The three-dimensional structure of humic substances and soil ...

Fresenius J Anal Chem (I995) 351:62-73 Fresenius' Journal of
© Springer-Verlag 1995
The three-dimensional structure of humic substances
and soil organic matter studied by computational analytical chemistry
H.-R. Schulten
Fachhochschule Fresenius, Department of Trace Analysis, Dambachtal 20, D-65193, Wiesbaden, Germany
Received: 8 August 1994/Revised: 29 August 1994/Accepted: 31 August 1994
Abstract. A novel three-dimensional structural concept
for humic substances and soil organic matter (SOM) is
proposed which is based on previously published, com-
prehensive investigations combining geochemical, wet-
chemical, biochemical, spectroscopic, agricultural and
ecological data with analytical pyrolysis. Direct, tempera-
ture-programmed pyrolysis in the ion-source of the mass
spectrometer and soft ionization in very high electric
fields (Py-FIMS) and Curie-point pyrolysis-gas chroma-
tography/mass spectrometry (Py-GC/MS) were the main
applied thermal methods. Emphasis is laid on molecular
modelling and geometry optimization of complex, poly-
disperse structures of biomacromolecules using modern
PC software (HyperChem®). Trapping and binding of
atrazine in an organo-mineral complex is introduced as a
first example of simulation experiments for soil processes
at atomic level (nanochemistry). Future applications of
semi-empirical calculations and molecular dynamics in
pyrolysis studies are outlined.
Introduction
Since practically all pyrolysis data so far have been ob-
tained and published as two-dimensional (2 D) plots, it is
of interest to illustrate recent progress in commercially
available, relatively low cost software and personal com-
puter (PC) equipment which allow three-dimensional
(3D) displays and computer-assisted design (CAD) of
chemical structures and model reactions. In particular
the possibilities for molecular modelling and geometry
optimizations of complex macromolecules, which are of-
ten the target of analytical pyrolysis, virtually open up a
new dimension. This is demonstrated in the following for
humic substances, one of the most complex naturally oc-
curring materials, as an example.
Dedicated to Professor Dr. Dieter Klockow on the occasion of his 60th
birthday
At the 5th International Meeting of the International
Humic Substances Society in Nagoya, Japan, a new
approach for a better understanding of the structure of
humic substances and soil organic matter (SOM) was in-
troduced [1]. A black and white copy of the authentic
transparency is shown in Fig. 1, and was the basis of three
essential statements.
First, a characteristic portion of the organic carbon
consists of alkylaromatics and forms a flexible carbon-
carbon network structure.
Second, the voids formed by this kind of three-dimen-
sional structure could trap and bind three major classes
of organic SOM constituents such as N-containing com-
pounds, aromatics and aliphatics.
Third, binding of the humic substances onto the min-
eral surface remains still an unsolved problem (see sche-
matic surface area and question mark in the left part of
Fig. 1).
At that time, typical constituents of humic substances
and soil organic matter in agricultural soils were assem-
~ Agricultural Soil
I . \ / 3
i ~ (CH2)6'"~CH2)>lO OH SOM
CH 3. ~ '~ \ ,CH2-CH
/
] Arom Lic~
@r
Fig. 1. Copy of the original co]our transparency shown at the 5th Meet-
ing of the International Humic Substances Society, August 6, 1990,
Nagoya, Japan

led for N-containing compounds (Table 1), aromatic
(Table 2) and aliphatic compounds (Table 3) which result-
ed from analytical pyrolysis studies using mainly Py-
FIMS and Py-GC/MS [1]. More than 170 characteristic,
fundamental structures were identified and are listed in
Tables 1 to 3 and, considering homologues and isomers,
these might well represent > 1000 chemical compounds.
This gave the basis for the proposed carbon-carbon net-
work structure of humic substances which has been
visualized with its voids and flexible covalent links [2]
and clearly demonstrated the complexity and variability
of the model. The latter was particularly emphasized and
indicated by the % symbols which stand for a wide range
Table 1. Nitrogen-containing compounds characteristic for soil organic matter in agricultural and
forest soils (status by Py-FIMS and Py-GC/MS, August 6, 1990) [1]. Molecular weights are given
in brackets (m/z)
NH3 ( 17 ) @ CH 3 ~ 0 C--CH3 ~ N CH2-CH20H
HC_=N
1181 (93)
11351 1161)
(27) H3C~-- ~ CHS ~ / C----N
0 H
C~3NH2 ~S/ ~CH ~ 0CH3
(311 (95) 3
~-- CHB ~NH2
~,o %N 2--cH3 113s) OH
H--C~-NH 2 11631
H3C C~.NH 2 H 1951 C~2--CH20H 11711
1591
~ OH 1137) ~CO--CH 3
~ C2Hs ~N'-CH3
H (95) H
(67) H (1731
H N ~ CH3
(68) 2 H CH 3 (175)
u 0 ~C00H
(79)
(113)
--CN
OH
0%C/H
(149) ~/~N'~CH3
H
( 175 )
CH3 ~ CH3 CH2--0
(811 (1171
H N~ 3Hv
+=0
CH3 ~ CH3
• 181) (124) (185)
~OH ~ N N'\0CH3 C12H25-C-N
1152) (195)
H
(55) . k-.

64
Table 2. Aromatic compounds characteristic for soil organic matter (see Table 1) [1]
CH=CH 2
H3C0 OCH 3
(78) v\CH3 0H
1118) 1134)
(154)
O%C--H ~J_C~N CH3
H 3 OH
(94) ~ CN2-CH3 (CH2)4-CH3
(104)
CH3 Y "0CH3
H3 OH CH=CH-CHa
OCH 3
1120) 0CH 3
(106) CH 3
CH3_C=CH 2
H 3 CH2-CH3 (CH3)2 OCH 3
1106) OH 1164)
(144) OC--CH 3
0CH3 1146) OCH3
(106) OH OH
~OH ~CH3 (124) ~--~CH2)3-CH 3 ~(164)
1108) (130) 1148) 1166)
CH3 CH 3 - CH=CH2 ~3
OH OH
(116) y "OCH 3
(134) OH
ed as a first draft 2D structure [3]. It had the elemental
composition of C308H328090N 5 with a molecular mass of
5 540.027 g tool -1 and an elemental analysis of 66.8% C,
6.0% H, 26.0°70 O, 1.3% N. Compared to isolated stan-
dard HAs with about 56.3070 C and 32.8% O, this pro-
posed HA structure is obviously too high in carbon and
too low in oxygen. However, carbohydrates have been re-
ported to account for about 10% of the HA weight and
a similar value has been suggested for proteinaceous ma-
terials in HA [3]. Thus, it has been assumed that one HA
molecule traps within its voids approximately 10% carbo-
hydrates and 10% proteinaceous materials. The resulting
HA complex has then an elemental composition of
C342H3880124N12 with a molecular mass of 6650.912g
mo1-1 and an elemental analysis of 61.8% C, 5.9% H,
_CH3 (CHe)s-CH3
(152) 1176)
29.8% O, 2.5% N. If more carbohydrates and/or pro-
teinaceous materials are added, the C content decreases
and the O content increases.
Thus, for a stereochemical understanding of the
structure of humic substances and their reactions with
biological and anthropogenic compounds three main,
urgent questions emerged:
1. What is a fast, reliable and versatile method to convert
the 2D structure into a 3D structure?
2. Is it possible to optimize the geometry of the chemical
3 D structure and to determine and minimize its en-
ergy?
3. How can chemical reactions of such complex biomac-

Table 2 (continued)
o
CH2-CH2-C~H
0H
CH2-CH:-CHO
H3C0 OCH 3
OCH3 OH
(210)
CH=CH2 (180) 22--(CH2) s-CH3
H3CO~0CH 3 ~ (218)
OKcI{2__CH3(180) ~ ~CH2) 7-cn3
/ ~ 0H (220)
H3CO OCH 3
OH ~H2--( CH2 ) 9-CH3
1182)
©
(232)
CH2--( CH 2 ) io-CH3
OH OK (186) ~--~H3
--( CH 2 ) ~-CH3
(246 )
(1901 ~ -(CH2) II--CH3
H O ~ 12601
1194) ~C6H14
CH2-CHO
TC6HI4
H3C0 0CH 3 (264)
OH
(196) ~--(CH2) 12-CH3
.~--( CH2 ) 7 -CH3
(204)
(274)
¢
CH2--(CH2) 13-CH3
12881
-4CH 2114-CH3
(302)
-qCH 2 ) 15-CH3
1316)
-~CH 2 ) 16-CH3
(330)
romolecules be simulated at atomic and molecular
level?
Experimental
Pyrolysis-fieM ionization mass spectrometry (Py-FIMS)
For temperature-resolved Py-FIMS, about 100~tg of
humic substances such as humic acid (HA), fulvic acid
(FA), humin or 5 mg of whole soil samples, respectively,
were thermally degraded in the ion source of a MAT 731
(Finnigan, 28127 Bremen, Germany) modified high-per-
formance (AMD Intectra GmbH, 27243 Harpstedt, Ger-
many) mass spectrometer. The samples were weighed be-
Table 3. Aliphatic compounds characteristic for soil organic matter
(see Table i) [11
CH2=CHCHO
CH3-C0--CH 3
CH3-C00H
CHa--CO--CHO
CH2-C--CO--CH 3
CN3-CH--CH--CH--CH--CH 3
3 H C ~ CH3
65
(56)
(58)
(601
(72)
(84)
(96)
0
(i12)
Qo 1114)
3HC-~ OH
11281
0 0 0
II II II
CH3-C--CH2-C--CH2-C--CH 3
11421
~H3 ?H3 ~H3
CH3-CH--(CH2) 3--CH--(CH2) 3-CH--CH2=CH--CH2 (224)
CH CH 3 CH 3
3 i i
CH3--CH--~CH2)3--CH---~CH 2 3~CH~--~CH2 2-C~3
CH3--~ CH 2 )IT"CH20H
CH 3 CH 3 CH 3 CH 3
I I I
CH3-CK--~CH 2 3-CN--CH 2 3-CH--CN 2 3-C=CH2
n=2-30 series
CH3-~ CH 2 )~--CH3
n=i-28 series
CH2=CH--( CH 2 )~--CH 3
CH3--( CH 2 )~4 COOH
(226)
12281
(266)
(58-450)
(56-4341
(256)
14101
fore and after Py-FIMS (error + 0.01 mg) to determine the
pyrolysis residue and the produced volatile matter. The
heatable/coolable direct introduction system with elec-
tronic temperature-programming, adjusted at the + 8 kV
potential of the ion source and the field ionization emit-
ter, was used. The slotted cathode plate serving as count-
er electrode was on - 6 kV potential. Thus, at 2 mm dis-
tance between the emitter tips and the cathode, in total a
potential difference of 14 kV is applied resulting in an ex-
tremely high electric field strength which is the essential
basis for soft ionization. All samples were heated in high
vacuum (1.3 × 10 -4 Pa) from 323 K to 973 K at a heating
rate of approximately 0.5Ks -1. About 60magnetic

66
(CH3)o-3
(CHs)o-2
0 ~
@O ~
(CH3) 0-5 @
OH
H O~'.1
oH°'f -° oH°-.-r "°H ,o,
OH OH
scans were recorded for the mass range 16 to 1000
Daltons. In general, at least three replicates were per-
formed for each sample. The total ion intensities (TII) of
the single spectra were normalized to 1 mg sample weight,
averaged for replicate runs, and plotted versus the
pyrolysis temperature, resulting in Py-FIMS thermo-
grams. For the selection of biomarkers and quantitative
evaluations, in particular of whole soils and soil particle-
size fractions, detailed descriptions of the method have
been given recently [6-9].
HO
OH
Curie-point pyrolysis-gas chromatography~mass
spectrometry (Py-GC/MS)
The humic substances and soils were pyrolyzed in a type
0316 Curie-point pyrolyzer (Fischer, 53340 Meckenheim,
Germany). The samples were not pretreated except drying
and milling. The final pyrolysis temperatures (Tf) em-
ployed were 573 K, 773 K and 973 K, respectively. The to-
tal heating time (THT) was varied between 3 and 9.9 s.
Following split injection (split ratio 1:3; flow rate
1 ml 20 s -1) the pyrolysis products were separated on a
gas chromatograph (Varian 3700, 64289 Darmstadt, Ger-
many), equipped with a 30 m capillary column (DB5),
coated with 0.25 gm film thickness and an inner diameter
of 0.32 mm. The starting temperature for the gas chro-
matographic temperature program was 313 K, and the
end temperature was 523 K, with a heating rate of
10 K min -1. The gas chromatograph was connected to a
nitrogen-selective, thermosensitive detector (TSD) and a
double-focusing Finnigan MAT 212 mass spectrometer.
Conditions for mass spectrometric detection in the elec-
tron ionization mode were +3 kV accelerating voltage,
70eV electron energy, 2.2kV multiplier voltage, 1.1 s/
OH
HO
HO
(CH3)0-2
/.(N ~.,.,2 (CH3)0-2
H
C=N
OH
~0/ ~C_. ?
O
HO =N
[~OH /CH2OH Fig. 2. Drawing of the proposed
humic acid structure using Indian
ink and a chemical stencil as published
in Ref. [3]
mass decade scan speed and a recorded mass range be-
tween m/z 50 and m/z 500. A detailed description of the
principle, potential and limitations of Py-GC/MS of
humic fractions and soils has been given [10]. Further-
more, studies of organic nitrogen-containing compounds
in soils using the TSD detector were reported [11] which
expanded the knowledge on the structure of 'unknown'
soil nitrogen beyond the status reported in Tables 1- 3.
Structural modelling and geometry optimization
For all described 2D- and 3 D-work, model construction,
chemical interaction studies and semi-empirical calcula-
tions, the HyperChem ® software (release 2 for windows)
[12] was used which is assisted by a comprehensive manu-
al. In the present text some main software commands are
indicated in brackets (in italics). The program is supplied
by Computer Aided Animation (CAA) GmbH, Kenner-
weg 15, 72622 Ntirtingen, Germany.
The employed PC consisted of the Escom local-bus
tower 486DX2/66, VLB 34 in combination with 8 MB
memory, Escom 17" colour monitor with AVGA VL
bus/1MB graphic card, Samsung 250 MB disk, 250 MB
powerstreamer, and peripheric hardware plus utility pro-
grams (Escom office, Rheinstr. 41, D-65185 Wiesbaden,
Germany).
Results and discussion
Building molecules
Building a 3 D model of the 2D humic acid structure
(Fig. 2) is performed in five steps using the HyperChem ®
software.

First, a preliminary 2 D structure of the C-C skeleton
is drawn by hand into the workspace using the drawing
tool and is far from perfect as shown in Fig. 3. In addi-
tion to carbon, the elements oxygen and nitrogen are in-
cluded (Default Element). In this manner 308 carbon-,
90 oxygen- and 5 nitrogen-atoms (403 atoms in total) are
positioned.
Second, hydrogen is attached with accurate bond
lengths and angles to the preliminary structure using the
software (Add Hydrogen) as shown in Fig. 4. Whereas in
the original publication the 2 D HA subunit structure [2]
was incomplete and was supposed to have a wide variety
of different bonding sequences (see :k-- symbols), the mol-
ecule in Fig. 4 represents a defined, intact HA monomer
(738 atoms). Thus, in order to obtain a complete chemi-
cal compound, it had to be supplemented by 7 hydrogen
atoms. The corresponding elemental composition then is
C308H335090N 5 with a molecular mass of 5547.004g
mol -~ and an elemental analysis of 66.69% C, 6.09% H,
25.96% O, and 1.26% N.
Third, building the 3 D structure of the selected HA
molecule (Model BuiM) with accurate bond distances
and bond angles, torsion angles, van der Waals forces,
and hydrogen bonds, is illustrated in Fig. 5. This prelimi-
nary, energetically still unoptimized structure shows
clearly the atomic distances and angles (Sticks) and gives
a first three-dimensional structure information. Even this
rough display indicates already the importance of
aliphatic chains as link between the aromatic moieties
and prerequisite for the formation of gaps and voids in
the HA structure.
Fourth, the (Sticks) structure in Fig. 5 is converted in-
to the 3 D structure (Disks, Perspective) shown in Fig. 6
which better reflects the space requirements of the 738 at-
oms. The white disks are hydrogen atoms, black disks ni-
trogen, the brighter shaded disks are carbons and the
darker ones oxygen. It is evident that even more complex
structures such as naturally occurring organomineral
complexes or trapping of biological and anthropogenic
substances in SOM will be difficult to evaluate in these
Fig. 3. Handdrawn two-dimensional version (2D) of the humic acid
draft structure using the drawing tool in the HyperChem ® workspace
-2
Fig. 4. Hydrogen attachment to the handdrawn humic acid draft struc-
ture performed by HyperChem ® (Add Hydrogen)
black and white plots. As will be illustrated in the follow-
ing fifth step, colourplots can be of great help and allow
a distinct view and facilitated evaluation of the complex,
large macromolecules.
Chemical calculations
After building the molecule from scratch (Fig. 3), the
atomic coordinates in the software files allow a wide
range of quantitative evaluations. Measuring structural
properties includes the determination of bond distances,
bond angles, torsion angles, non-bonded distances, and
hydrogen bonds. For minimizing the total energy of the
molecular system and thus increasing the stability of its
conformation, step-by-step instructions for performing
single point calculations, geometry optimization, and
molecular dynamics are given by HyperChem ®.
When one cycle of a single-point calculation of the
HA 3 D structure in Fig. 6 was performed using the algo-
rithm of Polak-Ribiere (conjugate gradient), the total en-
Fig. 5. Three-dimensional structure (3D) of the draft HA structure
shown in Fig. 4 using HyperChem ® for molecular modelling (Build
Model). Display of the structure in the sticks mode (Rendering; Sticks)
67

68
ergy of the molecular system was calculated with
6993.92 kJ 0.1 nm -1 mo1-1 and a gradient (derivative of
the energy with respect to all Cartesian coordinates) of
91.16 kJ 0.1 nm -1 mol -I. The termination conditions
for this calculation were 83.80 kJ nm- 1 mol - 1 and 11070
cycles maximum. After approximately 4700 calculation
Fig. 6. First approximation (1 cycle, 3 points, convergence limit of
83.80 kJ 0.1 nm -1 mol ~) for the geometrical optimization of the HA
structure (II) given in Fig. 5 (Compute; Single Point; Geometry Optimi-
zation) by semi-empirical calculations. Display of the structure in the
disks mode (Rendering; Disks)
~ J
Fig. 7. Geometry optimization
and energy minimization of the
3 D structure of HA (II, Fig. 6)
after 4700 calculation cycles and
a resulting total energy of
710.70 kJ nm -1 mo1-1 and a
convergence gradient of
0.037 kJ nm- 1 mol- 1
cycles with a convergence limit of 0.83 kJ 0.1 nm -1
tool-l, the energy minimization leads to a molecular en-
ergy of 752.57kJ 0.1 nm -1 tool -1 with a gradient of
0.71 kJ 0.1 nm -1 tool -1. As an illustration of the energy
minimization process in Fig. 7 the corresponding HA
structure is displayed. Rotation of the energy-minimized

HA 3 D version demonstrates the flexible network with
voids and hollows which offer binding and trapping of
biological and anthropogenic molecules [2]. Further-
more, long-continued calculation periods (> 6 days) give
energies in the order of 608 kJ 0.1 nm-1 mol-1 and gra-
dients

70
Fig. 9. Result of atrazine transport into the 3 D structural model of an organo-mineral complex in soil with atrazine trapped in the voids (V)
the atrazine molecule moves with decreasing energy and
increasing conformational stability step-by-step more
into the available space of the void. Finally, arriving with
the geometry optimization and energy minimization
close to a total energy of 10008.21 kJ nm -1 mo1-1 and a
gradient of 0.37 kJ nm-1 mol-t, the atrazine molecule is
immobilized in the void by formation of a hydrogen bond
from H (21, molecule I) to a HA carboxyl group of II (O
(606, molecule II)) which is an integral part of the
organo-mineral particle V now. Figure 10c shows the
space requirements of the atrazine molecule in the hollow
space, but also, may be more important, how much space
is left for chemical decomposition reactions.
Nanochemistry
For the biological and chemical reactivity of the trapped
anthropogenic compound it is highly interesting to deter-
mine exactly the distances between I and the surrounding
molecular complex of the HA II and the silica matrix IH.
As derived from Fig. 10a, the numbered atoms and
Fig. 10b the atoms labelled by element symbols, some

~749 ~54
Fig. 10a-e. The enlarged section of a void in an organo-mineral com-
plex (V; geometry and energy optimization: bonds and angles, torsions,
van de Waals forces, hydrogen bridges) is shown in which atrazine is
trapped and fixed via a distinct hydrogen bond and SOM is bound to
the mineral matrix via hydrogen bonding in addition to three covalent
bonds, a Section (Sticks, Atoms, Number) is shown with the displayed
selected distances between atrazine I and II and III are as
follows:
Starting from C1 (i, I) (atom 1, molecule I) the dis-
tances to H (256, III) are 0.7947 nm (HyperChem ® out-
put 7.947477 Angstrom), to H (255, III) 1.0466 nm, to O
(225, III) 0.3737 nm, O (187, III) 0.3431 nm, O (100, III)
0.8717 nm and to the hydrogens of the long aliphatic
chain (see lower part of V in Fig. 9) H (1004, II)
0.6051 nm and H (1011, II) 0.8632 nm. From H (3, I) to
H (950, II) a distance of 0.3074 nm, to H (996, II) of
0.2160 nm and to H (1004, II) of 0.4177 nm was measured
for the lower part of the void. In the upper part, for in-
stance between H (17, I) and H (902, II) 0.3074 nm, H
(749, II) 0.4468 nm, H (745, II) 0.6537 nm, C (400, II)
0.7601 rim, O (577, II) 0.4885 nm were obtained. Despite
the apparent proximity between H (19, I) and H (251, III)
a distance of 1.1614 nm was found and clearly demon-
strated the necessity of exactly calculated coordinate
systems. Moreover, the distance between H (21, I) and
O (606, II) was of interest, because it narrowed with
progressing geometry optimization from >1rim to
0.2221 nm generating a hydrogen bond which immobi-
lized the atrazine molecule in the HA void. The more the
total energy content of the organo-mineral complex V is
/
1
6
4
6~4 "~
71
atoms labelled by numbers; b Section (Sticks, Atoms, Symbols) is
shown with atoms labelled by element symbols; c Section (Rendering;
Disks, Perspective) is shown as colour plot illustrating the space re-
quirements of the pesticide and the available three-dimensional trap-
ping space
lowered, the more shrinking of the hollow space and con-
sequently stronger bonding of the atrazine is observed. In
summary, one can assume that without operator interfer-
ence energy minimization simulates aging processes.
The results of the three-dimensional structure and dis-
tance measurements furthermore allow to evaluate the
space between the trapped molecule and its host, the
organo-mineral complex. In particular the space filling of
trapped biological or anthropogenic substances is sup-
posed to have an essential influence on their presumable
fate in SOM. Clearly air (N2, O2) and water (HzO) and
small organic acids (CH3COOH) can invade the gaps be-
tween the pesticide and organic or inorganic soil surfaces.
Thus, non-metabolic decomposition processes are easily
possible at this stage. Metabolic processes, however, can
be excluded as microorganisms such as bacteria (> 200 to
1000 nm) and fungi (> 10000 nm) have no access into the
described voids and even enzymes are too large to react
with the trapped materials when the front and back side
of the void is closed by recalcitrant SOM or inorganic
substances. The latter is much more likely, as the organic
matter in agricultural soils is only around 3°70. Thus, in
nature the inorganic matrix represented here by the
hypothetic model III should be larger by at least a factor
of 30.

72
Fig. 10b
\
Fig. 10c

It is expected that computational chemistry in combi-
nation with analytical pyrolysis (and analytical chemistry
in general) will play an important role in future studies
not only in environmental studies [14] and ecology [15]
but also in soil science [16] and agriculture as described
recently [13, 17].
Conclusions
(1) The molecular-chemical structure of soil organic mat-
ter is mainly determined by the inorganic soil matrix, the
climate, plant precursors, and the dynamics and specific
activity of biomass.
(2) The resulting three-dimensional, organo-mineral
complexes of decomposition products, biologically syn-
thesized materials and soil mineral particles can be ana-
lyzed indirectly by Py-GC/MS and directly by in-source
Py-MS.
(3) Analytical pyrolysis of humic substances and
whole soils yields a vast variety of pyrolysis products.
Their intra- and intermolecular chemical properties are
only understood when fast, reliable and powerful com-
puter-assisted data handling and evaluating allows the
chemistry to evolve in the three dimensions of space un-
der the precise structural parameters such bond lengths,
angles, and torsions, van der Waals forces, hydrogen
bridges, etc.
(4) To reassemble the identified chemical subunits to
a hypothetic structure of soil organic matter that reflects
as closely as possible the genuine status, above all con-
structive interdisciplinary work and collaboration is
needed.
Acknowledgements. The author is grateful for the financial support by
the Deutsche Forschungsgemeinschaft (projects Schu 416/3; Schu 416/
18-1) and the Ministry of Science and Technology, Bonn-Bad Godes-
berg, and the Umweltbundesamt, Berlin, Germany. Thanks are due to
R. Hempfling, R. Mt~ller, C. Sorge and H. Wilken (Fresenius Group)
and P. Leinweber, University of Osnabrack for their help and coopera-
73
tion. To work with the "pioneer of humic substances" Dr. Morris
Schnitzer, Agriculture Canada, Ottawa, has been and is a unique occa-
sion which gives its merits and rewards by itself.
References
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Substances Society, August 6-10, Nagoya, Japan
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78:311
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