solution (from Rais, 2005)

Treatment

Plants No plants

No HMa HMa No HMa HMa

Topsoil 10 cm DOC /mmol litre �1 1288b 1100b 463b 430b

pH 7.6b 7.6b 8.1b 8.2b

Total Cu/mmol litre�1 0.344c 1.436c 0.110c 1.541c

Free Cu/mmol litre�1 — 7.65c — 2.75c

Retained fraction/% 11.4b 13.5b 15.6b 17.0b

Subsoil Acidic Calcareous Acidic Calcareous Acidic Calcareous Acidic Calcareous

40 cm DOC/mmol litre�1 292b 943b 230b 708 684 954 782 991

pH 6.1b 8.6 5.8b 8.5 7.7 8.6 7.9 8.6

Total Cu/mmol litre�1 0.0224c 0.1495 0.0216c 0.1334 0.0261 0.1778 0.045 0.1256

Retained fraction/% 19.9b 6.8 18.2b 12.9 7.3 16.5 7.8 13.3

70 cm DOC/mmol litre�1 188b 447 168b 341 785 1176 721 1009

pH 5.6b 8.5 5.8b 8.5 6.8 8.6 6.9 8.6

Total Cu/mmol litre�1 0.0132c 0.075 0.013c 0.1075 0.0242 0.1935 0.0328 0.1998

Retained fraction/% 23.9b 12.1 21.6b 18.8 8.4 13.7 8.5 12.6

aHM heavy metals.bAverage value of four samples.cValues measured in solution.


# 2007 The Authors


IMAC-retained Cu ligands with the total and free Cu concen-

tration in soil solution. Smaller values of free Cu2þ were found

in polluted topsoil without vegetation than in the topsoil with

vegetation (Table 1) in spite of the fact that total dissolved

copper tended to be greater (Rais, 2005). Obviously, the DOC

from plant-free soils had stronger Cu complexing properties

than the DOC from sites with vegetation. We found more

IMAC-retained ligands in the topsoil without vegetation than

with vegetation, an indication that the IMAC-retained fraction

might also be involved in Cu complexation in soil solution.

Conclusion

The findings demonstrate that:

1 IMAC is well suited for the separation and chemical charac-

terization of ligands able to form ternary complexes with Cu

in soil solution.

2 The soil type and the vegetation had the largest influence on

the quality and quantity of Cu ligands able to form ternary

complexes.

3 A larger amount of the protein-type ligands able to form

ternary complexes with Cu was found in soil with vegetation

compared with plant-free soil.

4 IMAC-retained ligands can be related to the biological

activity in soils.

Acknowledgements

The authors would like to thank D. Rais and J. Luster for pro-

viding samples of soil solution. The financial support of the Velux

foundation, Zurich, Switzerland, is gratefully acknowledged.

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