SOIL

ORGANIC
MATTER
PROCEEDINGS OF A
SYMPOSIUM
BRAUNSCHWEIG, 6-10 SEPTEMBER 1976
JOINTLY ORGANIZED BY THE
IAEA AND FAO
IN CO-OPERATION WITH AGROCHIMICA

ENERGY A G E N C Y , V IE N N A , 1 9 7 7
SOIL ORGANIC MATTER STUDIES

VOL. II
 PROCEEDINGS SERIES

SOIL ORGANIC MATTER STUDIES

PROCEEDINGS OF A SYMPOSIUM
ON SOIL ORGANIC MATTER STUDIES
JOINTLY ORGANIZED BY
THE INTERNATIONAL ATOMIC ENERGY AGENCY
AND
THE FOOD AND AGRICULTURE ORGANIZATION
OF THE UNITED NATIONS
IN CO-OPERATION WITH AGROCHIMICA
AND HELD IN BRAUNSCHWEIG, 6 -1 0 SEPTEMBER 1976

In tw o volum es

VOL.II

INTERNATIONAL ATOMIC ENERGY AGENCY

VIENNA, 1977
SOIL ORGANIC MATTER STUDIES
IAEA, VIENNA, 1977
STI/PUB/438
ISBN 92-0-010177-1

Printed by the IAEA in Austria

April 1977
 FOREWORD

The nature, content and behaviour of the organic matter, or humus, in soil are factors of
fundamental importance for soil productivity and the development of optimum conditions for
growth of crops under diverse temperate, tropical and arid climatic conditions. Unfortunately,
the study of soil organic matter presents some of the most complex problems with which the
soil specialist and his collaborators in soil biochemistry and soil microbiology have to deal. How­
ever, through the co-operation of scientists of various disciplines and the use of tracer and other
modem techniques in research, valuable contributions have been and are being made to a better
understanding of the behaviour and functions of organic matter in soil; this will, in turn, lead to
the increase in crop production that is sorely needed.
The Food and Agriculture Organization of the United Nations and the International Atomic
Energy Agency had jointly convened two international meetings, the first in 1963 and the Second
in 1968, in co-operation with the International Soil Science Society, to review the progress that
had been achieved through the use of tracer techniques in studies of soil organic matter, and the
ways in which these methods could contribute to the growth of better crops. In the present,
third symposium, as in the two preceding ones, due consideration was given to studies involving
the use of radioactive and stable isotopes, but this third symposium departed from previous
practice in that it was organized in co-operation with AGROCHIMICA and that non-isotopic
approaches to research on soil organic matter were included. The meeting was the first symposium
sponsored by the IAEA and the FAO to deal with the subject matter without any restriction
regarding specific techniques.
The symposium, held at Braunschweig from 6 to 10 September 1976, was attended by 146
participants from 33 countries and two international organizations. A total of 77 papers were
presented and discussed during nine sessions. To make this possible within the limited time of the
meeting some of the papers were presented by rapporteurs. A number of papers, dealing with the
behaviour and functions of organic matter, make a contribution to increasing agricultural pro­
duction by proposing improved management practices. Other papers discussed turnover o f plant
residues, release of plant nutrients through the biodegradation of organic compounds, and nitrogen
economy and the dynamics of transformation of organic forms of nitrogen. Among other topics
raised and discussed were: the biochemical transformation of organic matter, the characterization
of humic acids, carbon dating and the impact of modern techniques on soil organic matter
research.
It is hoped that these Proceedings, which include the papers presented and the discussions,
will lead to further knowledge of the ways in which soil organic matter, with appropriate manage­
ment practices, contributes to increased production of crops for food and industrial purposes.
The organizers of the symposium wish to express their gratitude to the Federal Ministry of
Research and Technology, the Federal Ministry of Food, Agriculture and Forestry, and the
Forschungsanstalt für Landwirtschaft. The staff of the latter organization’s Institut für Bio­
chemie des Bodens in Braunschweig contributed greatly to the success of the symposium.
 E D IT O R IA L N O T E

The papers an d discussions have been e d ite d b y th e editorial s ta f f o f th e In tern ational

A to m ic Energy A g en cy to th e e x te n t con sidered necessary f o r th e rea d er’s assistance. The view s
expressed an d th e general s ty le a d o p te d rem ain, h ow ever, th e respon sibility o f th e n am ed authors
o r participan ts. In a ddition , th e view s are n o t necessarily th ose o f th e g overn m en ts o f the
n om in atin g M em ber S ta tes or o f th e n om in atin g organizations.
Where papers have been in corporated in to these P roceedings w ith o u t resettin g b y th e A gency,
this has been d o n e w ith th e kn ow ledge o f th e au th ors and th eir govern m en t au th orities, an d their
co o p era tio n is gratefu lly ackn ow ledged. The Proceedings have been p rin te d b y com p o sitio n
typ in g a n d p h o to -o ffse t lith ograph y. Within th e lim itation s im posed b y this m eth od, every effo rt
has been m ade to m aintain a high editorial standard, in particular to achieve, w h erever practicable,
co n sisten cy o f u n its an d sy m b o ls an d c o n fo rm ity to th e standards re co m m en d ed b y c o m p e te n t
in tern ation al bodies.
The use in th ese Proceedings o f particu lar designations o f cou n tries o r territories does n o t
im p ly an y ju d g em en t b y th e publisher, th e IA E A , as to th e legal statu s o f such cou n tries or
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The m en tio n o f sp ecific com pan ies o r o f th eir p ro d u c ts or brand nam es does n o t im p ly an y
en d o rsem en t o r recom m en dation on th e p a rt o f th e IAEA.
A u th o rs are th em selves respon sible f o r obtain ing th e necessary perm ission to reprodu ce
co p yrig h t m aterial fro m o th e r sources.
 CONTENTS OF VOL. II

BIOCHEMICAL TRANSFORMATION OF ORGANIC MATTER (Session 6)

Factors affecting the biostability of metabolic materials in soil (IAEA-SM-211 /20)............ 3

L.H. Sorensen
Discussion.............................................................................................................................. 14
Stability constants of some complexes of Argentine humic acids and
micronutrients (IAEA-SM-211/51)..................................................................................... 15
R .A . R esell, A.M . Miglierina, L.Q. d e N ovilla
Discussion............................................................................................................................ 21
Decomposition in soil of specifically carbon-14-labelled DHP and corn stalk lignins,
model humic acid-type polymers and coniferyl alcohols (IAEA-SM-211/4).................... 23
J.P. M artin, K. H aider
Discussion.............................................................................................................................. 32
Transformation d’acides phenoliques simples en substances para-humiques par des
micro-organismes du sol (IAEA-SM-211 /41) ...................................................................... 33
J.-R. Bailly, V. N kundikije-D esseaux, K. A g b ek o
Discussion.............................................................................................................................. 41
Caracterisation et transformations en milieu mull d’un modele humique issu de
l’autoxydation du Systeme catechol-glycine et marque selectivement au carbone-14
(IAEA-SM-211/42)............................................................................................................... 43
F. A n dreu x, D o ro ta G olebiow ska, T herese Chone, F. Jacquin, M. M etch e
Discussion.............................................................................................................................. 57
Effect of nitrogen on the formation of pyrocatechin-humic acid and the nitrogen
linkage characteristic of this acid (IAEA-SM-211/4 3 )........................................................ 59
H. Ö zb ek
Chemical alterations of natural lignin by interactions with humic-like autoxidation
products of pyrogallol(1,2, 3-trihydroxybenzene)(IAEA-SM-211/44)............................ 67
T. W eichelt
Discussion ............................................................................................................................. 83
Possibilities of an economic and non-polluting utilization of lignin
(IAEA-SM-211/45)............................................................................................................... 85
W.H.M. Schweers, W. Vorher
Discussion.............................................................................................................................. 89
Oxidation of some phenolic substances as influenced by clay minerals
(IAEA-SM-211/46).................................................................................................................. 91
Z. Filip, W. Flaig, E. R ie tz

BITUMENS IN SOIL ORGANIC MATTER (Session 7a)

Lipids of microbial origin in soilorganic matter (IAEA-SM-211/4 7 ) .................................... 99

G. H. Wagner, E.I. M u zorew a
Analyse et röle des bitumes dans les sols sableux acides (IAEA-SM-211 /48)....................... 105
E. F ustec-M athon, P. Jam bu, G. Joly, R. Jacqu esy
Discussion................................................................................................................................ 114
CHARACTERIZATION OF HUMIC ACIDS (Session 7b)

Review paper:

Recent findings on the characterization of humic substances extracted from soils from
widely differing climaticzones (IAEA-SM-211/7)............................................................... 117
M. S ch n itzer
Discussion................................................................................. 131
Structure et genese des acides fulviques des sols des Landes du Medoc (France)
(IAEA-SM-211/3 9 )............................................................................................................... 133
D. R ight, P. Jam bu, T. D u pu is
Discussion.............................................................................................................................. 141
Aggregation-dispersion phenomena in humicsubstances (IAEA-SM-21T/72) ....................... 143
N. Senesi, Y. Chen, M. S ch n itzer
Structure chimique des acides humiques et fulviques du sol (IAEA-SM-211 /73)................. 157
J.-A. N eyro u d , M. S ch n itzer
Applications of computer techniques in humus research (IAEA-SM-211/49)...................... 171
B.R. Nagar

CARBON DATING (Session 7c)

Mesures d’activite specifique de fractions de matiere organique appliquees ä l’etude

de revolution des sols de Guyane (IAEA-SM-211 /69)....................................................... 179
J. L. Rapaire, J.F. Turenne
Datations par le carbone-14 naturel de la matiere organique d’horizons spodiques de
podzols des Landes du Medoc (France) (IAEA-SM-211 /7 0 )............................................. 187
D. Right, B. G uillet
The search for biologically inert and lithogenic carbon in recent soil organic matter
(IAEA-SM-211/71).............................................................................................................. 193
H.W. Scharpenseel
Discussion.................................................................................................................................. 201

MODERN TECHNIQUES AND TH EIR IMPACT ON SOIL ORGANIC M ATTER

RESEARCH (Session 8a)

Review paper:

Advantages and limitations of mass- and photo-spectrometry in nitrogen-15-aided

studies (IAEA-SM-211 /8 ).................................................................................................... 205
V. M id d elb o e
Discussion ............................................................................................................................. 210
Studies on soil humic compounds, fungal melanins and model polymers by pyrolysis
mass-spectrometry (IAEA-SM-211/53)............................................................................... 213
K. H aider, B.R. Nagar, C. Saiz, H .L.C. Meuzelaar, J.P. M artin
Discussion.............................................................................................................................. 219
Chromatography of humic substances on controlled pore glass (IAEA-SM-211 /54)............ 221
O. H. D anneberg
Discussion.............................................................................................................................. 228
Review paper:

Role of organic matter in volatilization of sulphur and nitrogen in soils

(IAEA-SM-211/9 )................................................................................................................ 229
J.M. B rem ner
Discussion ............................................................................................................................. 240
Application of high-pressure liquid chromatography to studies of extractable soil
organic matter:Porous silica packings (IAEA-SM-211/5 5 ) ................................................. 241
R.H . L o ep p ert, B.G. V olk
Discussion ............................................................................................................................. 249

SLUDGE (Session 8b)

Municipal sludge as organic fertilizer with special reference to the heavy metals
constituents (IAEA-SM-211/74)......................................................................................... 253
N. el-Bassam, C. Tietjen
Epandage de boues residuaires: repercussions sur l’azote du sol et le prelevement
par du ray-grass de Cd, Cr, Hg et Zn (IAEA-SM-211/7 5 ) .................................................. 259
J. C. Fardeau, G. Guiraud, J o celyn e Jappe, Gisele L lim ous
Determination de l’aptitude ä la biodegradation des boues residuaires d’origines
diverses —Action sur les proprietes physico-chimiques du sol (IAEA-SM-211/76).......... 265
J.L. M orel, F. Jacquin
Evaluation of municipal refuse from Dahomey (Benin) as an organic manure
(IAEA-SM-211/77)......................................................... 277
F.X. K o m a A lim u , I E . S oe A gnie, B.H. Janssen
Discussion.................................................................................................................................. 289
Variation in content of polycyclic aromatic hydrocarbons in soil and plants by using
municipal waste composts in agriculture (IAEA-SM-211/31) ........................................... 291
P.-Chr. E llw ardt

SOIL ORGANIC MATTER COMPONENTS AND PLANT METABOLISM

(Session 9a)

Metabolism of soil-related phenolic compounds in plants and cell suspension cultures

(IAEA-SM-211/56)............................................................................................................... 301
H. Harms
Estudio de la action ejercida sobre la planta de maiz por dos tipos de äcido humico
(IAEA-SM-211/57)............................................................................................................... 307
V. H ernando, В. C. Ortega, C. F ortun
Discussion.............................................................................................................................. 317
Effect of organic matter and salts on the activity of some soil enzymes
(IAEA-SM-211/58)............................................................................................................... 319
AS. A bdel-G haffar, M .H .A. el-Shakw eer, M .A. Barakat
Discussion.............................................................................................................................. 324
INTERACTION BETWEEN AGROCHEMICALS AND ORGANIC MATTER
(Session 9b)

Blocage de molecules s-triaziniques par la matiere organique (IAEA-SM-211/7 8 )............... 327

M. Schiavon, F. Jacquin, C. Goussault
Bound or adsorbed methabenzthiazuron residues in soil (IAEA-SM-211/7 9 )...................... 333
F. Führ, W. M ittelsta ed t, K. H aider
Discussion ................................................................................................................................ 339

PEAT (Session 9c)

Composition and content of amino acids in peat-forming plants and in peats

(IAEA-SM-211/81)............................................................................................................... 343
F. Maciak, H. Süchtig, W. Flaig
Stabilization of organic matter in sand mixed cultures (IAEA-SM-211/82)......................... 359
B. S ch effer
Organic nitrogen content of peat soils combined with different fractions of humic
substances (IAEA-SM-211/83)............................................................................................ 365
W. R ochu s

List of Chairmen of Sessions and Secretariat......................................................................... 371

List of participants.................................................................................................................... 373
Author ind ex ............................................................................................................................ 387
B IO C H E M IC A L T R A N S F O R M A T IO N
O F O R G A N IC M A T T E R
( S e s s io n 6 )
 IAEA-SM-211/20

FACTORS AFFECTING THE BIOSTABILITY

OF METABOLIC MATERIALS IN SOIL
L.H. S0RENSEN
Agricultural Research Department,
Research Establishment Risф,
Roskilde,
Denmark

Abstract

FACTORS A FFECTING THE BIOSTABILITY OF METABOLIC M ATERIALS IN SOIL.

A loam and a sandy soil, incubated w ith 14C-labelled straw for 3 and 12 years respectively, were used for
an investigation o f th e effec ts o f th e addition o f energy material (unlabelled glu cose) and air-drying and grinding
on th e ev olution o f labelled and native C 0 2-C, and on the biom ass. The flush o f CO 2 caused by fum igation with
chloroform was taken as a measure o f the biom ass. The addition o f 5 0 and 2 0 0 mg glucose-С /100 g soil respectively,
resulted in an evolution o f labelled C 0 2-C that was 1.6 and 3 .2 tim es the evolution from unam ended soil ( ‘prim ing’).
The priming was largest after th e sm all addition o f glucose-C when calculated per unit o f glucose-C added. One
addition o f glucose resulted in a decrease in the labelled biom ass in com parison w ith unam ended soil, whereas
after repeated additions it was approxim ately o f th e same size as in th e unam ended soil. Air-drying and grinding
caused an evolution o f labelled C 0 2-C that ranged from 1.2 to 5.2 tim es the evolution from untreated soil.
Grinding had the strongest effect. Air-drying had a m inor influence on the biom ass, whereas grinding had a'
strong influence. The results suggest th at th e increase in evolution o f C 0 2-C caused b y the treatm ents originated
partly from killed biom ass and partly from non-biom ass material. Air-drying and grinding influenced the
evolution o f native CO--C, w hich, on th e w h ole, was parallel to the in flu en ce on the labelled C 0 2-C.

INTRODUCTION

Metabolites, which are synthesized in soils during decomposition of labelled cellulose and
other decomposable materials added to the soils, decay slowly [1, 2]. Conditions such as irregular
chemical structure and interaction with other soil constituents might be the cause of this biostability.
The rate of decomposition of soil organic matter can be increased by certain treatments as,
for example, addition of fresh decomposable materials, drying of the soil, and disruption of the
soil structure [3—5].
The investigation described here is of a preliminary nature; it was performed with the aim
of exploring to what extent the rate of decay of a fraction of labelled materials in soils was
influenced by the above-mentioned treatments, and to what extent the biomass in the soils was
influenced. The flush of C02 caused by fumigation with the vapour of CHC13 was taken as a
measure of the biomass according to the method introduced by Jenkinson [6]. A series of five
papers was recently published by Jenkinson and collaborators [7—11 ]. Evidence is presented in
these papers that the flush of C02 caused by fumigation with CHC13 can be used as a measure of
the soil biomass. It was thus shown that there was a close agreement between biomass-C measured
by fumigation and soil biovolume measured by optical microscopy [9].
A loam and a sandy soil were used for the present investigation, each containing a fraction
of 14C-labelled materials originating from 14C-labelled straw incubated in the soils for 3 and 12 years
respectively.

3
4 S0RENSEN

MATERIALS AND METHODS

Sandy soil: pH 5.8; organic C, 1.9%; N,0.18%; particles <0.02 mm, 15%.
Loam soil: pH 8.1; organic C, 1.0%; N,0.11%; particles <0.02 mm, 32%.
The sandy soil was incubated in the field for 12 years after the addition of 14CTabelled
straw; this was added in 1964 in an amount corresponding to 350 mg C/100g soil [12]. Three
lots of the soil were used for the present investigation: lots 1 and 2 were removed from the
field plot after 8 years, lot 1 was incubated in the laboratory for 4 years moistened to 60% of
the water-holding capacity; lot 2 was air-dried and stored in this condition for 3 years when it
was remoistened; lot 3 was removed from the field plot in April 1976. At the beginning of the
experiments described here lots 1—3 contained 53, 55 and 50 mg labelled C/100 g soil respectively.
About 21% of the labelled C in the soil was in the form of amino acids.
The loam soil was incubated in the laboratory for 3 years after the addition of the labelled
straw, which was added in amounts corresponding to 880 mg C/100 g soil. At the beginning of
the present experiment the soil contained 223 mg labelled C/100 g soil. About 17% of the labelled
C was in the form of amino acids.
Analytical methods and the preparation of the labelled straw have been described in previous
publications [2].

Treatments

Air-drying was performed by placing thin layers of the moist soil on aluminium foil at about
25°C in a slow air stream for 24 h. Oven-drying was performed by placing the air-dried soil in an
oven at 80°C for 24 h. The dried soils were remoistened by adding the required amount of water
followed by gentle stirring with a spatula. Gradual moistening was carried out by placing the air-
dried sample over a layer of water in a closed jar for 16 h; the weight of the soil increased 2%
during this time. The soil sample was then placed on moistened filter paper for 6 h; during this
time the soil absorbed half the required amount of water, the remainder being added as described
above.
Grinding of air-dried soil was performed by means of an electrically powered mortar and
pestle until 95% of the material was < 0.125 mm.
Unlabelled glucose was added to soil samples dissolved in 3 ml of water. The moist soil
samples were left in open jars in the air until they had lost about 3 g in weight, the glucose was
then added and the sample was gently stirred.

Incubation and C02 determination

Samples corresponding to 40—50 g oven-dried soil were placed in wide-mouth 100-ml

Erlenmeyer flasks. Water was added in amounts corresponding to 60% of the water-holding
capacity. To the ground samples was added an amount of water corresponding to 60% of the
water-holding capacity of the same weight of normal soil. The untreated control samples consisted
of soil which had been incubated moist for at least 3 months before the beginning of the present
experiment. The soil was weighed out moist, and dry matter determinations were performed
at the same time.
The flasks were fitted to an aeration system yielding a slow stream of moist C02-free air.
The C02 evolved from the samples was trapped in absorption towers containing 0.2N KOH and
determined by titration. Radioactivity was determined by scintillation counting. The incubation
temperature was 20°C. The flasks were weighed every month and evaporated water was restored;
the loss in weight rarely exceeded 1 g. For further details, see Ref. [12]. All determinations were
performed in duplicate. All results are expressed on an oven-dried soil basis (105°C for 24 h) unless
otherwise stated.
 IAEA-SM-2II/20 5

Biomass determination

At the end of the incubation period that followed the treatments, the flasks containing the
soil samples were removed from the aeration system and placed in a desiccator containing a;beaker
with ethanol-free chloroform. The desiccator was evacuated until the chloroform began to boil,
and then it was closed and left in the dark for 40 h. When opened, the beaker was removed from
the desiccator, which was then evacuated and opened several times to remove traces of chloroform.
The fumigated soil samples were inoculated by the addition of 1 ml of a soil suspension prepared
by shaking 1 g of fresh unlabelled soil with 100 ml of water. The flasks were then connected to
the aeration system, and the amount of C02-C evolved during 10 days of incubation was determined.
The increase in C02-C evolution caused by the fumigation was taken as the amount calculated by
deducting the amount of C02-C evolved from the sample during 10 days of incubation preceding
the fumigation (these values are not shown in the tables) from the value observed after fumigation.
Or it was taken as the difference between the amount evolved from the fumigated soil and the
amount evolved from non-fumigated soil during the second incubation period ranging from the
10th to the 20th day. The first-mentioned procedure was used only when the samples had been
incubated uninterruptedly for 2 months or more. Jenkinson [10] reported that on an average 50%
of the carbon in killed biomass was mineralized to C02 by the 10th day of incubation after
fumigation; this value was used for the calculation of biomass-C in the present investigation.

RESULTS AND DISCUSSION

Rate of decomposition and biomass in untreated soil

The untreated samples of the sandy soil evolved during 285 days of incubation 1.057 mg
labelled C02-C and 37.69 mg native CO2-C/100 g soil (Tables I and II, Fig.l). This represented

TABLE I. EVOLUTION OF LABELLED C02-C FROM THE SANDY SOIL (Lot 1) AFTER
ADDITION OF UNLABELLED GLUCOSE, AND AFTER FUMIGATION WITH CHC13
F um igation was p e rfo rm e d a fter in cu bation f o r 2 8 5 days; biom ass-C was calcu lated fro m these
values

Labelled

COj-C evolved after

T reatm ent3
Biomass-C
Treatment Fum igation
(2 8 5 days) (1 0 days)

m g /100 g %b m g /100 g m g /1 0 0 g %c

N one 1.057 2.0 0 .3 1 2 0 .5 4 2 1.0

135 mg non-labelled
glucose-C added 1.651 3.1 0 .2 8 3 0 .4 7 2 0.9
6 7 5 mg non-labelled
glucose-C added 3.3 5 5 6.3 0 .4 4 6 0 .6 4 8 1.3

a T he glucose was added in 3 portions each corresponding to 45 and 225 mg g lu cose-C /100 g soil respectively;
th e first p ortion was added at th e beginning, th e second and third after incubation periods o f 9 0 and 180 days
respectively.
b Percentage o f labelled C in soil at th e start.
c Percentage o f labelled C remaining in soil after treatm ent and incubation.
6 S0RENSEN

TABLE II. EVOLUTION OF NATIVE AND NON-LABELLED C02-C AFTER REPEATED

ADDITIONS OF NON-LABELLED GLUCOSE TO THE SANDY SOIL (Lot 1)
For fu r th e r explan ation s see Table 1

N ative and non-labelled

C 0 2-C evolved after

Treatment
Biomass-C
Treatment Fum igation
(2 8 5 days) (1 0 days)

m g /1 0 0 g %a m g /100 g m g /1 0 0 g % b

N one 3 7 .69 2.1 2 .98 5 .0 2 2 0.3

135 mg non-labelled
glucose-C added 1 4 0.54 76.2 8.55 1 4 .4 2 0 29.2
675 mg non-labelled
glucose-C added 509.87 7 0 .0 2 1 .0 9 3 0 .0 9 0 12.4

a Percentage o f native C in soil at the beginning, or o f non-labelled glucose-C added (see tex t).
^ Percentage o f native soil-C or non-labelled glucose-C remaining in soil after treatm ent and incubation (see tex t).

PERI OD OF I NCUBA T I ON ( DAYS)

F IG .l. C u m u la tive e v o lu tio n o f labelled CO y C fr o m th e sa n d y so il ( lo t 1} a fte r rep ea ted a d d itio n s o f n o n -

labelled glucose-C. T he g lu c o se was a d d ed in three p o r tio n s each co rresp o n d in g to 4 5 ( V ) a n d 2 2 5 m g (£s)
g lu c o se -C jl 00 g so il respectively; th e fir s t p o r tio n was a d d ed a t th e beginning, th e s e c o n d a n d th ird as in d ica te d
b y arrow s; (O ) u n a m e n d e d soil. C om pare w ith Table /.
 IAEA-SM-211/20 7

TABLE III. EVOLUTION OF LABELLED C02-C FROM THE SANDY SOIL (Lot 2) AFTER
VARIOUS TREATMENTS, AND AFTER FUMIGATION WITH CHC13 ;
F um igation w as p e rfo rm e d a fter in cu bation fo r 77 d a ys fo llo w in g th e treatm en ts; biom assrC
was calcu la ted fr o m th ese values

Labelled '

CO 2 -C evolved after
T reatm ent B iom ass-C ,
Treatment Fum igation
( 1 1 days) (1 0 days)

m g /1 0 0 g %a m g /100 g m g /1 0 0 g % b

N one 0 .9 4 0 1.7 0.4 9 2 0 .7 6 4 . 1.4

Air-drying
norm al m oistening 1.064 1.9 0.4 0 7 0 .6 3 3 ! 1 .2

Air-drying
gradual m oistening 1.085 2 .0 0.3 7 5 0 .5 7 8 ; 1.1

Grinding 2 .2 3 4 4.1 0.251 0 .2 1 6 ; 0.4

a Percentage o f labelled C in soil at the beginning.

k Percentage o f labelled C remaining in soil after treatm ent and incubation.

TABLE IV. NATIVE C02-C EVOLVED FROM THE SANDY SOIL (Lot 2) AFTER VARIOUS
TREATMENTS, AND AFTER FUMIGATION WITH CHC13
F um igation was p e rfo rm e d a fte r in cu bation f o r 77 d a ys fo llo w in g th e treatm en ts; biom ass-C
was calcu la ted fr o m th ese values

Native

CO 2 -C evolved after

Treatm ent Biomass-C

Treatment Fum igation
(7 7 days) (1 0 days)

mg / 1 0 0 g %a mg / 1 0 0 g mg / 1 0 0 g % b

N one 16.69 0.9 5.62 7.36 0.4

Air-drying
norm al m oistening 17.62 1 .0 5.05 6.11 0 .3

Air-drying
gradual m oistening 18.57 1 .0 4 .9 8 6 .4 4 : 0 .4

Grinding 3 0 .3 7 1.7 4 .5 0 4 .4 5 ; 0 .3

a Percentage o f native C in soil at the beginning.

^ Percentage o f native C remaining in soil after treatm ent and incubation.
 S0RENSEN

TABLE V. EVOLUTION OF LABELLED C02-C FROM THE LOAM SOIL AFTER VARIOUS
TREATMENTS, AND AFTER FUMIGATION WITH CHC13
F um igation was p e rfo rm e d a fter incubation fo r 5 8 days fo llo w in g treatm en ts; biom ass-C was
calcu la ted fr o m these values

Labelled

C 0 2-C evolved after

Treatment Biomass-C
Treatment Fum igation
(5 8 days) ( 1 0 days)

m g /100 g %a m g /100 g m g /100 g %b

N one 4 .2 9 0 1.9 5 .737 1 0 .270 4.7

Air-drying 9.061 4.1 5 .6 9 4 9 .9 7 8 4.7
Oven-drying 14.744 6.6 3 .1 1 9 3.971 1.9
Grinding 2 2 .4 3 0 10.1 1.874 1.166 0.6
50 mg non-labelled
glucose-C added 7 .077 3.2 5.275 8 .8 7 6 4.1
200 mg non-labelled
glucose-C added 13.400 6.0 4.951 6 .9 7 6 3.3

3 Percentage o f labelled C present in the soil at the beginning,

k Percentage o f labelled C remaining in the soil after treatm ent and incubation.

TABLE VI. NATIVE AND NON-LABELLED COr C EVOLVED FROM THE LOAM SOIL
AFTER VARIOUS TREATMENTS, AND AFTER FUMIGATION WITH CHCI3
F or fu rth er explan ation see Table V

Native and non-labelled

C 0 2-C evolved after

Treatment Biomass-C
Treatment Fum igation
(5 8 days) (1 0 days)

m g /100 g %a m g /100 g m g /1 0 0 g %b

N one 8.80 0.9 6 .9 4 11.04 1.1

Air-drying 10.22 1.0 6 .97 11.54 1.2

Oven-drying 18.70 1.9 5 .38 6 .9 0 0.7

Grinding 36.43 3.6 5.16 3 .8 0 0.4

50 mg non-labelled
glucose-C added 4 3 .1 4 68.7 10.52 16.23 33.1

200 mg non-labelled
glucose-C added 162.73 7 7 .0 13.96 19.32 18.0

a Percentage o f native C in soil at th e beginning, or o f non-labelled glucose-C added (see tex t).
^ Percentage o f native soil-C or non-labelled glucose-C remaining in soil after treatm ent and incubation (see tex t).
 IAEA-SM-211/20 9

an average of two replicas, the deviation between the two replicas being 4.2 and 6.8% of the
mean for the labelled and the native C respectively. This rate of decomposition corresponds
to a half-life of 25—27 years for both the labelled and the native carbon in the soil. The values
were calculated by means of the formula Ti = О.ЗТ/log (A0/A), where A0 represents the amount
of substance at zero time, and A the amount after time T [13]. The values are considered only
to indicate the order of the size, a longer period of incubation being necessary to establish a
more exact value. The decomposition in lot 2 of the sandy soil proceeds more rapidly (Tables III
and IV). The half-life of the labelled and the native carbon, calculated as above, corresponded to
9 and 17 years respectively. The reason for this difference is that, after removal from the field,
lot 2 of the sandy soil was stored air-dried for 3 years and only remoistened 3 months before the
initiation of the present experiment. The rate of decomposition in the loam soil (Tables Vand VI)
corresponded to a half-life of 6 years for the labelled carbon and 13 years for the native carbon.
At the end of the incubation periods described above, the biomass in the soils was determined.
It accounted for 1.0 and 1.4% of the labelled carbon remaining in lots 1 and 2 of the sandy soil
respectively; of the native carbon, 0.3 and 0.4% were in the biomass. The biomass in a sample
of the sandy soil removed from the field plot in April 1976 (lot 3), and thus incubated uninter­
ruptedly in the field for 12 years, accounted for 2.9 and 0.7% of the labelled and native carbon
respectively. The biomass in the loam soil accounted for 4.7 and 1.1% of the labelled and native
carbon respectively (Tables I—VI).

The priming effect

The addition of unlabelled glucose increased the rate of decomposition of labelled materials
both in the sandy and in the loam soils; the larger the addition, the larger the increase —the
‘priming effect’ (Tables I and V, Fig. 1). The evolution of labelled C02-C ranged from 1.6 to
3.2 times the evolution from unamended samples for both soils. The priming effect, however,
decreased per unit added glucose. The addition of 135 and 675 mg glucose-C, respectively;
to 100 g of the sandy soil resulted thus in a priming of 0.44 and 0.34 mg labelled C02-C per 100 g
of glucose-C added. The corresponding figures for the addition to the loam soil of 50 and 200 mg
glucose-C, respectively, were 5.6 and 4.6 mg labelled C02-C (Table VII). The repeated addition
of glucose to the sandy soil showed that the first addition had the strongest effect, whereas the
third addition did not increase the rate of decomposition of labelled materials initiated by the
second addition. The rate was, however, roughly maintained, indicating that the labelled materials
susceptible to priming were not exhausted. !
The addition of glucose, and the conditions in the soil during the period of incubation
resulting from this, diminished the size of the labelled biomass in the loam soil in comparison
with the unamended soil, and the largest addition caused the largest reduction (Table V). Per
unit of added glucose, however, the reduction caused by the small addition was the largest,
0.028 and 0.016 mg, respectively, per mg of glucose-C after the addition of 50 and 200 mg
glucose C/l 00 g soil (Table VII). Stated as a percentage of the previous increase in evolution of
labelled C02-C, the small addition also had the largest effect on the biomass. The decrease caused
by the addition of 50 mg glucose-C corresponded to 50% of the increase in labelled C02-C,
whereas the decrease caused by the addition of 200 mg glucose-C corresponded to only 36%
of the increase.
The labelled biomass in the sandy soil after repeated additions of glucose was approximately
of the same size as in the unamended soil; in the sample that had received the large addition it
was even slightly larger (Table I). This indicates either that the biomass was not diminished in
this case, or, what is more likely, that repeated additions resulted in such conditions in the soil
that the labelled biomass was able to regain its original size. A low addition of energy material
such as glucose might primarily result in an increased death rate of the biomass, whereas larger
additions might also result in an abundant production of enzymes which render non-biomass
10 S0RENSEN

TABLE VII. INCREASE IN C02 EVOLUTION AND THE CORRESPONDING DECREASE

IN BIOMASS CAUSED BY THE TREATMENTS

Increase in Decrease in
Decrease in
evolution in biomass-C
biomass-C^
c o 2- c a as % o f
Soil Treatment U161Cd5C ill
m g /100 g soil CO 2 evolution

Labelled Native Labelled Native Labelled Native

Air-drying
norm al m oistening 0 .1 2 4 0 .9 3 0 0 .1 3 2 1.250 106 134
Sandy soil Air-drying
(lo t 2) gradual m oistening 0.145 1.880 0 .1 8 6 0 .9 2 0 128 49

Grinding 1.294 13.680 0 .5 4 8 2 .9 1 0 42 21

Air-drying 4.771 1.420 0 .2 9 2 - 0 .5 0 0 6 ND

Oven-drying 10.454 9 .9 0 0 6 .3 0 0 4 .1 4 0 60 42
Grinding 18.140 2 7 .6 3 0 9 .1 0 4 7 .2 4 0 50 26
Loam soil
50 mg non-labelled
glucose-C added 2.787 ND 1.3 9 4 ND 50 ND
2 0 0 mg non-labelled
glucose-C added 9 .1 1 0 ND 3 .2 9 4 ND 36 ND'

135 mg non-labelled
Sandy soil glucose-C added 0 .5 9 4 ND 0 .0 7 0 ND 12 ND
(lo t 1) 675 mg non-labelled
glucose-C added 2.2 9 8 ND - 0 .1 0 6 ND ND ND

a The evolution from untreated soils deducted from the evolution o f C 0 2-C during the period o f incubation
follow ing th e treatm ents.
k Biomass-C in treated soils deducted from biomass-C in untreated soils.

material more susceptible to microbial attack. These aspects have been discussed in greater
detail elsewhere [14]. After repeated additions, the labelled biomass might therefore eventually
regain its original size, or even become larger than in the unamended soil.
A tentative calculation was made of the evolution of non-labelled glucose-C in C02 and the
percentage of glucose-C remaining in the soil and present in the biomass. For this calculation it
was assumed that the evolution of native C02-C and the native biomass in the samples amended
with non-labelled glucose were of the same size as in the unamended soil; these values were
deducted from the total values. These assumptions are not entirely correct since the decomposition
of the native soil organic matter was presumably increased by the added glucose (priming), and the
biomass reduced. The results are consequently only considered to indicate the order of the
size. Of the 135 and 675 mg glucose-C added to 100 g of the sandy soil, 76 and 70% respectively
were evolved as C02, and of the glucose-C remaining in the soil, 29 and 12% respectively were in
the biomass (Table II). The loam soil was amended with 50 and 200 mg glucose-C/100 g; 69 and
77% of this were evolved as C02, and of the amount remaining in the soil, 33 and 18% respectively,
were in the biomass (Table VI).
 IAEA-SM-211/20 11

TREATMENTS

0 U N T R E A T E D V 50 mg
о -------„ -------- Д 200 N O N -L A B E L L E D
+ AIR DRYIN G
▼ 135 G LU C O SE - C

▲ 675_ A D D E D / 100 g SO IL
• OVEN DR YIN G
□ G RINDING

о
(л LA BELLED С NATIVE С
o> 12 12
LOAM 0+ LOAM
о
о |-° +
V
SOIL SOIL
8 8
д
Ol А |-
А -
Е
О О 0 _i
10 20 30 10 20 30 АО
со
со
<
s: 1. 2 12Г
SA N D Y SA N D Y
о
- SO IL - SO IL
m
0.8 8 -
0 О
Н*
t ▼
-
0. A A -
о -

0 ............................ 0 -
1. 0 2.0 3.0 10 20 30 АО

С 0 2-С mg / Ю О д S OI L

F IG .2. T he relation b e tw e e n C 0 2-C ev o lved during th e in c u b a tio n fo llo w in g th e tr e a tm e n ts a n d th e biom ass

p re se n t in th e soils a t th e te rm in a tio n o f in cu b a tio n . T he s y m b o l f o r u n tr e a te d so il ( Q j refers to th e sa n d y
so il flo t 1} th a t received rep ea ted a d d itio n s o f п о п -labelled glucose (T a b le I). T h e s y m b o ls (+ X J refe r fojair­
d ryin g fo llo w e d b y n o rm a l an d gradual m o iste n in g respectively. 1

The effect of drying and grinding

The evolution of labelled C02-C after air-drying was 1.2 and 2.1 times as large as the evolution
from the untreated soil in the case of the sandy and the loam soils, respectively; grinding increased
the evolution 2.4 and 5.2 times. Only the loam soil was subjected to oven-drying, and this
increased the evolution of labelled C02-C 3.4 times (Tables V and VI).
The increase in C02 evolution during the incubations subsequent to the treatments was
followed by a reduction in biomass in comparison with untreated soils (Fig.2). In the sandy soil
this decrease was slightly larger than the previous increase in C02 evolution; it corresponded to
106 and 128% of the increase for air-drying followed by normal and gradual moistening respectively
(Table VII). In the loam soil the decrease in biomass caused by air-drying was insignificant.
It corresponded to only 6% of the previous increase in C02 evolution, whereas oven-drying was
followed by a reduction corresponding to 60%. The decrease in biomass that followed after
grinding corresponded to 42 and 50% of the previous increase in C02 evolution (Table VII).
12 S0RENSEN

T A B L E V III. T H E D IR E C T IN F L U E N C E O F A IR - D R Y IN G A N D G R IN D IN G O N T H E
B IO M A S S

Labelled Native

CO 2 "Ca CO 2 -Ca
Soil Treatm ent *5 Bio- Bio-
mass-C mass-C
A lone + CHC13 A lone + CHCI3

mg / 1 0 0 g soil

No N one 0 .1 2 4 0 .8 5 0 1.452 4 .4 6 0 11.1 3 0 13.3 4 0

Sandy soil
Air-drying 0.3 2 7 0 .972 1.2 9 0 6 .8 1 0 1 1 .5 9 0 9 .5 6 0
(lo t 3)

Grinding 1.231 1.176 ND 17.4 8 0 1 6 .1 3 0 ND

N one 0 .6 1 0 5.821 1 0 .420 0 .7 2 7 5 .8 7 0 10.2 9 0

Loam soil Air-drying 3 .7 1 4 8.921 1 0 .410 6 .4 0 0 9 .3 7 0 5 .9 4 0

Grinding 11.541 11.636 0 .1 9 0 16.4 8 0 1 4 .5 8 0 ND

The evolution o f C 0 2 was determ ined during 10 days o f incubation fo llow ing treatm ent alone or treatm ent plus
fum igation.
^ Fum igation w ith CHC13 was performed im m ediately after treatm ent and rem oistening.

G r a d u a l m o is t e n in g o f t h e s a n d y s o il r e s u lte d i n a s l i g h t l y la r g e r e v o l u t io n o f la b e lle d C 0 2-C

t h a n n o r m a l m o is t e n in g . T h e o p p o s it e r e s u lt h a d b e e n e x p e c t e d s in c e i t w a s b e lie v e d t h a t n o r m a l ,
r a p i d m o is t e n in g m ig h t c a u s e s o m e r u p t u r e o f s o il c r u m b s , a n d t h u s r e n d e r m o r e o f t h e m a t e r ia l
s u s c e p t ib le t o d e c o m p o s it i o n . T h e d if f e r e n c e b e t w e e n t h e t w o t r e a t m e n t s w a s o b s e r v e d a f t e r
t h e f i r s t 1 2 d a y s o f in c u b a t io n .
T h e in f l u e n c e o f d r y in g a n d g r in d in g o n t h e e v o l u t i o n o f n a t iv e C 0 2-C a n d o n n a t iv e b io m a s s
w a s o n t h e w h o l e p a r a lle l t o t h e e f f e c t o n t h e la b e lle d m a t e r ia ls ( T a b le s I V , V I , V I I a n d F ig . 2 ) .

T h e d i r e c t e f f e c t o f a ir - d r y in g a n d g r in d in g o n t h e b io m a s s

T h e v a r ia t i o n s i n s iz e o f t h e b io m a s s a f t e r t h e in c u b a t i o n t h a t f o l l o w e d t h e t r e a t m e n t s a re
n o t n e c e s s a r ily a t r u e m e a s u r e o f t h e in f l u e n c e o f t h e t r e a t m e n t s o n t h e b io m a s s , s in c e n e w
g e n e r a t io n s o f m ic r o - o r g a n is m s m u s t h a v e d e v e lo p e d d u r i n g t h e p e r i o d o f i n c u b a t io n w h e n th e
m a t e r ia l e x p o s e d b y t h e t r e a t m e n t s w a s d e c o m p o s e d . T h e d ir e c t e f f e c t o f a ir - d r y in g a n d g r in d in g
o n t h e b io m a s s i n s o ils w a s t h e r e f o r e in v e s tig a te d b y f u m i g a t i o n im m e d ia t e ly a f t e r t h e t r e a t m e n t s
a n d r e m o is t e n in g . I t w a s a s s u m e d t h a t t h e e f f e c t o f f u m i g a t i o n w o u l d b e t h e s a m e i n r e c e n t ly
t r e a t e d s o ils as i n s o ils w h e r e t h e b io lo g ic a l c o n d i t io n s w e r e s ta b iliz e d d u r i n g a p e r io d o f u n i n t e r ­
r u p t e d i n c u b a t io n . A n in c r e a s e i n t h e e v o l u t io n o f C 0 2 a f t e r f u m ig a t i o n , i n c o m p a r is o n w i t h th e
e v o l u t i o n f r o m t r e a t e d b u t n o t f u m ig a t e d s o ils , s h o u ld t h u s b e a m e a s u r e o f t h e b io m a s s t h a t h a d
s u r v iv e d t h e t r e a t m e n t .
T h e r e s u lt s in d ic a t e d t h a t a ir - d r y in g h a d s o m e e f f e c t o n t h e la b e lle d b io m a s s i n t h e s a n d y
s o il; i t w a s r e d u c e d t o 8 9 % o f t h a t i n u n t r e a t e d s o il, w h e r e a s t h e la b e lle d b io m a s s i n t h e lo a m
s o il w a s u n a f f e c t e d ( T a b le V I I I ) . T h e s e f i n d i n g s a re i n a c c o r d a n c e w i t h t h e p r e v io u s o b s e r v a t io n s
f o r t h e lo a m s o il, w h e r e , a f t e r a ir - d r y in g a n d s u b s e q u e n t in c u b a t i o n , t h e la b e lle d b io m a s s w a s
p r a c t ic a ll y o f t h e s a m e s iz e as t h a t i n u n t r e a t e d s o il ( T a b le V I I ) . T h e in c r e a s e i n C 0 2 e v o l u t io n
c a u s e d b y a ir - d r y in g o f t h e s a n d y s o il w a s f o ll o w e d b y a d e c re a s e in b io m a s s o f a p p r o x im a t e ly
 IAEA-SM-211/20 13

t h e s a m e s iz e ( T a b le V I I ) . T h e o b s e r v a t io n o f t h e d ir e c t e f f e c t is s o m e w h a t c o n t r a r y t o t h a t ,
h o w e v e r ; b o t h o b s e r v a t io n s s u g g e s t t h a t t h e la b e lle d b io m a s s i n t h e s a n d y s o il w a s m o r e s u s c e p t ib le
t o t h e t r e a t m e n t t h a n t h e b io m a s s i n t h e lo a m s o il.
T h e d ir e c t e f f e c t o f a ir - d r y in g o n t h e n a t iv e b io m a s s w a s s t r o n g e r t h a n o n t h e la b e lle d ; th e
n a t iv e b io m a s s w a s r e d u c e d t o 7 2 a n d 5 8 % o f t h a t i n t h e u n t r e a t e d s a m p le s f o r t h e s a n d y s o il
a n d t h e lo a m s o il r e s p e c t iv e ly .
O n t h e w h o le , t h e r e s u lt s s u g g e s t t h a t t h e m a t e r ia l i n t h e s o ils r e n d e r e d d e c o m p o s a b le b y
a ir - d r y in g w a s o n l y p a r t l y d e r iv e d f r o m k i l l e d b io m a s s , a n d le a s t f o r t h e la b e lle d b io m a s s i n th e
lo a m s o il. T h is is i n a g r e e m e n t w i t h o b s e r v a t io n s b y T u c k w e l l a n d J e n k in s o n [ 1 5 ] , w h o f o u n d t h a t
t h e in c r e a s e i n e v o l u t io n o f C 0 2 a f t e r a ir - d r y in g o r ig in a t e d p a r t l y f r o m k i l l e d b io m a s s a n d p a r t l y
f r o m a n o n - b io m a s s c o m p o n e n t . S h ie ld s , P a u l a n d L o w e [ 1 6 ] c o n lu d e d t h a t t h e in c r e a s e d e v o l u t io n
o f la b e lle d C 0 2- C a f t e r p h y s ic a l t r e a t m e n t s c o u l d n o t b e a s c r ib e d s t r i c t l y t o b io m a s s .
T h e n u m b e r s o f v ia b le o r g a n is m s , d e t e r m in e d b y p la t e c o u n t s , h a v e b e e n f o u n d b y s o m e
in v e s t ig a t o r s [ 1 7 , 1 8 ] t o b e l o w e r i n t h e a ir - d r ie d a n d r e m o is t e n e d s o il t h a n i n fr e s h s o il, w h e r e a s
o t h e r w o r k e r s s ta te t h a t a ir - d r y in g h a s n o m a r k e d e f f e c t o n t h e n u m b e r s o f e it h e r f u n g i o r
b a c t e r ia [ 1 9 ] .
G r in d in g h a d a s tr o n g e ffe c t; t h e e v o l u t i o n o f C 0 2 a f t e r f u m ig a t i o n w a s i n t h r e e cases
o u t o f f o u r l o w e r t h a n f r o m t h e g r o u n d b u t u n f u m ig a t e d s o il ( T a b le V I I I ) . T h is s u g g e s ts t h a t
t h e b io m a s s w a s a lm o s t c o m p l e t e ly k i l l e d b y t h e t r e a t m e n t . T h e b io m a s s o b s e r v e d i n t h e g r o u n d
s a m p le s a f t e r t h e s u b s e q u e n t in c u b a t io n , a n d s h o w n i n T a b le s I I I , I V , V a n d V I , s h o u ld c o n s e q u e n t ly
b e n e w b io m a s s d e v e lo p e d d u r i n g t h e d e c o m p o s it i o n o f k i l l e d b io m a s s a n d n o n - b io m a s s m a t e r ia l
e x p o s e d b y th e tr e a tm e n t.
T h e la b e lle d b io m a s s i n t h e u n t r e a t e d s a n d y s o il ( 0 . 7 6 4 m g , T a b le I I I ) c o r r e s p o n d s t o 5 9 %
o f t h e in c r e a s e i n C 0 2 e v o l u t io n c a u s e d b y t h e g r in d in g ( 1 . 2 9 4 m g , T a b le V I I ) . T h e b io m a s s in
t h e u n t r e a t e d lo a m s o il ( 1 0 . 2 7 0 m g , T a b le V ) c o r r e s p o n d s t o 5 7 % o f t h e in c r e a s e c a u s e d b y
g r in d in g ( 1 8 . 1 4 0 m g , T a b le V I I ) . T h is s u g g e s ts t h a t a b o u t 4 0 % o f t h e C 0 2-C re le a s e d as a r e s u lt
o f t h e g r in d in g o r ig in a t e d f r o m n o n - b io m a s s m a t e r ia l.
T h e c o r r e s p o n d in g fig u r e s f o r t h e n a t iv e s o il- C w e r e 5 4 % f o r t h e s a n d y s o il ( in c r e a s e 1 3 . 6 8 0 m g ,
b io m a s s 7 . 3 6 0 m g ) a n d 4 0 % f o r t h e lo a m s o il ( in c r e a s e 2 7 . 6 3 0 m g , b io m a s s 1 1 . 0 4 0 m g ) .
J e n k in s o n [ 2 0 ] c o n c lu d e d t h a t t h e f l u s h o f d e c o m p o s it i o n c a u s e d b y u l t r a s o n ic d is p e r s io n
w a s t o o la r g e t o d e r iv e s o le ly f r o m k i l l e d o r g a n is m s , a n d t h a t n o n - l iv in g p a r t s o f s o il o r g a n ic
m a t t e r m u s t a ls o h a v e b e e n e x p o s e d t o a t t a c k .

REFERENCES

[ 1 ] JEN K INSO N , D.J., Soil Sei. 111 (1 9 7 1 ) 64.

[2 ] S 0R E N S E N , L.H., Soil Biol. Biochem . 4 (1 9 7 2 ) 245.
[3 ] JEN K INSO N , D .S., “ The priming action ” , The Use o f Isotop es in Soil Organic Matter Studies, Pergamon
Press, Oxford (1 9 6 6 ) 199.
[4 ] BIRCH, H.P., Plant Soil 10 (1 9 5 8 ) 9.
[5 ] CRASWELL, E.T., W ARING, S.A ., Soil Biol. B iochem . 4 (1 9 7 2 ) 4 2 7 .
[ 6 ] JENKINSON, D .S., J. Soil Sei. 17 (1 9 6 6 ) 280.
[7 ] JENKINSON, D .S., POWLSON, D .S., Soil Biol. B iochem . 8 (1 9 7 6 ) 167.
[ 8 ] POWLSON, D .S., JEN K INSO N , D .S., Soil Biol. B iochem . 8 ( 1 9 7 6 ) 179.
[9 ] JENKINSON, D .S., POWLSON, D .S., W ED DER BU RN , R.W.M., Soil Biol. B iochem . 8 (1 9 7 6 ) 189.
[1 0 ] JENKINSON, D .S., Soil Biol. B iochem . 8 (1 9 7 6 ) 203.
[1 1 ] JEN K INSO N , D .S., POWLSON, D .S., Sod Biol. B iochem . 8 (1 9 7 6 ) 2 09.
[1 2 ] S 0 R E N S E N , L.H., Soil Biol. B iochem . 6 (1 9 7 4 ) 287.
[1 3 ] COMAR, C.L., R adioisotopes in B iology and Agriculture, McGraw-Hill, N ew York (1 9 5 5 ) 19.
[1 4 ] S 0R E N S E N , L.H., Soil Sei. Soc. A m ., Proc. 39 (1 9 7 5 ) 809.
[1 5 ] TUCKWELL, S.B., JENKINSON, D .S., “ E ffects o f air-drying on th e release o f organically-bound nutrients
by soils” , R otham sted Experim ental Station R eport for 1 9 74, Part I (1 9 7 5 ) 197.
14 S0RENSEN

[1 6 ] SHIELDS, J.A ., PAUL, E.A ., LOWE, W.E., Soil Biol. B iochem . 6 (1 9 7 4 ) 31.
[1 7 ] STEVENSO N , I.L., Plant Soil 8 (1 9 5 6 ) 170.
[1 8 ] SOULIDES, D .A ., ALLISO N, F.E., Soil Sei. 91 (1 9 6 1 ) 291.
[1 9 ] MACK, A .R ., Can. J. Soil. Sei. 4 3 (1 9 6 3 ) 316.
[2 0 ] JENKINSON, D .S., “ The effects o f m echanical disturbance o n th e decom p osition o f soil organic m atter” ,
R otham sted Experim ental Station R eport for 19 7 4 , Part I (1 9 7 5 ) 197.

D IS C U S S IO N

D .R . S A U E R B E C K : Y o u s p e a k o f p r im in g e f f e c t s m e a s u r e d f o r t w o m o n t h s a f t e r t h e
p a r t ic u la r t r e a t m e n t . I n o u r e x p e r ie n c e t h i s a c c e le r a t e d m in e r a liz a t i o n la s ts f o r o n l y a f e w d a y s ,
so t h a t o n p l o t t i n g t h e ra te s o f C 0 2 e v o l u t io n w e g e t a s te p - s h a p e d c u r v e w h i c h s h o w s a b r i e f
in c r e a s e im m e d ia t e ly a f t e r t h e t r e a t m e n t b u t q u i c k l y r e t u r n s t o it s f o r m e r s h a p e .
L .H . S O R E N S E N : W e , t o o , h a v e o b s e r v e d t h a t m o s t o f t h e a c c e le r a t e d e v o l u t io n o f la b e lle d
C 0 2 o c c u r r e d d u r i n g t h e f i r s t f iv e d a y s o f i n c u b a t io n a f t e r t h e a d d i t i o n o f t h e u n la b e lle d g lu c o s e .
T h e r e a f t e r t h e a c c e le r a t e d p r o d u c t i o n o f la b e lle d C 0 2 le v e lle d o f f , a n d a f t e r 3 0 d a y s o f in c u b a t io n
t h e c u r v e w a s a lm o s t p a r a lle l w i t h t h e c u r v e f o r t h e u n a m e n d e d s o il.
 IAEA-SM-211/51

STABILITY CONSTANTS OF SOME COMPLEXES OF

ARGENTINE HUMIC ACIDS AND MICRONUTRIENTS
R . A . R O S E L L * , A . M . M I G L I E R I N A , L .Q . D E N O V I L L A
U n iv e r s id a d N a t i o n a l d e l S u r ,
B a h ia B la n c a ,
A r g e n t in a

A b s tr a c t

STABILITY C O N STA N TS OF SOME COMPLEXES O F ARG ENTIN E HUMIC ACIDS A N D M ICRONUTRIENTS.

One o f th e m ain aims in th e stud y o f th e com p lexes o f hum ic substances and plant mineral nutrients
in th e soil is to ob tain therm odynam ic data w hich, com pared w ith know n system s, can give th e necessary inform ation
to calculate th e elem en t activity in solu tion . This inform ation is essential for predicting th e dynam ics o f such
nutrients in th e soil solu tion , sin ce th e latter is o n e o f th e main location s o f fundam ental reactions related to soil
genesis and fertility. The distribution o f radioactive 6 5 Zn and non-radioactive Cu b etw een a cation exchange resin
and th e hum ic system s w as used to calculate th e stability constants o f th ese elem en ts and the hum ic acids (H A )
extracted from various A rgentine soils. O n th e basis o f th ese stability con stants, th e fo llow ing con clusion s were
obtained: (1 ) C opper w as m ore strongly com p lexed than zinc b y th e hum ic acid o f a S olod soil. Copper had
also alm ost th e sam e value o f th e logarithm o f the stability constant for several soils (8.1 t o 9 .1 ); (2 ) T he value
o f the stability con stant o f th e Zn-HA co m p le x was higher for a C hestnut so il (Petrocalcic Paleustoll) than fo r the
o th er soils studied; (3 ) Since HA is an im portant in solu b le fraction in norm al soil con d ition s, it is suggested that
th e m o b ility o f Cu is lo w in th e soil solu tion o f th ese soils. Therefore, a Cu d eficien cy m ay be predicted or exp ected.

IN T R O D U C T IO N

T h e c a p a c i t y o f h u m i c s u b s ta n c e s t o f o r m s ta b le c o m p o u n d s w i t h m e t a ll ic c a t io n s i n t h e s o il
s o l u t i o n h a s a h ig h p e d o lo g ic a l a n d a g r o n o m ic v a lu e , b u t i t is n e i t h e r w e l l k n o w n n o r u n d e r s t o o d .
O f m o s t i m p o r t a n c e is t h e r e la t i o n s h ip b e t w e e n t h e m a in e s s e n t ia l p l a n t m i c r o n u t r i e n t s a n d t h e
f u l v i c a n d h u m i c a c id s . T h e s e h u m i c f r a c t i o n s a re p o l y e l e c t r o l i t i c m a c r o m o le c u le s , m a in ly a c id ic ,
w h i c h f o r m m e t a ll ic s e q u e s tr a te s a n d c h e la te s t h a t p a r t ic i p a t e i n i m p o r t a n t r e a c t io n s d u r i n g th e
g e n e s is a n d d e v e l o p m e n t o f s o ils . F u r t h e r m o r e , th e s e c o m p le x e s r e g u la t e t h e m o v e m e n t o f s o il
n u t r i e n t s a n d t h e i r a v a i l a b i l i t y t o p la n t s .
T a b le I p r e s e n ts t h e l o g a r it h m o f t h e s t a b i l i t y c o n s t a n t o f s o m e c o m p le x e s o f s o il h u m i c
m a t e r ia ls a n d t h e m i c r o n u t r i e n t s Z n a n d C u . T h e v a lu e o f t h e s t a b i l i t y c o n s t a n t d e p e n d s o n th e
t y p e o f h u m i c s u b s ta n c e a n d t h e p H o f t h e d e t e r m i n a t io n .
T h e o b j e c t o f t h is r e s e a r c h w a s t o e s ta b lis h t h e s t a b i l i t y c o n s t a n t o f c o m p le x e s o f Z n a n d C u
w i t h h u m i c a c id s e x t r a c t e d f r o m s e v e ra l A r g e n t in e s o ils . T h e d i s t r i b u t i o n o f r a d io a c t iv e 65 Z n
a n d n o n - r a d io a c t iv e C u b e t w e e n a c a t io n e x c h a n g e r e s in a n d t h e h u m i c s y s te m w a s u s e d t o c a lc u la t e
th e s t a b ilit y c o n s ta n t. T h is i n f o r m a t i o n is u s e f u l i n c o m p a r in g t h e r e la t iv e s o il c o m p l e x in g p o w e r ,
a n d it s i n f l u e n c e o n t h e d y n a m ic s o f m i c r o n u t r i e n t s in t h e s o il s o lu t i o n .

M A T E R IA L S A N D M E T H O D S

S o ils a n d h u m i c a c id s

T h e h u m i c a c id s w e r e e x t r a c t e d w i t h O .S N N a O H f r o m t h e A 1 h o r i z o n s o f B r u n i z e m , S o lo d
a n d S o lo n e t z s o ils o f B a lc a r c e , A r g e n t in a [ 1 0 ] , a n d f r o m a C h e s t n u t s o il o f B a h ia B la n c a , A r g e n t in a
[11, 12].

* A t present A. von H um boldt Stiftu ng fello w and guest-professor at the Institu t für B iochem ie des Bodens
der Forschungsanstalt für Landwirtschaft, Braunschw eig-Völkenrode, Federal R epublic o f Germ any.

15
16 ROSELL et al.

T A B L E I. L O G A R IT H M O F T H E S T A B IL IT Y C O N S T A N T O F
V A R IO U S O R G A N IC M A T E R IA L S A N D C O P P E R A N D Z IN C

Type o f com p lex Log К (pH ) Reference

Cu-peat 6.5 tu
Cu-fulvic acid 3 .2 3 (3 .5 ) [2]
Cu-humic acid 7 .0 0 (5 .0 ) [2]
Cu-non-dialysable hum us
in th e soil solu tion (N D H SS) 5.5 [3]
Cu-fulvic acid 5.78 (3 .5 ), 8 .6 9 (5 .0 ) [4]
Zn-organic m atter 3 .4 (4 .5 ), 5 .6 (7 .0 ) [5]
Zn-manure (aq ueous extr.) 7.8 (7 .0 ) [6]
Zn-hum ic acid 4 .4 (3 .6 ), 6 .2 (5 .6 ), 6 .8 (7 .0 ) [7]
Zn-fulvic acid 2.8 3 (3 .5 ) [2]
Zn-hum ic acid 2.8 7 (5 .0 ) [2]
Zn-NDHSS 4.3 [3]
Zn-fulvic acid 1 .7 3 (3 .5 ), 2 .3 4 (5 .0 ) [4]

Zn-fulvic acid (in 32 soils) 3.9 to 9 .3 , average 6 .2 (7 .0 ) [8]

Zn-hum ic acid (in 29 soils) 4 .2 to 10.8, average 7.3 (7 .0 ) [8]

Zn-hum ic acid (in 5 soils) 3 .1 3 to 5 .1 3 (6 .5 ) [9]

C a t io n - e x c h a n g e m e t h o d

T h e c a tio n - e x c h a n g e m e t h o d , f i r s t d e v e lo p e d b y S c h u b e r t [ 1 3 ] , a n d a p p lie d b y m o s t o f th e
a u t h o r s in d ic a t e d i n T a b le I , is b a s e d o n t h e r e s in s e le c t i v it y t o a d s o r b fr e e m e t a ll ic c a t io n s ( M e )
f r o m e q u i l i b r i u m s o lu t io n s . I n t h e p r e s e n c e o f a c o m p l e x in g a g e n t ( R ) , t h e m e t a ll ic c a t io n is
d i s t r i b u t e d b e t w e e n t h e r e s in a n d t h e e q u i l i b r i u m s o lu t i o n , d e p e n d in g o n t h e c o n c e n t r a t i o n a n d
c o m p l e x in g p o w e r o f ( R ) .
T h e e q u i l i b r i u m r e a c t io n a n d t h e f o r m a t i o n o f s t a b i l i t y c o n s t a n t ( K ) c a n b e f o r m u la t e d
as f o l l o w s [ 2 ] :

Me + xR = M eRx (1 )

(M e R x )
K. U )
(M e )(R )x "

w h e re (M e ) is t h e a c t i v i t y o f t h e fr e e m e t a ll ic c a t io n a t e q u i l i b r i u m ,

(R ) is t h e a c t i v i t y o f t h e f r e e c o m p l e x in g a g e n t a t e q u i l i b r i u m ,
(M e R x ) is t h e a c t i v i t y o f t h e c o m p l e x , a n d
X is t h e n u m b e r o f m o le c u le s o f t h e c o m p l e x in g a g e n t c o m b in e d w i t h o n e m e t a ll ic
c a t io n .
W o r k in g a t a c o n s t a n t i o n i c s t r e n g t h , b y a d d in g a n e le c t r o li t e t o t h e e q u i l i b r i u m s o lu t i o n , i t is
p o s s ib le t o u s e m o la r c o n c e n t r a t i o n s in s t e a d o f a c t iv i t i e s w i t h o u t a lt e r i n g t h e t h e r m o d y n a m ic
v a lu e a n d m e a n in g o f E q . ( 2 ) .
 IAEA-SM-211/51 17

T h e v a lu e s o f t h e u n k n o w n p a r a m e te r s x a n d ( R ) m u s t b e e s t im a t e d . T h e d is tr ib u t io n
c o n s t a n t A is d e f in e d b y

(M e ) + (M e R x )

w h e re m is t h e a m o u n t o f m e t a ll ic c a t io n b o u n d t o t h e r e s in , a n d
(M e ) + (M e R x ) is t h e t o t a l m e t a ll ic c a t io n in t h e e q u i l i b r i u m s o l u t i o n , M .
I n t h e a b s e n c e o f a c o m p l e x in g a g e n t t h e d i s t r i b u t i o n c o n s t a n t is A 0 :

m
Aq - (4 )
(M e )

E x p r e s s in g E q s ( 2 ) , ( 3 ) a n d ( 4 ) i n l o g a r i t h m i c f o r m a n d e lim i n a t in g te r m s , t h e r e la t i o n o b t a in e d
b y M a r t e l l a n d C a lv in [ 1 4 ] is :

lo g ] = lo g К + x l o g ( R ) (5 )

T h e p l o t lo g v e r s u s lo g ( R ) g iv e s t h e v a lu e s o f x a n d lo g K .

S in c e ( R ) is u n k n o w n a n d v e r y d i f f i c u l t t o o b t a in , th e v a lu e o f lo g ( R ) w a s p l o t t e d u s in g
r e la t iv e c o n c e n t r a t i o n s f o l l o w i n g R a n d h a w a a n d B r o a d b e n t [ 7 ] . T h e f u n c t i o n t h u s o b t a in e d
h a s t h e s a m e s lo p e as E q . ( 5 ) , a n d a llo w s x ( F i g . 1 ) t o b e c a lc u la t e d .

F I G .l. D e te r m in a tio n o f th e n u m b e r o f m o le c u le s (X ) o fa S o lo d h u m ic a c id c o m b in e d w ith o n e z in c c a tio n .

 T A B L E II. D E T E R M IN A T IO N O F T H E T A B L E III. M O L A R C O N C E N T R A T IO N O R
M O L A R C O N C E N T R A T IO N O R A C T IV IT Y (R ) A C T IV I T Y ( R ) O F H U M IC A C ID S O L U T IO N S
O F T H E A l- S O L O D H U M IC A C ID
HA fro m M olar c o n c e n tra tio n , M
C o u n ts/m in
T re a tm e n t m g Z n in s o lu tio n A l-B runizem 4 .81 X 1 0 '4
A ctu a l A verage A l-Solod 9 .0 6 X 1 0“4

A I-Solonetz 9 .5 4 X 1 0 '4
B lank 6581
6668 10
B lank 6756 A l-C h estn u t 12.3 6 X 10~4
W ith HA 5089
5031 7 .5 4
W ith H A 4973

R O SE L L e t al.
T A B L E IV . S T A B IL IT Y C O N S T A N T O F T H E Z n -H A C O M P L E X A T p H 5
Soil HA, A l-Solod

HA
Z n in
0.5 m g /m l (H A ) ^•0 Xo
log (H A ) so lu tio n C o u n ts /m in <*0 *0 X — 1 log — - 1 X log К
s o lu tio n M X 1 0 's X X
(m b )
(m l)

B lank - 8743
0 0 - 29 .8 879 90Л 455 - - - - -
5 9.1 0 .9 5 9 0 4 4 1 .0 1207 8 6 .4 3 1 7 .5 0.43 1 .6 3 3 4 7 1.21 4.5

10 18.0 1 .2 5527 51.1 1504 8 3 .0 2 4 4 .0 0 .8 6 1 .9 3 4 5 0 4.5

15 27.1 1 .4 3297 68 .0 2000 7 7 .4 171.0 1.66 0 .2 2 0 1 1 4.5

20 ' 36 .2 1.55871 69.7 2050 76.8 165.5 1.74 0 .2 4 0 5 5 4 .4

In itia l ac tiv ity o f th e m e tallic so lu tio n : 8 7 4 3 c o u n ts/m in = 3 0 0 p g Z n /flask 4 .4 7

 IAEA-SM-211/51 19

( R ) is c a lc u la t e d b y d e t e r m i n in g t h e m a x im u m c o m p l e x in g c a p a c it y o f t h e H A . T h is is
o b t a in e d b y e s t a b lis h in g t h e a m o u n t o f t h e m e t a ll ic c a t io n t h a t c o m b in e s w i t h a H A s o lu t i o n
o f k n o w n c o n c e n tr a tio n [2 ]. S in c e i t is a ls o k n o w n t h a t x m o le c u le s o f H A a re c o m b in e d w i t h a
m e t a ll ic a t o m ( E q . ( l ) ) , i t is p o s s ib le t o c a lc u la t e t h e n u m b e r o f m o le c u le s o f H A c o n t a in e d in
th e s o lu tio n a n d , th e r e fo r e , it s a c tiv ity (R ) .

Determination o f x

T o 5 0 - m l v o l u m e t r i c f la s k s t h e f o l l o w i n g s o lu t io n s a re a d d e d :
V a r ia b le a m o u n t o f H A s o l u t i o n ( 0 . 5 m g H A / m l )
D i s t i lle d w a t e r ( D W ) u p t o a b o u t 3 5 m l
5 m l 0 .1 N N a C l
3 m l o f m e t a ll ic c h lo r id e s o l u t i o n ( 1 0 0 p g m e t a l / m l ) , w h i c h is ta g g e d w i t h a r a d i o t r a c e r w h e n p o s s ib le
A d j u s t p H t o 5 w i t h d i l u t e H C 1 o r N a O H a n d c o m p le t e t o 5 0 m l v o lu m e w i t h D W .
T h e s o lu t i o n s a re t r a n s f e r r e d t o 1 2 5 - m l e r le n m e y e r fla s k s c o n t a in in g 0 . 5 g D o w e x 5 0 W
( 2 0 — 5 0 m e s h ) r e s in i n t h e N a - f o r m , a n d a re t h e n s h a k e n f o r 2 h i n a c o n s t a n t t e m p e r a t u r e ( 2 5 ° C )
b a th . I n t h e s u p e r n a t a n t t h e c o n c e n t r a t i o n o f f r e e a n d c o m p le x e d m e t a ll ic c a t io n s is d e t e r m i n e d b y
a t o m i c a b s o r p t i o n f o r C u a n d r a d i o a c t i v i t y c o u n t in g f o r 65 Z n .

Distribution constants

T h e f o l l o w i n g p r o c e d u r e is f o l l o w e d :

«о V
(6)
0 _ 1 0 0 -a o X P

w h e re a0 is t h e p e r c e n ta g e o f t h e m e t a ll ic c a t io n c o m b in e d w i t h t h e r e s in
100 - a0 is t h e p e r c e n ta g e o f t h e m e t a ll ic c a t io n r e m a i n in g i n s o lu t i o n
V is t h e v o lu m e o f t h e e q u i l i b r i u m s o lu t i o n , 5 0 m l
P is t h e r e s in w e i g h t , 0 . 5 g.
X is c a lc u la t e d as X 0 , i n t h e p r e s e n c e o f v a r ia b le a m o u n t s o f H A .

Molar concentration or activity (R ) o fH A s

D u p l ic a t e s o lu t i o n s a re p r e p a r e d as f o ll o w s :
5 m l o f c o n e . H A s o lu tio n (5 m g H A / m l)
5 m l O . lN N a C l
1 0 m l o f m e t a ll ic c h lo r id e s o l u t i o n (1 m g m e t a l / m l )
A d ju s t p H to 5
A d u p l ic a t e b la n k ( w i t h o u t H A ) is r u n t o o b t a in t h e i n i t i a l m e t a ll ic c a t io n c o n c e n t r a t i o n .
T h e s o lu t i o n s a re s h a k e n f o r 2 h a t 2 5 ° C , c e n t r if u g e d t o s e p a r a te t h e m e t a ll ic h u m a t e , a n d
a n a ly s e d t o o b t a in t h e r e m a i n in g m e t a ll ic c a t io n i n s o lu t i o n . I n T a b le I I t h e r e s u lt s o b t a in e d w i t h
t h e c o m p l e x b e t w e e n A l - S o l o d H A a n d Z n a re p r e s e n te d ( 2 . 4 6 m g Z n w e r e c o m b i n e d o r c o m p le x e d
b y 2 5 m g H A ).
T a b le I I I p r e s e n ts t h e m o l a r c o n c e n t r a t i o n o r a c t i v i t y ( R ) o f t h e v a r io u s H A s .

Calculation o f stability constants

T a b le I V s h o w s t h e p r o c e d u r e u s e d t o c a lc u la t e th e s t a b i l i t y c o n s t a n t o f t h e c o m p l e x b e t w e e n
t h e A l - S o l o d H A a n d t h e m e t a ll ic c a t io n Z n , a c c o r d in g t o E q . ( 5 ) .
20 ROSELL et al.

TABLE V. S T A B IL IT Y C O N S T A N T S O F
V A R IO U S C O M P L E X E S O F M IC R O N U T R IE N T S
A N D H U M IC A C ID S F R O M A R G E N T IN E S O IL S

L og К (p H 5)
H um ic acid
from soil
Cu Zn

Al-Brunizem 8.4 6.1

A l-Solod 9.1 4.5
Al-Solonetz 9.0 9.5

Al-Chestnut 8.1 9.7

R E S U L T S A N D D IS C U S S IO N

T a b le V p r e s e n ts t h e l o g a r it h m o f t h e s t a b i l i t y c o n s t a n t s o f t h e c o m p le x e s o f m i c r o n u t r ie n t s
( C u a n d Z n ) a n d t h e h u m i c a c id s e x t r a c t e d f r o m s e v e r a l A r g e n t in e s o ils . T h e f o l l o w i n g c o m m e n t s
can be m ade:
1. T h e lo g o f t h e s t a b i l i t y c o n s t a n t s o f t h e C u - H A c o m p le x e s a r e h ig h a n d o f t h e s a m e
o r d e r o f m a g n it u d e ( f r o m 8 .1 t o 9 . 1 ) . T h e S o lo d s o il s h o w s t h e h ig h e s t v a lu e a n d t h e C h e s t n u t
s o il t h e lo w e s t . S in c e H A is a n i m p o r t a n t in s o lu b le f r a c t i o n i n n o r m a l s o il c o n d i t io n s , i t is
s u g g e s te d t h a t t h e a v a i l a b i l i t y o f C u is l o w i n t h e s o il s o l u t i o n o f th e s e s o ils . T h e r e f o r e a C u
d e f ic i e n c y m a y b e e x p e c t e d o r p r e d i c t e d . G o o d m a n a n d C h e s h ir e [ 1 5 ] h a v e r e c e n t ly r e p o r t e d
C u - f i x a t i o n i n o r g a n ic m a t e r ia ls f r o m s t u d y in g t h e e le c t r o n p a r a m a g n e t ic r e s o n a n c e o f t h e c o ­
o r d i n a t i o n c o m p le x e s b e t w e e n C u a n d t h e n i t r o g e n i n p o r p h y r i n d e r iv a t iv e s o f p e a t h u m i c a c id s .
2 . T h e s t a b i l i t y c o n s t a n t s o f t h e Z n - H A c o m p le x e s o f A r g e n t in e s o ils a re h ig h e r t h a n t h e
d a t a r e p o r t e d i n t h e l i t e r a t u r e ( T a b le I ) . T h e v a lu e s a re h ig h e r f o r t h e C h e s t n u t s o il a n d lo w e r f o r
t h e S o lo d s o il, i n in v e r s e o r d e r t o t h a t o f t h e C u c a t io n .
3 . T h e p H d e p e n d e n c y o f t h e s t a b i l i t y c o n s t a n t v a lu e s ( T a b le I ) s u g g e s ts t h e c o n v e n ie n c e
o f s t u d y in g t h e b e h a v io u r o f t h e m a in m ic r o n u t r i e n t s i n s im i la r n a t u r a l c o n d i t io n s t o t h o s e
( c o n c e n t r a t i o n , a c i d i t y , s o lu b le a n d in s o lu b le o r g a n ic m a t t e r , e t c . ) o f t h e s o il s o lu t i o n . I t w o u ld
b e o f s p e c ia l in t e r e s t t o e s ta b lis h t h e s t a b i l i t y c o n s t a n t s o f t h e p l a n t m i c r o n u t r i e n t s a n d t h e
v a r io u s c o m p l e x in g s y s te m s , s u c h as t h e s o lu b le a n d in s o lu b le o r g a n ic m a t t e r , i n t h e s o il s o lu t i o n .
T h is t h e r m o d y n a m ic i n f o r m a t i o n c o u l d h e lp t o o b t a in a b e t t e r a n d c le a r e r p i c t u r e o f t h e g e n e r a l
d y n a m ic s o f m in e r a l c o m p o n e n t s w h i c h p la y a f u n d a m e n t a l r o le i n t h e g e n e s is , d e v e lo p m e n t
a n d f e r t i l i t y o f s o ils .

ACKNO W LEDG EM ENT

T h e C o n s e jo N a c io n a l d e In v e s tig a c io n e s C ie n t if ic a s у T e c n ic a s ( C O N I C E T ) , A r g e n t in a ,
is t h a n k e d f o r f i n a n c ia l h e lp .

REFERENCES

[ 1] C O L E M A N , N.T., M c C L U N G , A.C., M O O R E , D.P., Science 123 (1 9 5 6 ) 330.

[2 ] C O U R P R O N , C., A nn. Agron. 18 (1 9 6 7 ) 623.
[3 ] G E E R IN G , H.R., H O D G S O N , J.F., Soil Sei. Soc. Am., Proc. 33 (1 9 6 9 ) 54.
[4 ] S C H N IT Z E R , M., Soil Sei. Soc. Am., Proc. 33 (1 9 6 9 ) 75.
 IAEA-SM-211/5I 21

[ 5 ] H IM E S , F.L., B A R B E R , S.A., Soil Sei. Soc. Am., Proc. 21 (1 9 5 7 ) 368.

[6 ] M I L L E R , M.H., O H L R O G G E , A.J., Soil Sei. Soc. Am., Proc. 2 2 ( 1 9 5 8 ) 225.
[ 7 ] R A N D H A W A , N.S., B R O A D B E N T , F.E., Soil Sei. 99 (1 9 6 5 ) 362.
[8 ] M A T S U D A , K., IT O , S., Soil Sei. Plant. Nutr. 16 (1 9 7 0 ) 1.
[9 ] A R D A K A N I , M.S., S T E V E N S O N , F.J., Soil Sei. Soc. Am., Proc. 36 (1 9 7 2 ) 884.
[ 1 0 ] R O S E L L , R.A., A L L A N , A.L., A G U L L O , E „ G E L O S , B., L A Z Z A R I , M.A., Turrialba 22 3 (1 9 7 2 ) 327.
[ 11 ] C O N IG L IO , R., R O S E L L , R.A., Ciencia e Investigaeiön (Argentina) 28 (1 9 7 2 ) 271.
[ 12] R O S E L L , R.A., A L L A N , A.L., A G U L L O , E „ G E L O S , B., L A Z Z A R I , M.A., Turrialba 22 3 (1 9 7 2 ) 333.
[ 1 3 ] S C H U B E R T , J., J. Phys. Coll. Chem. 52 (1 9 4 8 ) 340.
[ 14] M A R T E L L , E.A., C A L V IN , M., Chem istry of Metal Chelates C om pounds, Prentice Hall, N ew Y o r k (1952).
[ 15] G O O D M A N , B.A., C H E S H IR E , M.V., Nature (L o n d o n ) N ew Biol. 2 4 4 (1 9 7 3 ) 158.

D IS C U S S IO N

C h. JU S TE : E q u a t i o n ( 1 ) , w h i c h y o u u s e f o r d e t e r m i n in g t h e a p p a r e n t s t a b i l i t y c o n s t a n t s ,
s u g g e s ts t h a t y o u c o n s id e r t h e c o m p le x e s f o r m e d t o b e m o n o n u c le a r . A r e y o u c e r t a in o f t h is ,
f o r i f p o l y n u c l e a r c o m p le x e s a re f o r m e d , r e l a t i o n ( 2 ) is n o l o n g e r a p p lic a b le ?
R .A . R O S E L L : N o , I d o n o t k n o w th e ty p e s o f c o m p le x fo r m e d . I a m a w a re o f th e lim ita tio n s
o f t h e m e t h o d , w h i c h h a v e b e e n p o i n t e d o u t b y s e v e ra l a u t h o r s (e .g . R e f . [ 9 ] ) , b u t o u r in t e r e s t w a s
i n o b t a in in g i n f o r m a t i o n o n t h e r e la t iv e c o m p l e x in g p o w e r o f t h e h u m i c a c id s f r o m d if f e r e n t
s o ils a n d o f t h e v a r io u s e s s e n tia l m ic r o n u t r ie n t s .
C h. J U S T E : D o y o u h a v e a n y h y p o t h e s e s f o r e x p l a in in g t h e d if f e r e n c e s o b s e r v e d i n th e
d e t e r m i n a t io n o f s t a b i l i t y c o n s t a n t s w h e n t h e h u m i c a c id s d i f f e r i n o r ig in ?
R .A . R O S E L L : W e h a v e m a d e n o h y p o t h e s e s a b o u t th e s e d if f e r e n c e s . W e t h i n k t h e y a re
d u e t o d i f f e r e n t f u n c t i o n a l g r o u p s a n d s t r u c t u r a l c o m p o s i t io n s o f t h e h u m i c a c id s . W e p la n t o
s tu d y th is p r o b le m in th e n e a r fu tu r e .
M. S C H N IT Z E R : T h e r e is a t p r e s e n t n o s a t is f a c t o r y m e t h o d f o r d e t e r m i n in g m e t a l- o r g a n ic
m a t t e r c o m p le x e s . M r . R o s e ll h a s d o n e t h e b e s t h e c o u l d c o n s id e r in g t h e k n o w le d g e t h a t is c u r r e n t l y
a v a ila b le . M o r e r e s e a r c h i n t h is f i e l d is n e e d e d t o d e v e lo p b e t t e r m e t h o d s .
\
 IAEA-SM-211/4

DECOMPOSITION IN SOIL OF SPECIFICALLY

CARBON-14-LABELLED DHP AND CORN STALK LIGNINS,
MODEL HUMIC ACID-TYPE POLYMERS
AND CONIFERYL ALCOHOLS
J. P. M A R T I N
D e p a r t m e n t o f S o il a n d E n v ir o n m e n t a l S c ie n c e s ,
U n i v e r s i t y o f C a l if o r n ia ,
R iv e r s id e ,
U n i t e d S ta te s o f A m e r ic a

K . H A ID E R
I n s t i t u t f ü r B io c h e m ie d e s B o d e n s d e r
F o r s c h u n g s a n s t a lt f ü r L a n d w ir t s c h a f t ,
B r a u n s c h w e ig - V ö lk e n r o d e ,
F e d e r a l R e p u b lic o f G e r m a n y

Abstract

DECOMPOSITION IN SOIL OF SPECIFICALLY CARBON-14-LABELLED DHP A ND CORN STALK LIGNINS,

MODEL HUMIC ACID-TYPE POLYM ERS A N D CON IFERY L ALCOHOLS.
Side chain 2-I4C, ring-14C and 0 14CH3-labelled coniferyl alcohols were synthesized. T hese were linked into
m odel DHP lignin using peroxidase and hydrogen peroxide, and in to m odel hum ic acid-type polym ers using m ush­
room phenolase and m ixtures o f h yd roxytolu en es, hydroxyph en ols and h y droxybenzoic and cinnam ic acids.
Labelled corn stalk lignins w ere prepared by injecting side-chain 1 ,2 and 3 14C, ring 14C and 0 14CH3 feruhc acids
as lignin precursors in to th e growing stalks. The labelled con iferyl alcohols, polym ers and lignins were incubated in
G reenfield sandy loam (pH 7 .0 ) adjusted to 60% m oisture capacity. The to ta l C 0 2 and th e 14C 0 2 evolved were
measured over a 28-w eek incubation period. Compared w ith other com m on p henolic substances tested , the coniferyl
alcohol carbons w ere relatively resistant to m icrobial utilization in soil. A fter 28 w eeks, about 4 9 , 4 6 and 66%
respectively o f th e ring-14C, 2-14C (side chain) and 0 14CH3 carbons had evolved as 14C 0 2 . Linkage o f coniferyl
alcohol in to m odel lignins (D H P), plant lignins, and especially in to m od el hum ic polym ers, reduced avaüability. The
carbons in th e m od el hum ic acids were m ost resistant. A fter 28 w eeks, about 5, 6 and 14% respectively o f th e ring,
2- side chain and OCH3 carbons had evolved as C 0 2. T he sam e carbons in the m odel lignins were decom posed to a
greater ex ten t, nam ely, 2 0 , 22 and 45% for th e ring, 2-side chain and OCH3 carbons respectively. The related carbons
in the corn stalk lignin were utilized to a slightly greater ex ten t than th o se in the DHP lignins. Loss o f ring, 1 ,2 and
3- side chain and OCH3 carbons after 28 w eek s w ere about 2 8 , 4 2 , 2 8 , 32 and 52% respectively.

IN T R O D U C T IO N

L ig n in , a r e la t i v e l y s ta b le p h e n o lic p o l y m e r , is t h e s e c o n d m o s t a b u n d a n t p o l y m e r s y n th e s iz e d
b y p la n t s [ 1 — 3 ] . F r o m t h e e c o lo g ic a l p o i n t o f v ie w , i t is i m p o r t a n t t h a t t h e c a r b o n p r e s e n t in
l i g n i n b e re le a s e d as C 0 2 f o r u s e b y n e w g e n e r a t io n s o f li v in g p la n t s [ 4 , 5 ] . A l t h o u g h r e la t i v e l y
r e s is t a n t t o b io d e g r a d a t io n , li g n i n is d e c o m p o s e d i n n a t u r e [ 2 , 6 , 7 ] . A g r o u p o f b a s id io m y c e t e s , th e
w h i t e r o t f u n g i, a re p a r t i c u l a r l y a c t iv e i n li g n i n u t i l i z a t i o n , a n d h a v e r e c e iv e d m o s t a t t e n t i o n in
l i g n i n d e c o m p o s it i o n s tu d ie s [ 2 , 5 , 7 — 9 ] . S o m e o f th e s e f u n g i c a n d e g r a d e u p t o 8 0 % o f a n a v a ila b le
lig n in s o u rc e . O t h e r f u n g i, in c lu d in g Fungi imperfecti and Ascomycetes, a n d b a c t e r ia a re a ls o a b le
t o d e c o m p o s e li g n i n [ 2 , 9 ] . V e r y l i t t l e is k n o w n , h o w e v e r , a b o u t t h e b io c h e m i c a l m e c h a n is m s
in v o lv e d i n t h e d e g r a d a t io n p ro c e s s e s .
L i g n i n is c o n s id e r e d a n i m p o r t a n t s o u r c e o f s t r u c t u r a l u n i t s f o r f o r m a t i o n o f s o il h u m u s
[1 0 — 1 3 ]. I t c o u l d c o n t r i b u t e t o h u m u s f o r m a t i o n i n o n e o r m o r e o f a t le a s t t h r e e w a y s : ( 1 ) th e
li g n i n m o le c u le s c o u l d b e a lt e r e d o r p a r t i a l l y d e g r a d e d t h r o u g h m i c r o b i a l a c t i v i t y , a n d o t h e r o r g a n ic

23
24 MARTIN and HAIDER

c o n s t it u e n t s s u c h as p r o t e in s , p e p t id e s , a m in o a c id s a n d p o ly s a c c h a r id e s w i t h a m in o s u g a r u n it s
c o u l d b e l i n k e d t o t h e a lt e r e d r e s id u e s t h r o u g h e n z y m a t ic o r a u t o x id a t iv e p o l y m e r iz a t io n r e a c t io n s ;
(2 ) p h e n o lic d e g r a d a t io n p r o d u c t s o f l i g n i n t o g e t h e r w i t h m i c r o b i a l l y s y n th e s iz e d a r o m a t i c a n d
o t h e r r e a c t iv e c o m p o u n d s c o u l d u n d e r g o a u t o x id a t iv e o r e n z y m a t ic p o l y m e r i z a t i o n r e a c t io n s e it h e r
e x t r a c e llu la r ly o r w i t h i n m ic r o b i a l c e ll w a lls t o f o r m h u m i c m o le c u le s ; a n d ( 3 ) t h e li g n i n m o le c u le s
in c lu d in g t h e b e n z e n e r in g s c o u ld b e d e g r a d e d t o s im p le a l ip h a t ic c o m p o u n d s , s o m e o f w h i c h
c o u l d b e u t i l i z e d f o r t h e m ic r o b i a l s y n th e s is o f r e a c t iv e p h e n o ls a n d o t h e r s u b s ta n c e s w h i c h c o u ld
b e p o l y m e r iz e d t o f o r m h u m i c m o le c u le s [ 1 0 — 1 8 ] . S e v e ra l in v e s t ig a t o r s b e lie v e t h a t t h e s e c o n d
p a t h w a y m a y b e r e la t i v e l y m o r e i m p o r t a n t t h a n t h e o t h e r t w o [ 1 0 , 1 2 , 1 5 ] .
D H P ( d e h y d r o p o l y m e r ) li g n i n s s y n th e s iz e d b y t h e a c t io n o f p e r o x id a s e a n d li g n i n a lc o h o ls
a p p e a r t o b e s im i la r t o i f n o t i d e n t ic a l w i t h t r u e p la n t lig n in s [ 1 , 5 , 1 9 — 2 1 ]. B y u s in g s p e c i f i c a l ly
14C - la b e lle d li g n i n a lc o h o ls i t is p o s s ib le t o s y n th e s iz e s p e c i f i c a l ly 14C 4 a b e lle d D H P lig n in s . T h e s e
lig n in s , h o w e v e r , a re n o t a s s o c ia te d w i t h c e llu lo s e a n d o t h e r p o ly m e r s as a re t h e li g n i n s i n p la n t s .
B y in je c t in g s p e c i f i c a l ly 14C 4 a b e lle d f e r u l i c a c id s as li g n i n p r e c u r s o r s [ 1 , 2 0 ] i n t o p la n t s te m s , i t is
p o s s ib le t o o b t a in p la n t m a t e r ia l w i t h s p e c i f i c a l ly 14C 4 a b e lle d li g n i n [ 8 ] .
U s e o f s p e c i f i c a l ly 14C 4 a b e lle d li g n i n s f o r b io d e g r a d a t io n s tu d ie s m a k e s i t p o s s ib le t o
d e t e r m in e w h i c h o f th e c o n s t it u e n t u n i t c a r b o n s a re u t i l i z e d m o s t r e a d i ly b y t h e s o il m ic r o b e s , a n d
w h i c h a r e r e la t i v e l y m o r e i m p o r t a n t i n s o il h u m u s f o r m a t i o n . P r e v io u s s tu d ie s w i t h U n k a g e o f
14C 4 a b e lle d p h e n o lic a n d c in n a m ic a c id d e r iv a tiv e s , a m in o a c id s , p e p t id e s , a m in o s u g a rs a n d
p o ly s a c c h a r id e s w i t h a m in o s u g a r c o n s t it u e n t u n i t s i n t o m o d e l h u m i c a c id p o ly m e r s h a v e s h o w n
t h a t a ll t h e c a r b o n s a re s t a b iliz e d t o a c o n s id e r a b le d e g re e [ 1 4 , 1 6 — 1 8 ] . H o w e v e r , s id e c h a in a n d
C O O H g r o u p s , a n d li n k e d p e p t id e s , p r o t e in s , a m in o a c id s , a m in o s u g a rs a n d p o ly s a c c h a r id e c a r b o n s
a re a l i t t l e m o r e s u s c e p t ib le t o d e g r a d a t io n t h a n a re t h e a r o m a t i c r in g c a r b o n s .
T h e p u r p o s e o f t h e s tu d ie s r e p o r t e d w a s t o c o m p a r e t h e d e c o m p o s it i o n i n s o il o f s p e c i f i c a l ly
14C 4 a b e lle d c o n i f e r y l a lc o h o ls i n t h e fr e e s ta te a n d a f t e r lin k a g e i n t o D H P lig n in s , c o r n s t a lk lig n in s
a n d m o d e l h u m i c a c id - t y p e p o ly m e r s i n o r d e r t o b e t t e r u n d e r s t a n d t h e p ro c e s s e s in v o lv e d i n th e
t r a n s f o r m a t io n o f l i g n i n t o s o il h u m i c p o ly m e r s .

M ETHODS

T h e m e t h o d s f o r t h e s y n th e s is o f I4 C - la b e lle d f e r u l i c a c id s h a v e b e e n d e s c r ib e d [ 8 , 2 2 — 2 5 ] .
T h e f e r u l i c a c id s w e r e a c e t y la t e d , c o n v e r t e d t o t h e a c id c h lo r id e s a n d r e d u c e d w i t h L i A l H 4 t o th e
c o n i f e r y l a lc o h o ls . U n la b e lle d c o n i f e r y l a lc o h o l w a s p u r c h a s e d f r o m F l u k a A G , S w it z e r la n d . T h e
n u m b e r in g o f t h e s id e c h a in c a r b o n s w a s b e g u n w i t h t h e a lc o h o l g r o u p [ 3 ] .
F o r p r e p a r a t io n o f m o d e l h u m i c a c id s , 1 .5 m m o l o f 2 ,3 - , 2 , 6 - a n d 3 , 4 - d i h y d r o x y t o lu e n e s ,
c a t e c h o l, p - c r e s o l, o r c i n o l , p h o r o g lu c in o l, p y r o g a ll o l , r e s o r c i n o l , a n d c a f f e i c , 2 , 4 - d i h y d r o x y b e n z o ic ,
3 , 5 - d i h y d r o x y b e n z o ic , g a l li c , p - h y d r o x y b e n z o ic , p - h y d r o x y c in n a m i c , p r o t o c a t e c h u ic , s a lic y l ic a n d
2 , 4 , 6 - t r i h y d r o x y b e n z o i c a c id s w e r e d is s o lv e d i n 1 5 0 0 m l o f 0 . 0 5 M K H 2P 0 4 s o lu t i o n , a n d t h e
m i x t u r e a d ju s te d t o p H 6 . 5 w i t h N a O H . T h r e e m m o l o f t h e d e s ir e d 14C - la b e lle d c o n i f e r y l a lc o h o l
a n d 2 0 0 m g ( 1 0 0 0 0 0 u n i t s ) o f m u s h r o o m p h e n o la s e , o b t a in e d f r o m I C N , I n c . , w e r e a d d e d , a n d
th e m ix t u r e a e ra te d f o r fiv e d a y s .
T h e d a r k s o lu t i o n s w e r e a c id if ie d t o p H 1 .5 w i t h 6 N H C 1 a n d a llo w e d t o s ta n d o v e r n ig h t . T h e
h u m i c a c id p r e c i p it a t e s w e r e r e c o v e r e d b y c e n t r if u g a t io n , w a s h in g w i t h d i s t i ll e d w a t e r a d ju s te d
t o p H 2 . 0 w i t h H C 1 , a n d f r e e z e - d r y in g . T o o b t a i n t h e f u l v i c a c id f r a c t i o n s t h e s u p e r n a t a n t s w e r e
a d ju s t e d t o p H 7 . 0 , c o n c e n t r a t e d t o 4 0 0 m l, a d ju s te d t o p H 2 , d ia ly s e d i n t u b in g s w i t h a 6 0 0 0 m o le ­
c u la r w e i g h t c u t - o f f a g a in s t f r e q u e n t c h a n g e s o f d i s t i ll e d w a t e r , a n d f r e e z e - d r ie d .
T h e m o d e l lig n in s w e r e p r e p a r e d a c c o r d in g t o t h e ‘ Z u t r o p f m e t h o d o f F r e u d e n b e r g a n d
N im z [ 1 9 ] , u s in g h i g h l y p u r i f i e d h o r s e - r a d is h p e r o x id a s e o b t a in e d f r o m N u t r i t i o n a l B io c h e m ic a ls ,
In c . F o r la b e lli n g t h e l i g n i n o f c o r n p la n t s , 1 m l o f s o lu t i o n s c o n t a in in g 1 .5 m g o f la b e lle d f e r u l i c
a c id s w a s in je c t e d i n t o t h e b a s e o f y o u n g c o m s ta lk s b y m e a n s o f a s y r in g e w i t h t h e p is t o n r e m o v e d .
 IAEA-SM-211/4 25

T h e l i q u i d w a s t a k e n u p b y t h e p la n t w i t h i n t w o t o t h r e e d a y s . T h is p r o c e d u r e w a s r e p e a te d th r e e
t im e s o v e r a t w o - w e e k p e r i o d . T h e p la n t s w e r e a llo w e d t o g r o w f o r a n o t h e r th r e e w e e k s . T h e t o p s
w e r e g r o u n d i n l i q u i d N 2 , f r e e z e - d r ie d , e x t r a c t e d th r e e t im e s w i t h 8 0 % b o i l i n g e t h a n o l, a n d d r ie d [ 8 ] .
T h e m a t e r ia l h a d a li g n i n c o n t e n t o f 7 - 8 % w h i c h c o n t a in e d m o r e t h a n 8 0 % o f t h e t o t a l r a d i o a c t i v i t y .
F o r d e c o m p o s it i o n te s ts , G r e e n f ie ld s a n d y lo a m t o p s o il w a s a ir - d r ie d , p a s s e d t h r o u g h a 2 - m m
s c re e n a n d w e ig h e d i n t o 2 5 0 - m l e r le n m e y e r fla s k s i n 1 0 0 g p o r t io n s . T h is s o il h a d a p H o f 7 . 0 ,
a n e x c h a n g e c a p a c i t y o f 11 m e q 1 0 0 g a t p H 7 , a n d c o n t a in e d 1 .2 % o r g a n ic m a t t e r . T h e la b e lle d
c o n i f e r y l a lc o h o l a t c o n c e n t r a t i o n s o f 1 0 0 a n d 1 0 0 0 p p m , a n d t h e la b e lle d lig n in s , m o d e l h u m i c
a c id s a n d c o r n s t a lk s a t c o n c e n t r a t i o n s o f 1 0 0 0 o r 2 0 0 0 p p m , w e r e t h o r o u g h l y m ix e d w i t h t h e d r y
s o ils . T h e m o is t u r e c o n t e n t w a s a d ju s t e d t o 6 0 % o f t h e H ilg a r d c u p m o is t u r e c a p a c i t y ( 1 / 3 b a r ) , t h e
fla s k s a t t a c h e d t o a c lo s e d s y s te m , a n d s l o w l y a e r a t e d w i t h a c o n s t a n t s t r e a m o f C 0 2- fr e e a n d
h u m i d i f i e d a ir . W i t h t h is p r o c e d u r e 0 2 d o e s n o t b e c o m e a l i m i t i n g f a c t o r , a n d t h e m o is t u r e c o n t e n t
o f t h e s o il r e m a in s v i r t u a l l y c o n s t a n t f o r lo n g p e r io d s o f t i m e . M o s t tre a tm e n ts w e re r u n in
d u p l ic a t e . T h e a c t i v i t y o f t h e c o n i f e r y l a lc o h o ls a p p lie d r a n g e d f r o m a b o u t 1 0 0 0 0 t o
5 0 0 0 0 d is / m i n p e r m g . T h e a c t i v i t y o f t h e p o ly m e r s , t h e D H P li g n i n s , a n d t h e c o m s t a lk p r e p a ­
r a tio n ra n g e d f r o m a b o u t 5 0 0 t o 2 0 0 0 d is /m in p e r m g .
T h e C 0 2 e v o lv e d f r o m t h e in c u b a t in g s o ils w a s c o lle c t e d i n K O H s o lu t i o n , a n d t h e t o t a l C 0 2
d e t e r m in e d a t in t e r v a ls b y t i t r a t i o n o f a n a li q u o t w i t h s ta n d a r d H C 1 a f t e r a d d i t io n s o f B a C l2
s o lu t i o n . T h e 14C 0 2 w a s d e t e r m i n e d b y a c i d i f y i n g a n a li q u o t a n d c o lle c t in g t h e I4 C 0 2 r e le a s e d in
2 m l o f N C S r e a g e n t i n a s c i n t i l l a t i o n v ia l f i t t e d w i t h a n a d s o r p t i o n t o w e r . T h e to w e r c o n te n ts
w e r e w a s h e d i n t o t h e v ia l w i t h P P O t o lu e n e c o c k t a i l ( 0 . 5 % w t / w t , 2 , 5 - d ip h e n y lo x a z o l e i n t o l u e n e ) ,
a n d t h e a c t i v i t y d e t e r m in e d i n a B e c k m a n l i q u i d s c i n t i l l a t i o n s y s te m . T h e a c t i v i t y o f t h e c o n i f e r y l
a lc o h o ls , D H P lig n in s , c o m s t a lk s , m o d e l h u m i c a c id s , r e s id u a l s o ils a n d s o il f r a c t i o n s w a s d e t e r m in e d
b y c o m b u s t io n a t 9 7 0 C i n t h e p r e s e n c e o f a m e t a l c a t a ly s t a n d a s tr e a m o f p u r e 0 2 . T h e 14C 0 2
r e le a s e d w a s c o lle c t e d i n N C S r e a g e n t a n d t h e a c t i v i t y d e t e r m i n e d as in d ic a t e d a b o v e .
P o r t io n s o f t h e in c u b a t e d s o ils w e r e e x t r a c t e d w i t h 0 . 5 N N a O H , a n d t h e h u m i c a n d f u l v i c
a c id f r a c t i o n s o b t a in e d b y t h e c la s s ic a l p r o c e d u r e [ 2 6 ] . B e f o r e e x t r a c t i o n a n d c o m b u s t io n t h e
e n t ir e 1 0 0 g p o r t i o n s o f s o il w e r e f i n e l y g r o u n d i n a m e c h a n ic a l g r in d e r . P o r t io n s o f s o il w e r e a ls o
h y d r o l y s e d u n d e r r e f l u x w i t h 6 N H C 1 f o r 1 8 h , a n d t h e a c t i v i t y i n t h e h y d r o l y s e d s o ils a n d i n t h e
h y d r o ly s a t e s d e t e r m in e d .

R ESULTS

D e c o m p o s it i o n p e r c e n ta g e s f o r t h e 14C - la b e lle d c o n i f e r y l a lc o h o ls a re g iv e n i n T a b le I . B ased

o n t o t a l C 0 2 e v o lv e d , a b o u t 5 6 % o f t h e a d d e d c o n i f e r y l a lc o h o l C w a s lo s t d u r i n g t h e 2 8 w e e k s ’
in c u b a t i o n p e r i o d . B a s e d o n e v o l u t i o n o f 14C 0 2 , f r o m 4 6 t o 7 3 % o f t h e s p e c i f i c a l ly 14C - la b e lle d
c a r b o n s w a s lo s t d u r i n g t h is p e r i o d . I n a l l t e s ts a g r e a t e r lo s s o c c u r r e d f r o m t h e 1 0 0 0 p p m t h a n
f r o m t h e 1 0 0 p p m a d d i t io n s . T h e 2 - 14C ( s id e c h a in ) a n d r i n g - 14C w e r e d e g r a d e d a t a b o u t t h e s a m e
r a t e ( 4 6 t o 5 5 % ) w h e r e a s t h e 0 ,4 C H 3 c a r b o n s w e r e u t i l i z e d t o a g r e a t e r e x t e n t ( 6 0 t o 7 3 % ) .
A c c o r d i n g t o T a b le I I , t h e c o n i f e r y l a lc o h o l c a r b o n s w e r e h i g h l y s t a b iliz e d a g a in s t m i c r o b i a l
d e g r a d a t io n t h r o u g h lin k a g e i n t o m o d e l h u m i c - t y p e p o ly m e r s . O n l y 5 t o 7 % o f t h e r in g a n d 2 - s id e
c h a in c a r b o n s w e r e e v o lv e d as 14C 0 2 o v e r t h e 2 8 - w e e k in c u b a t i o n p e r i o d . T h e O C H 3 c a r b o n s w e r e
s l i g h t l y m o r e s u s c e p t ib le t o m i c r o b i a l u t i l i z a t i o n . A b o u t 1 4 % o f th e s e c a r b o n s w a s lo s t as 14C 0 2 .
T h e p e r c e n ta g e r e c o v e r y o f t h e a p p lie d 14C a c t i v i t y r a n g e d f r o m 9 8 t o 1 0 8 % .
T a b le I I I r e c o r d s t h e p e r c e n ta g e lo s s o f t h e s p e c i f i c a l ly 14C - la b e lle d c o n i f e r y l a lc o h o ls a f t e r
lin k a g e i n t o D H P lig n in s . A b o u t t h r e e t o f o u r t im e s as m u c h o f t h e a p p lie d C w a s e v o lv e d as w a s
n o t e d f o r t h e s a m e c a r b o n s l i n k e d i n t o t h e m o d e l h u m i c a c id s . L o s s e s o f t h e 2 - ( s id e c h a i n ) a n d
r in g - c a r b o n s r a n g e d f r o m 2 0 t o 2 3 % , a n d lo s s e s o f t h e 0 14C H 3 c a r b o n f r o m 4 4 t o 5 1 % . R e c o v e r ie s
o f t h e a p p lie d a c t i v i t y v a r ie d f r o m 9 9 t o 1 0 8 % .
26 MARTIN and HAIDER

T A B L E I. D E C O M P O S IT IO N O F S P E C IF IC A L L Y C A R B O N - 1 4 - L A B E L L E D C O N IF E R Y L
A L C O H O L S IN G R E E N F IE L D S A N D Y L O A M

C oncentration D ecom p osition after (w eeks)

14C label
(ppm ) 'l 2 4 8 12 20 28

Percentage o f 14C evolved as 14C 0 2 a

0 14CH3 100 38 43 47 53 56 59 60
1000 54 60 63 67 70 73 73
2-14C 100 17 21 25 32 38 42 43
(side chain) 1000 i6 31 37 43 47 49 50
Ring-14C 100 18 23 28 32 36 44 46
1000 20 25 32 36 40 48 52

Percentage o f total added C evolved as CC>2 b

0 14CH3 1000 26 44 48 49 52 54 56
2-14C 1000 25 43 47 49 50 52 55
(side chain)

Ring-14C 1000 40 45 50 53 55 57 58

a Percentage o f recovered 14C ( 14C 0 2 + residual 14C in soil) evolved as 14C 0 2.

b Based on total C 0 2 evolved minus C 0 2 evolved from controls.

T A B L E II. D E C O M P O S IT IO N I N G R E E N F IE L D S A N D Y L O A M O F S P E C IF IC A L L Y
C A R B O N - 14 - L A B E L L E D C O N I F E R Y L A L C O H O L S L I N K E D I N T O M O D E L P H E N O L A S E
H U M IC P O L Y M E R S 3

D ecom p osition after (w eek s)b T otal 14C activity

I4C label
1 12 recovered0
2 4 8 20 28

‘H um ic acid’ fraction

0 14CH3 4 6 8 10 12 14 15 102
3 5 7 9 11 13 14 100
to

1 2 2 3 4 5 6 102
(sid e chain) 1 1 2 3 4 5 6 108

Ring-14C 1 2 3 4 5 6 7 106
1 2 2 3 4 5 6 99

‘Fulvic acid’ fraction

o 14c h 3 4 6 8 9 10 12 13 104
3 5 7 10 11 13 14 100
2-I4C 1 2 3 4 5 6 6 104
(side chain) 1 2 3 3 4 5 6 110
Ring-14C 1 2 3 4 5 6 6 105
1 2 2 3 4 5 5 98

a Polym ers from a m ixture o f 18 h ydroxytolu en es, p henols and ben zoic acid derivatives; soil addition
1 0 0 0 ppm .
b Percentage o f to ta l recovered activity evolved as 14C 0 2.
c 14C 0 2 evolved + residual 14C in soil
X 1 00 .
to ta l 14C activity added
 IAEA-SM-211/4 27

T A B L E III. D E C O M P O S IT IO N I N G R E E N F IE L D S A N D Y L O A M O F S P E C IF IC A L L Y
C A R B O N - 1 4 - L A B E L L E D C O N I F E R Y L A L C O H O L S L I N K E D I N T O D H P L I G N I N S 3*

Percentage decom p osition after (w eek s)b T otal 14C activity

14C label
1 2 4 8 12 20 28 recovered6*

0 14CH3 (a) 10 16 21 29 34 39 44 99
(a) 8 14 20 29 34 38 44 99
(b) 10 21 30 38 42 47 51 108
(b) 10 21 30 36 39 44 47 104
2-j4C 2 4 7 11 14 19 22 106
2 4 7 12 15 20 23 106
Ring-14C 2 4 7 12 15 18 21 110
2 5 7 12 14 17 20 109

a Soil addition 1 0 0 0 ppm.

b Percentage o f to ta l 14C recovered evolved as l4C 0 2.
c 14CO evolved + 14C in soil
---------- TJ--------------------------- X 1 0 0 0 .
to ta l C activity added

T A B L E IV . D E C O M P O S IT IO N I N G R E E N F IE L D S A N D Y L O A M O F S P E C IF IC A L L Y
C A R B O N - 14 - L A B E L L E D C O N I F E R Y L A L C O H O L S L I N K E D I N T O C O R N S T A L K L I G N I N S ,
L A B E L L E D B Y A B S O R P T I O N O F 0 14C H 3 , S I D E C H A I N O R R I N G L A B E L L E D F E R U L I C
A C ID R E S P E C T IV E L Y 3

Percentage decom p osition after (w eek s)3 T otal 14C activity

1 2 4 8 12 20 28 recovered6

o 14c h 3 7 18 30 40 46 51 54 98
7 17 28 38 43 48 50 97
l- l4C (CH2OH) 7 12 22 27 32 37 40 102
(side chain) 12 17 26 31 36 41 44 101
10 15 23 32 36 37 40 112
10 15 23 33 37 38 41 112
2 -i4C 4 7 9 12 15 23 25 108
(sid e chain) 4 6 9 13 18 24 28 108
i 3 4 5 15 26 28 94
i 4 11 17 21 27 30 96
3-14C 6 8 12 i6 19 24 27 102
(sid e chain) 6 9 13 17 20 25 28 111
7 12 17 23 27 33 35 104
7 12 17 22 26 32 34 98
Ring-14C 3 5 11 19 22 26 29 99
3 5 10 18 21 25 28 101

3 S o il addition 2 0 0 0 ppm .
b Percentage o f to ta l 14C recovered evolved as I4C 0 2.
c 14C 0 2 evolved + 14C in soil
----------- 7 2 -------------:-------------- X 100.
to ta l C activity added
28 MARTIN and HAIDER

TA B LE V. D E C O M P O S IT IO N O F M O D E L P H E N O L A S E P O L Y M E R S ,
D H P L IG N IN S A N D C O R N S T A L K S B A S E D O N T O T A L A D D E D
C A R B O N E V O L V E D A S C 0 2a

Percentage d ecom p osition after (w eek s)b Range at 28 weeks

1 2 4 8 12 20 28 (%>

DHP lignins

5 13 18 23 27 29 30 2 7 -3 4

M odel phenolase polym ers — hum ic acid fractions

1 2 3 3 4 4 5 3 -7

M odel phenolase polym ers — fulvic acid fractions

1 2 3 4 4 5 5 2 -7

Corn stalks

25 36 45 57 57 63 64 6 0 -6 6

a T otal C evolved as C 0 2 m inus C 0 2 evolved from controls.

b Averages o f all polym er and corn stalk treatm ents.

T h e r e la t e d c a r b o n s i n t h e c o r n s t a lk lig n in s w e r e s l i g h t l y m o r e a v a ila b le t o t h e s o il m ic r o b e s
( T a b le I V ) t h a n w e r e t h e s a m e c a r b o n s i n t h e D H P lig n in s . L o s s e s o f r in g - a n d 2 - c a r b o n s ra n g e d
fro m 25 to 30% . A b o u t 4 2 , 3 0 a n d 5 2 % o f t h e 1 - 14C ( C H 2O H ) , 3 - 14C a n d 0 14C H 3 c a r b o n s ,
r e s p e c t iv e ly , w e r e e v o lv e d as 14C 0 2 . T h e r e s id u a l 14C i n t h e in c u b a t e d s o ils p lu s t h a t e v o lv e d as
14C 0 2 c o n s t it u t e d f r o m 9 4 t o 1 1 2 % o f t h e a c t i v i t y a p p lie d . D e c o m p o s it i o n p e r c e n ta g e s b a s e d o n
t o t a l C 0 2 e v o lv e d s u p p o r t e d t h e 14C 0 2 e v o l u t io n d a t a ( T a b le V ) , o r w e r e c o m p a r a b le w i t h
p r e v io u s te s ts .
T h e d i s t r i b u t i o n o f r e s id u a l a c t i v i t y i n t h e s o il h u m i c a n d f u l v i c a c id s , a n d e x t r a c t e d s o il
f r a c t i o n s , a f t e r in c u b a t i o n o f t h e la b e lle d fr e e a lc o h o ls w a s e v e n ly d is t r i b u t e d i n t h e h u m i c a c id ,
f u l v i c a c id a n d e x t r a c t e d s o il f r a c t i o n s ( T a b le V I ) . W i t h a ll t h e p o ly m e r s a n d lig n in s t h e b u l k o f t h e
r e s id u a l a c t i v i t y w a s r e c o v e r e d i n t h e h u m i c a c id f r a c t i o n s . F r o m 3 6 t o 5 8 % o f t h e a p p lie d a c t i v i t y
w a s p r e s e n t i n t h e h u m i c a c id f r a c t i o n a f t e r in c u b a t i o n w i t h t h e D H P lig n in s . F o r t h e c o r n s t a lk
li g n i n a n d m o d e l h u m i c p o l y m e r s , t h e h u m i c a c id f r a c t i o n c o n t a in e d 2 5 t o 5 6 % o f t h e a p p lie d
a c tiv ity . T h e lo w e s t p e r c e n ta g e s w e r e r e p r e s e n te d b y t h e 0 14C H 3 c a r b o n s f o r t h e D H P lig n in s a n d
m o d e l h u m i c p o l y m e r s , a n d 1 - 14C a n d 0 14C H 3 c a r b o n s f o r t h e c o m s t a lk lig n in s .
T h e lo s s o f a c t i v i t y u p o n 6 N H C 1 h y d r o l y s is o f s e le c te d s o ils a f t e r i n c u b a t io n w i t h th e
s p e c i f i c a l ly 14C - la b e lle d p r o d u c t s is g iv e n i n T a b le V I I . T h e g r e a t e s t lo s s e s , 3 8 t o 4 7 % o f th e
r e s id u a l a c t i v i t y , w e r e f r o m t h e s o ils in c u b a t e d w i t h t h e fr e e a lc o h o ls . L o s s e s f r o m t h e s o ils
a m e n d e d w i t h D H P a n d c o m s t a lk li g n i n v a r ie d f r o m 1 8 t o 3 5 % a n d 2 7 t o 3 7 % r e s p e c t iv e ly . In each
g r o u p t h e le a s t lo s s o c c u r r e d f r o m t h e s o ils r e c e iv in g t h e r i n g - 14C - la b e lle d m a t e r ia l.

D IS C U S S I O N

I n c o m p a r is o n w i t h m a n y o t h e r p h e n o lic s u b s ta n c e s te s te d [ 1 6 , 1 7 ] , c o n i f e r y l a lc o h o l is
r e la t i v e l y r e s is t a n t t o m i c r o b i a l d e g r a d a t io n i n s o il. I n a 2 8 - w e e k i n c u b a t io n p e r i o d o n l y a b o u t
5 0 % o f t h e 2 - 14C ( s id e c h a in ) a n d r i n g - 14C , a n d 6 6 % o f t h e 0 I4 C H 3 c a r b o n s , w e r e e v o lv e d as 14C 0 2 .
T h is c o m p a r e s w i t h lo s s e s o f a b o u t 5 3 , 6 9 a n d 7 5 % r e s p e c t iv e ly o f c o m p a r a b le c a r b o n s i n f e r u l i c
a c id o v e r o n l y a 1 2 - w e e k i n c u b a t io n [ 1 7 ] , a n d w i t h g r e a t e r C lo s s e s f r o m b e n z o ic a n d v e r a t r ic
a c id s [ 1 6 ] .
 IAEA-SM-211/4 29

T A B L E V I. D IS T R IB U T IO N O F C A R B O N -1 4 A C T IV IT Y A F T E R S O IL IN C U B A T IO N W IT H
S P E C I F I C A L L Y C A R B O N - 14 - L A B E L L E D C O N I F E R Y L A L C O H O L S , F R E E A N D L I N K E D
IN T O D H P L IG N IN S , C O R N S T A L K L IG N IN S A N D M O D E L H U M IC P O L Y M E R S 3

D istribution o f activity15
Label l4C 0 2 Humic acid Fulvic acid E xtracted soil

Free alcohols
0 14CH3 73 9 9 9
2-14C (side chain) 50 17 16 17
Ring-I4C 52 18 14 16

M odel phenolase hum ic polym ers — hum ic acid fractions

s

15 46 14 25
o
о
X

2-14C (side chain) 6 50 21 23

Ring-14C 6 52 21 21

M odel phenolase hum ic polym ers — fulvic acid fractions

0 14CH3 14 47 13 26
2-14C (side chain) 6 55 20 19
Ring-I4C 5 56 19 20

DHP lignins
0 ,4 CH3 47 36 8 9
2-14C (sid e chain) 22 53 11 14
Ring-14C 21 58 11 10

Corn stalk lignins

0 14CH3 52 24 9 17
1-14C (side chain) 42 29 8 21
2-14C (sid e chain) 28 38 13 21
3-14C (side chain) 32 40 14 14
Ring-14C 28 41 13 18

a 1 0 0 0 ppm additions.
b Based on total activity recovered = 100%.

S in c e c o n i f e r y l a lc o h o l is a la b il e a n d v e r y e a s ily o x id iz e d c o m p o u n d , i t is p r o b a b le t h a t a
p o r t i o n o f t h e w h o l e c o n i f e r y l a lc o h o l m o le c u le s o r s o m e o f t h e e a r l y t r a n s f o r m a t io n p r o d u c t s a re
s t a b iliz e d i n s o il h u m i c f r a c t i o n s , o r i n m ic r o b ia l m e la n in s [ 1 2 , 1 7 , 2 7 , 2 8 ] . M ic r o b ia l m e t a b o lis m
w o u l d in v o lv e t h e f o r m a t i o n o f s im p le a l i p h a t ic c o m p o u n d s s u c h as a c e t ic a n d s u c c in ic a c id s . I f
a ll t h e a p p lie d c o n i f e r y l a lc o h o l w e r e m e t a b o liz e d t h r o u g h o x i d a t i o n t o f e r u l i c a n d v a n i ll ic a c id s ,
d e m e t h o x y l a t i o n t o c a f f e i c a n d p r o t o c a t e c h u ic a c id s a n d r in g c le a v a g e , m o r e t h a n 8 0 % o f t h e
a d d e d c a r b o n w o u l d h a v e e v o lv e d as 14C 0 2 f r o m t h e s o il [ 1 7 ] .
C o n i f e r y l a lc o h o l li n k e d i n t o m o d e l h u m i c a c id p o ly m e r s o r D H P a n d c o m s t a lk li g n i n s w e r e
m u c h le ss a v a ila b le t o t h e s o il o r g a n is m s . C e r t a in c a r b o n s , h o w e v e r , w e r e s t i l l m o r e r e a d i ly u t i l i z e d
th a n o th e rs . T h e c o n i f e r y l a lc o h o l , l i n k e d i n t o t h e m o d e l h u m i c a c id s m a d e f r o m a r e la t i v e l y
30 MARTIN and HAIDER

T A B L E V II. L O S S O F C A R B O N -1 4 A C T IV I T Y U P O N 6 N HC1
H Y D R O L Y S IS O F S O IL S A F T E R IN C U B A T IO N W IT H
S P E C I F I C A L L Y C A R B O N - 14 - L A B E L L E D C O N I F E R Y L A L C O H O L S
A N D L IG N IN S
The figures give percentage values o f the total activity remaining in
the soil after incubation

A ctivity lost upon 6N HC1 hydrolysis

Label
(%)

Free alcohols

0 14CH3 45

2-14C (sid e chain) 47

Ring-14C 38

DHP lignins

0 14CH3 35

2-14C (side chain) 26

Ring-14C 18

Corn stalk lignins

0 14CH3 37

2-i4C (sid e chain) 35

Ring-14C 27

c o m p l e x m i x t u r e o f p h e n o lic s u b s ta n c e s , w a s m o s t r e s is t a n t . A ft e r 2 8 w e e ks, o n ly a b o u t 6% o f
t h e r in g - a n d 2 - ( s id e c h a in ) c a r b o n s a n d 1 4 % o f t h e 0 14C H 3 c a r b o n s h a d e v o lv e d as l4 C 0 2 , a n d th e
b u l k o f t h e e v o lv e d C w a s lo s t d u r i n g t h e f i r s t 1 2 w e e k s . C o n i f e r y l a lc o h o l c a r b o n s li n k e d i n t o
lig n in s w e r e m o r e r e a d i ly a v a ila b le . A f t e r 2 8 w e e k s , 2 0 t o 3 0 % o f t h e r in g a n d 2 - s id e c h a in D H P
li g n i n c a r b o n s , a n d a b o u t 4 5 t o 5 0 % o f t h e O C H 3 c a r b o n s , h a d b e e n lo s t as C 0 2 , a n d d e g r a d a t io n
w a s c o n t i n u i n g a t a s te a d y b u t s lo w r a te . T h e p la n t l i g n i n d e c o m p o s e d a t a s l i g h t l y f a s t e r r a te
t h a n t h e D H P l i g n i n , w h i c h m a y b e e i t h e r r e la t e d t o t h e c lo s e a s s o c ia tio n o f t h e p la n t li g n i n w i t h
c e llu lo s e a n d o t h e r p l a n t p o l y m e r s o r t o a g r e a t e r a v a i la b ili t y o f t h e g ra s s li g n i n t y p e c o m p a r e d w i t h
t h a t f r o m c o n if e r s . I n t h e l i g n i n o f g ra s s e s , a ll t h r e e l i g n i n a lc o h o ls a re r e p r e s e n te d , w h e r e a s t h e
li g n i n o f c o n if e r s is p r i m a r i l y a p o l y m e r o f c o n i f e r y l a lc o h o l . T h e s e o b s e r v a t io n s s u g g e s t t h a t t h e
li g n i n , a lt h o u g h r e la t i v e l y r e s is t a n t t o m i c r o b i a l d e g r a d a t io n , w i l l c o n t in u e t o d e c o m p o s e s lo w ly ,
a n d t h a t g r e a t e r s t a b il iz a t io n o f t h e c o n s t it u e n t u n i t s o c c u r s w h e n t h e y a re l i n k e d i n t o t h e s o il
h u m i c m o le c u le s t h a t c o n t a in a m u c h g r e a t e r v a r ie t y o f c o n s t it u e n t u n i t s .
T h e b u l k o f t h e r e s id u a l c o n i f e r y l a lc o h o l c a r b o n s i n t h e lig n in s w a s r e c o v e r e d i n t h e h u m i c
a c id f r a c t i o n s . T h e s e o b s e r v a t io n s in d ic a t e t h a t b e f o r e l i g n i n h a s b e e n f u l l y d e c o m p o s e d o r
c o n v e r t e d t o h u m i c a c id i t w i l l b e s o lu b le i n d i l u t e N a O H s o lu t i o n s , a n d w i l l b e p r e c i p it a t e d w i t h
a c id s o t h a t t h e p r o c e d u r e , e s p e c ia lly f r o m s o ils r e c e n t ly a m e n d e d w i t h lig n a c e o u s r e s id u e s , w i l l
y i e l d h u m i c a c id t o g e t h e r w i t h p a r t l y d e c o m p o s e d li g n i n . T h e g r e a t e r u t i l i z a t i o n o f t h e O C H 3
g r o u p s b y t h e s o il o r g a n is m w a s r e f le c t e d i n a s m a lle r a m o u n t o f th e s e c a r b o n s i n t h e h u m i c a c id
a n d o t h e r s o il f r a c t i o n s w h e r e a s r in g c a r b o n s w e r e s o m e w h a t e n r ic h e d .
S o m e w h a t s m a lle r a m o u n t s o f t h e r e s id u a l r in g c a r b o n s o f t h e c o n i f e r y l a lc o h o ls w e r e lo s t
u p o n 6 N H C 1 h y d r o l y s is o f t h e i n c u b a t e d s o ils t h a n w e r e lo s t f r o m t h e O C H 3 a n d 2 - ( s id e c h a in )
c a rb o n s . T h is s u g g e s ts t h a t m o r e o f t h e l a t t e r l i g n i n fr a g m e n t s w e r e p r e s e n t i n m i c r o b i a l p r o d u c t s
s u c h as p r o t e in s a n d p o ly s a c c h a r id e s w h e r e a s m o r e o f t h e r in g c a r b o n s w e r e p r e s e n t i n t h e n o n -
 IAEA-SM-211/4 31

h y d r o l is a b l e p a r t . M a y a u d o n a n d B a t is t ic [ 2 9 ] r e p o r t e d f r o m s tu d ie s o n t h e b io lo g ic a l d e g r a d a t io n
o f u n i f o r m l y la b e lle d K la s o n ’s li g n i n t h a t t h is li g n i n d o e s n o t u n d e r g o a s e le c tiv e d e m e t h o x y la t i o n
d u r in g th e d e c a y p ro c e s s . H o w e v e r , K la s o n li g n i n is a h i g h l y d e n a t u r e d li g n i n w h i c h d o e s h o t
n e c e s s a r ily u n d e r g o t h e s a m e d e g r a d a t io n p ro c e s s e s as n a t u r a l l i g n i n p r e p a r a t io n s . G e n e r a l ly , s o il
h u m i c a c id s a re l o w i n m e t h o x y l g r o u p s [ 2 9 ] , a n d a ls o o n l y s m a ll a m o u n t s o f c o m p o u n d s a re
p r e s e n t w h i c h c a n b e d i r e c t l y tr a c e d b a c k t o li g n i n [ 3 0 , 3 1 ]. T h e p r e s e n t s t u d y a ls o s u g g e s ts t h a t
l i g n i n as s u c h , o r e v e n c h u n k s o f s t i l l h i g h l y m e t h o x y la t e d f r a g m e n t s , a re n o t s t a b iliz e d i n t h e
h u m ic c o m p o u n d s .
P u r e c u lt u r e s o f l i g n o l y t i c f u n g i , e s p e c ia lly w h i t e r o t f u n g i, c o m p l e t e ly m e t a b o liz e l i g n i n a n d
u s e i t as a s o u r c e o f c a r b o n a n d e n e r g y [ 9 , 3 2 ] . S im i la r o b s e r v a t io n s w e r e r e p o r t e d r e c e n t ly f o r
c e r t a in l i g n o l y t i c Fungi imperfecti is o la t e d f r o m s o il [ 6 , 8 ] . B r o w n r o t f u n g i, s e v e r a l f u n g i f r o m
a r a b le s o ils a n d s o m e b a c t e r ia [ 9 , T r o ja n o w s k i a n d H a id e r , u n p u b lis h e d ] e f f e c t i v e l y d e m e t h y la t e
l i g n i n , i n t r o d u c e a d d i t io n a l h y d r o x y l g r o u p s a n d in c re a s e t h e c a r b o n y l a n d c a r b o x y l c o n t e n t s
w h i c h le a d s t o a g r e a t e r s o l u b i l i t y [ 9 , 3 3 ] w i t h o u t a r a d ic a l d e g r a d a t io n o f t h e a r o m a t i c f r a m e w o r k .
O u r r e s u lt s i n d ic a t e t h a t s im i la r t y p e s o f r e a c t io n s m a y o c c u r a ls o d u r i n g t h e li g n i n d e g r a d a t io n i n
s o il. K i r k a n d c o - w o r k e r s [ 5 ] a ls o h a v e r e c e n t ly p r e p a r e d s p e c i f i c a l ly 14C T a b e lle d D H P lig n in s ,
a n d f o l l o w e d t h e re le a s e o f th e s e li g n i n c a r b o n s f r o m a f o r e s t s o il. D u r i n g a 2 5 - d a y i n c u b a t io n
p e r i o d , o n l y 4 t o 5 % o f t h e a p p lie d 14C w a s r e c o v e r e d as 14C 0 2 . I n o u r s tu d ie s , 7 % o f t h e 2 - s id e
c h a in a n d r i n g c a r b o n s a n d a b o u t 2 5 % o f t h e G C H 3- c a r b o n s w e r e r e le a s e d d u r i n g t h e f i r s t f o u r
w e e k s o f t h e 2 8 w e e k s ’ in c u b a t i o n p e r i o d . T h e s e d if f e r e n c e s c o u ld b e r e la t e d t o s o il p r o p e r t ie s
in c lu d in g t h e n a t u r e o f t h e m i c r o b i a l p o p u la t i o n . F u r t h e r te s ts a re n e e d e d u s in g a v a r ie t y o f s o ils
a n d w i t h v a r ia t i o n s i n t h e in c u b a t i o n c o n d i t io n s .

AC KNO W LEDG EM ENTS

T h e a u t h o r s t h a n k J .O . E r v in , H . L e m k e a n d E . P le is s f o r t e c h n ic a l a s s is ta n c e .

REFERENCES

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[3] SA R K A N EN , K .V ., LUDWIG, C.H. (E ds), Lignins, W iley-Interscience, N ew York (1 9 7 1 ) 1.
[4] D A G LEY , S „ Am . Sei. 6 3 ( 1 9 7 5 ) 68 1 .
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[6 ] H A ID ER , K ., DOMSCH, K .H ., Arch. M ikrobiol. 6 4 (1 9 6 9 ) 338.
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[9] KIRK, T .K ., “Chem istry o f lignin degradation b y w ood destroying fungi” , B iological T ransform ation o f
Wood b y Microorganisms (LIESE , W., Ed.), Springer-Verlag, Berlin, Heidelberg, N ew Y ork (1 9 7 5 ) 153.
[1 0 ] FELBECK, G .T ., Adv. Agron. 17 ( 1 9 6 5 ) 3 2 7 .
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substances” , Soil C om pon en ts 1: Organic C om ponents (GIESEKING, J.E ., E d .), Springer-Verlag, N ew York,
Heidelberg, Berlin (1 9 7 5 ) 1.
[1 2 ] H A ID ER , K., M ARTIN, J.P., FILIP, Z., “Hum us biochem istry” , Soil B iochem istry 4 (PA U L , E .A .,
M cLAREN, A .D ., Eds), Marcel D ekker, N ew York (1 9 7 5 ) 195. '
[1 3 ] M ARTIN, J.P., H A ID ER , K„ S oil Sei. 111 (1 9 7 1 ) 54.
[1 4 ] BO NDIETTI, E „ M ARTIN, J.P., H A ID ER , K ., S oil Sei. Soc. A m ., Proc. 3 6 (1 9 7 2 ) 5 97.
[1 5 ] KO NO NO VA , M.M., Sov. Soil S ei., N o. 7 (1 9 7 2 ) 27. j
[1 6 ] H A IDER , K., M ARTIN, J.P., S oil Sei. Soc. A m ., Proc. 39 (1 9 7 5 ) 6 5 7 .
[1 7 ] M ARTIN, J.P., H A ID ER , K„ Soil Sei. Soc. A m ., Proc. 4 0 ( 1 9 7 6 ) 3 7 7 . '
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[18] VERM A, L„ M ARTIN, J.P., H A ID ER , K ., Soil Sei. Soc. A m ., Proc. 39 (1 9 7 5 ) 2 79.

[19] FR EU D EN BER G , K., NIMZ, H„ Chem. C om m un. 1966 132.
[2 0 ] FR EU D EN BER G , K ., “T he con stitu tion and biosynthesis o f lignin” , C onstitution and B iosynthesis o f Lignin
(FR E U D E N B E R G , К ., NEISH, A .C ., Eds), Springer-Verlag, N ew Y ork, Heidelberg, Berlin (1 9 6 8 ) 1.
[21] NIMZ, H„ M OGHARAB, I., LÜDEM AN N , H .D ., M akromol. Chem. 175 (1 9 7 4 ) 2 5 6 3 .
[2 2 ] H A IDER , K .,J . Labelled Compd. 2 (1 9 6 6 ) 174.
[2 3 ] H A ID ER , K „ LIM, S., J. LabeUed Compd. 1 (1 9 6 5 ) 294.
[24] KR A TZL, K „ BILLEK, G., H olzforschung 7 (1 9 5 3 ) 66.
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[26] STEVENSO N , F.J., “ Gross chem ical fractionation of-organic m atter” , M ethods o f Soil A nalysis 2
(BLACK, C .A ., Ed.), Am. Soc. A gronom y, Madison (1 9 6 5 ) 1404.
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[2 8 ] ELLIS, D .H ., G R IFFITH S, D .A ., Can. J. M icrobiol. 2 0 ( 1 9 7 4 ) 1 379.
[2 9 ] M A Y AU D O N , J., BATISTIC, L., Ann. Inst. Pasteur 1 1 8 ( 1 9 7 0 ) 191.
[3 0 ] SCHNITZER, M., K H AN, S.U ., H um ic Substances in th e Environm ent, M. D ekker, N ew York (1 9 7 2 ).
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[3 2 ] W ILDUNG, R .E., CHESTER, G., BREM NER, D .E ., Plant Soil 3 2 (1 9 7 0 ) 2 2 1 .
[33] KIRK, T .K ., CHANG, H„ H olzforschung 28 (1 9 7 4 ) 217; ibid. 2 9 (1 9 7 5 ) 56.

D IS C U S S IO N

F. A N D R E U X : W h e n y o u c o m p a r e t h e d e c o m p o s it i o n o f c o n i f e r y l a lc o h o ls i n t r o d u c e d i n t o
t h e s o il e it h e r i n t h e f r e e s ta te o r i n c o m b in e d f o r m , i n s y n t h e t ic o r n a t u r a l p o ly m e r s , th e r e a re
s t r i k i n g d if f e r e n c e s i n t h e k in e t i c s o f C 0 2 re le a s e b e t w e e n t h e f i r s t case a n d t h e s e c o n d .
I w o u l d l i k e t o k n o w w h e t h e r t h e d e c re a s e i n t h e i n i t i a l r a te o f d e c o m p o s it i o n o f c o m b in e d
p r o d u c t s is d u e s o le ly t o t h e i r s t a b il iz a t io n b y r e s is t a n t c h e m ic a l b o n d s , o r w h e t h e r y o u h a v e
a ls o o b s e r v e d a d e p r e s s iv e o r t o x i c e f f e c t o f th e s e p o l y m e r s o n m i c r o b i a l d e v e lo p m e n t ,
e s p e c ia lly o n t o t a l s o il r e s p ir a t io n .
J .P . M A R T I N : N u m e r o u s te s ts h a v e in d ic a t e d t h a t i n t h e q u a n t i t ie s u s e d t h e p h e n o ls a n d
m o d e l p o l y m e r s d o n o t e x e r t a t o x i c e f f e c t o n t h e s o il m ic r o b io lo g ic a l a c t i v i t y . W h e n s u b s ta n c e s
s u c h as g lu c o s e a n d v a r io u s p o ly s a c c h a r id e s a re i n t i m a t e l y a s s o c ia te d w i t h t h e p o l y m e r s t h e y
d e c o m p o s e j u s t as f a s t i n t h e s o il as w h e n a d d e d a lo n e . T h e fig u r e s a t t h e b o t t o m o f T a b le I
r e p r e s e n t t h e a v e ra g e c a r b o n lo s t , a n d a ll th r e e c a n b e r e g a r d e d as r e p lic a t e s . T h e 4 0 % f ig u r e
f o r o n e w e e k a p p e a rs t o b e o u t o f lin e .
 IAEA-SM-211/41

TRANSFORMATION D’ACIDES PHENOLIQUES SIMPLES

EN SUBSTANCES PARA-HUMIQUES
PAR DES MICRO-ORGANISMES DU SOL
J .- R . B A I L L Y , V . N K U N D I K I J E - D E S S E A U X , K . A G B E K O
L a b o r a t o ir e d e p e d o lo g ie ,
U n i v e r s i t e P a u l S a b a tie r ,
T o u lo u s e ,
F ra n c e

Abstract-Rdsumd

TRAN SFO R M A TIO N OF SIMPLE PHENOLIC ACIDS INTO PARAHUM IC SUBSTANCES BY SOIL
MICRO-ORGANISMS.
During the cu ltivation o f soil m icroflora in selective culture media based on sim ple p henolic acids, the
occurrence o f brow n substances resem bling hum ic acids was observed. The in flu en ce o f certain culture conditions
was studied, such as th e nature and concentration o f the phenolic com p ou nd , aeration and so forth , on this effect.
S om e o f the data obtained from biochem ical analysis (hydrolysis and infra-red spectroscop y) confirm the
relationship betw een these brown substances and som e o f the natural hum ic acids. More especially, th e results
obtained by infra-red spectroscop y show that the brown substances obtained by cu ltivation d iffer from natural
hum ic acids on ly by the fact that th e bands for the -C H 2 - , -C H 3 groups substituted in the Ф rings are o f low er
in ten sity and th e fact that -O H alcohol I and alcohol II are present in addition to -O H alcohol III and/or phenol.
Lastly, the m icro-organisms responsible for the reaction were studied. T w en ty-tw o strains producing black
substances were isolated. A high proportion belonged to the genus P s e u d o m o n a s , probably because o f its m obility
and fast reproduction rate, but oth er m icrobial groups were present. On the w hole, th e soil micro-organisms can
produce from sim ple p henolic com pounds, under q uite norm al con d ition s, substances that can be termed
‘parahum ic’.

T R AN SFO R M A TIO N D’ACIDES PHENOLIQUES SIMPLES EN SUBSTANCES PARA-HUM IQUES PAR

DES MICRO-ORGANISMES DU SOL.
Au cours de cultures de m icroflore de sol sur des m ilieu x selectifs ä base d’acides phenoliques sim ples, on
a observt l’apparition de substances brunes ressem blant ä des acides hum iques. On a exam ine l’in flu en ce de
certaines con d ition s de culture (nature e t concentration du co m p o st p h tn o liq u e, aeration, e tc .) sur ce p htn om en e.
Certains resultats d’analyse biochim ique (h ydrolyses, spectroscopie i.r.) confirm ent la parente entre ces substances
brunes e t certains acides hum iques naturels. En particulier, les resultats ob ten us par spectroscopie i.r. m ontrent
que les substances brunes ob ten ues par culture ne different de ces acides hum iques naturels que par une m oindre
m ten site des bandes correspondant aux groupem ents -C H 2“ , “ CH 3 sub stitu te sur les cycles Ф et par la presence
de -O H alcool I e t II en plus des -O H alcool III et/o u p henol. Enfin, on a aborde l’etu d e des m icro-organism es
responsables de la reaction. V ingt-deux souches productrices de substances noires on t e t t isolees. U ne forte
proportion appartient au genre P s e u d o m o n a s , probablem ent ä cause de la m o b ilitt et de la rapidite de m ultiplication
de ces germes, mais d’autres groupes m icrobiens sont reprtsen tts. Au tota l, des m icro-organism es du sol peuvent,
dans des con d ition s qui n’on t rien d’excep tion n el, produire ä partir de com p oses phenoliques sim ples des
substances que Гоп peut appeler para-humiques.

IN T R O D U C T IO N

L ’ h y d r o l y s e d e s a c id e s h u m iq u e s lib e r e c e r te s d e p e t it e s q u a n t i t e s d ’ a c id e s a m in e s e t d e
g lu c id e s , m a is e ile lib e r e p r in c ip a le m e n t d e s c o m p o s e s p h e n o liq u e s [ 1 ] . L e s a c id e s h u m iq u e s
a p p a r a is s e n t d e p lu s e n p lu s c o n s t it u e s d e c o m p o s e s p h e n o liq u e s d iv e r s e m e n t c o n d e n s e s , p o ly m e r is e s
et oxydes.

33
34 BAILLY et al.

C ’ e s t p o u r q u o i o n a e x a m in e i c i la p o s s i b ili t y d e n e o - f o r m a t i o n d e c o m p o s e s h u m iq u e s ä
p a r t i r d ’ a c id e s p h e n o liq u e s s im p le s ( m o n o m e r e s ) p a r d e s m ic r o - o r g a n is m e s d u s o l.

L e s p r e m ie r e s c u lt u r e s a y a n t a b o u t i ä la f o r m a t i o n d e s u b s ta n c e s b r u n e s , o n a s u c c e s s iv e m e n t:
- p r e c is e c e r ta in e s c o n d i t io n s p h y s ic o - c h im iq u e s d u b r u n is s e m e n t ,
- a b o r d c l ’ e t u d e b io c h im iq u e d e s s u b s ta n c e s b r u n e s o b t e n u e s ,
- is o le e t c t u d ie q u e lq u e s m ic r o - o r g a n is m e s c a p a b le s d e p r o d u ir e ces r e a c t io n s .

1. F O R M A T IO N D E S U B S T A N C E S B R U N E S P A R L E S M IC R O - O R G A N IS M E S D U S O L

O n a e x a m in e le s d e u x s e rie s d ’ a c id e s p h e n o liq u e s s im p le s s u iv a n te s :

S e rie C 6- C , S e rie C 6- C 3

A c id e c in n a m iq u e
A c id e o - O H - b e n z o lq u e A c id e o - O H - c in n a m iq u e
A c id e m - O H - b e n z o 'iq u e A c id e m - O H - c in n a m iq u e
A c id e p - O H - b e n z o i'q u e A c id e p - O H - c in n a m iq u e
A c id e 3 ,5 d i- O H - b e n z o lq u e

O n r e a lis e le m il ie u d e c u lt u r e c i-d e s s o u s , d e p H = 7 ,0 , q u i , e x c e p t e le c o m p o s e p h e n o liq u e ,

e s f u n iq u e m e n t m in e r a l:

S o lu t i o n s a lin e s ta n d a r d d e W in o g r a d s k y [ 2 ] 50 m l
S o lu t i o n d ’ o lig o - e le m e n t s 2 m l
N itr a te d ’ a m m o n iu m 2 g
C o m p o s e p h e n o liq u e en g e n e ra l 3 g
E a u d is t i ll d e — q u a n t i t e s ü f f is a n t e p o u r 1000 m l

L e m il ie u s a n s l ’ a c id e p h e n o liq u e e s t n e u t r a lis e p u is s t e r ilis e ä l ’ a u t o c la v e . L ’ a c id e p h e n o liq u e

m is e n s o l u t i o n e t n e u t r a lis e e s t s t e r ilis e p a r f i l t r a t i o n e t a jo u t e a s e p t iq u e m e n t . C e m il ie u p e u t ,
e v e n t u e lle m e n t , e t r e g e lo s e ä 1 5 g/1.
L e c o m p o s e p h e n o liq u e c o n s t it u e la s e u le s o u r c e ä la f o is d e c a r b o n e e t d ’ e n e r g ie ( c u lt u r e s
s e le c tiv e s s e lo n W i n o g r a d s k y [ 3 ] e t P o c h o n [ 4 ] ) .
C om m e inoculum, o n a u t il is e d ’ u n e p a r t l ’ h o r i z o n A , d ’ u n s o l f o r e s t ie r a c id e d e v e lo p p e
s u r te r r a s s e a llu v ia le e t la l i t i e r e v e g e ta le c o r r e s p o n d a n t e ( f o r e t ä d o m in a n c e d e Quercus pubescens ) ,
d ’ a u t r e p a r t u n s o l n e u t r e c u lt i v e s u r m o la s s e e t u n e l i t ie r e f o r e s t ie r e p r o c h e ( d o m in a n c eQuercus
pubescens) t o u j o u r s s u r m o la s s e , s o i t e n t o u t q u a t r e e c h a n t illo n s .
L e s m i l i e u x r e p a r t is e n f i o l e s d e R o u x o u d ’ E r le n m e y e r ( 3 0 0 m l / f i o l e ) s o n t e n s e m e n c e s a
p a r t i r d e 1 g d e s o l o u d e l i t i e r e f o r e s t ie r e ; c e la c o n s t it u e la c u lt u r e I. A p r e s d e v e lo p p e m e n t
m i c r o b ie n e t d is p a r it i o n d e la p lu s g r a n d e p a r t d u c o m p o s e p h e n o liq u e ( d o s a g e s e lo n F o li n -
C io c u lt e u [ 5 ] m o d i f i e p a r M o n t r e a u [ 6 ] , o n r e p i q u e s u r le m e m e m il ie u e t d a n s le s m e m e s c o n d i t io n s
u n e p r e m ie r e f o is ( c u l t u r e I I ) , p u is u n e s e c o n d e f o is ( c u lt u r e I I I ) a f i n d ’ f i lim i n e r le s s u b s ta n c e s
a p p o r te e s p a r Г inoculum i n i t i a l .
S a u f ca s p a r t ic u li e r , t o u t e s le s c u lt u r e s s o n t re a lis e e s ä 3 0 ° C . T o u s le s r e s u lt a t s e t d is c u s s io n s
u lt e r i e u r s p o r t e r o n t u n i q u e m e n t s u r le s c u lt u r e s I I I p r e c e d e m m e n t d e f in ie s .
A p r e s u n te m p s d e c u lt u r e d e 2 ä 5 jo u r s , o n c o n s t a t e :
— u n e m u l t i p l i c a t i o n m ic r o b ie n n e ,
— u n e c o l o r a t i o n m a r r o n p lu s o u m o in s in te n s e , m a is t o u jo u r s b e a u c o u p p lu s f o r t e e t p lu s
r a p i d e m e n t a p p a r u e q u e c e lle q u e Г о п p e u t o b s e r v e r d a n s le s t 6 m o in s n o n e n s e m e n c 6 s ,
— la d is p a r it i o n , o u d u m o in s la f o r t e d i m i n u t i o n , d e la t e n e u r e n c o m p o s e s p h e n o liq u e s [ 5 , 6 ] .
 IAEA-SM-211/41 35

On doit done admettre que l’acide phenolique sert a la fois au developpement microbien
et a la formation des substances brunes.
Un certain nombre de parametres peuvent influer sur les resultats:
— La nature du compose phenolique: l’acide o-OH-benzoique et l’acide o-OH-cinnamique
sont, parmi les composes examines, ceux qui donnent generalement la coloration la plus intense;
e’est pourquoi on les a beaucoup utilises par la suite; des essais paralleles ont montre que la
tyrosine donne aussi des resultats interessante.
— La concentration du compose phenolique: dans l’intervalle 1—6 g/1 d’acide o-OH-benzoique
ou d’acide o-OH-cinnamique, plus la concentration initiale est elevde, plus la diminution de cette
concentration est lente.
— L’apport d’eau de levure (10 ml/1) accelere le brunissement qui se produit 2 ä 3 jours
plus tot, mais ne modifie pas la coloration finalement obtenue.
— Dans l’intervalle 20—50°C, la temperature la plus favorable au developpement microbien
et au brunissement est d’environ 30°C, ce qui confirme bien l’importance du phenomene biologique,
puisqu’une oxydation chimique serait plus intense ä 50°C.

Mais un p h en o m en e sem b le p a rticu lierem en t interessant: Pour etudier l’influence de l’aeration,

au moins d’une maniere qualitative sommaire, on a realise des cultures dans trois conditions
differentes, toutes autres choses etant identiques:
Condition 1: 6paisseur du milieu = 7 cm, culture non agit6e
Condition 2: epaisseur du milieu = 2 cm, culture non agitee
Condition 3: epaisseur du milieu = 2 cm, culture agitee (80 mouvements/min).
On constate que la condition 2, correspondant ä une aeration moderee, permet d’obtenir la
teinte la plus intense.
II semble que cela doive etre mis en relation avec le fait [7] que dans l’oxydation des composes
phenoliques, Poxygene intervient non seulement comme accepteur d’electrons, mais aussi comme
substrat enzymatique. Et, en presence d’une grande quantite d’oxygene, les composes phenoliques
sont en grande partie oxydes jusqu’au terme ultime.
Par ailleurs, des numerations microbiennes, realisees aussi bien par nephelometrie que par
culture sur agar-agar, ont montre que le developpement microbien 6tait plus intense dans les
cultures agitees, oh le brunissement est faible.
Le brunissement maximal semble done lie a une aeration moderee, en correlation avec un
developpement microbien egalement modere.

2. ETUDE BIOCHIMIQUE DES SUBSTANCES BRUNES PRODUITES

2.1. Purification

Le schema de purification est presente ä la figure 1; on peut voir que les substances brunes
ne dialysent pas: ce sont done de grosses molecules; on a constate par ailleurs qu’elles ргё-
cipitent en milieu acide.

2.2. Hydrolyses

Le tableau I indique les echantillons trait es.

On a effectue des hydrolyses acides selon Bohm et Towers [8] (HCl 2N, 1 h, 100°C) qui
attaquent les liaisons ether-oxydes, et des hydrolyses alcalines [8, 9] (OHNa 2N, 12 h, temperature
ambiante, atmosphere d’azote) qui attaquent la liaison ester.
Les composes phenoliques liberes sont extraits par Tether ethylique en milieu acide [10],
separes par Chromatographie en couche mince [ 11 ] et identifies ä la fois par leur Rf (position
36 BAILLY et al.

CULTURE BRUNE

F I G .l. P u r ific a tio n d e s s u b s ta n c e s b r u n e s fo r m e e s d a n s d e s c u ltu r e s m ic r o b ie n n e s .

relative sur le Chromatogramme) et diverses reactions colorees [12]. Le tableau II indique les
resultats obtenus.
Alors que les micro-organismes n’ont initialement ä leur disposition qu’un seul compose
phenolique, on retrouve dans Thydrolysat plusieurs monomeres differents. Sous Taction des micro-
organismes, se produisent done des reactions qui ne sont pas une simple polymerisation du
compose initial:
— Des echantillons, pourtant assez proches par leurs conditions de formation, ont donne par
hydrolyse des listes differentes de monomeres; les conditions experimentales imposees laissent
done encore aux micro-organismes quelques degres de liberte; plus exactement, la selection des
micro-organismes par ces milieux pourtant assez rigoureux doit permettre la survie de micro-
organismes divers, capables done de produire des substances brunes quelque peu differentes.
— Les composes phenoliques liberes par hydrolyse des matieres brunes sont assez semblables
ä ceux que Ton obtient par hydrolyse d’acides humiques naturels [ 1]. Les micro-organismes du
sol sont done capables ä partir d’un acide phenolique de produire des matieres brunes qui, par ce
caractere, sont apparentees aux acides humiques naturels.
 IAEA-SM-211/41 37

TABLEAU I. CONDITIONS D’OBTENTION DES SUBSTANCES BRÜNES ET

ANALYSES EFFECTUEES

A nalyses effec tu ee s
S u b stan ce
E c h an tillo n ca rb o n e e In o cu lu m Eau de levure H y d ro ly se H y d ro ly se S p ectro sco p ie
initiale acide alcaline i.r.

c A cide A i, sol avec + +

o-O H -benzoique fo restier
2 s/1 acide

D A cide A b sol avec + + +

o-O H -benzo'ique fo re s tie r
2 g/1 acide
E A cide A b sol sans + + +
o-O H -benzoique fo restier
2 g/1 acide

F A cide L itie re A q, avec + + +

o-O H -cinnam ique sol fo restier
2 g/1 acide

TABLEAU II. COMPOSES PHENOLIQUES MIS EN EVIDENCE

DANS LES HYDROLYSATS DES SUBSTANCES BRUNES

E chan tillo n
C om pose p h en o liq u e c D E F

A cide o-O H -benzoique X

A cide p -O H -benzoique + + +

A cide 3 ,4 di-O H -benzoique X +

A cide 2,5 di-O H -benzoique +
H y d ro q u in o n e X X

P yrogallol + +
G uaiacol +
D im e th y lp h e n o l n o n precise + X X + +
C orps n o n identifi6s 1 3 2 2

x: H y drolyse alcaline ä fro id

• : H y d ro ly se acide ä ch a u d

2.3. Spectroscopie infrarouge

On a utilise la technique des pastilles de KBr et un spectrophotometre UNICAM SP 200 G

ä rdseau.
La figure 2 presente les spectres des substances brunes obtenues par des cultures microbiennes
et les spectres de la fraction «molecules moyennes» d’acides humiques naturels fractionnes sur
gel de dextrane [13, 14].
38 BAILLY et al.

ГУ.

F I G .2 . S p e c t r e s I r . d 'a c id e s h u m i q u e s n a tu r e ls d e d im e n s io n s m o le c u la ir e s «m o y e n n e s » [1 3 , 1 4 ] e t d e s u b s ta n c e s

b r u n e s o b te n u e s p a r c u ltu r e s s u r a c id e p h e n o liq u e . 1: a c id e s h u m iq u e s e x tr a i ts d 'u n s o l g r is f o r e s tie r 1 6 - 2 9 cm ,

2: a c id e s h u m iq u e s e x tr a i ts d 'u n tc h e r n o z io m e 2 0 - 3 5 cm , D , E : c u ltu r e s s u r a c id e o -O H -b e n z o iq u e (v o ir

ta b le a u I ), F : c u l t u r e s u r a c i d e o - O H - c i n n a m i q u e f v o i r ta b l e a u 1 ). L e s s p e c tr e s s o n t e n r e g is tr e s e n p o u r c e n ta g e

d e lu m ie r e tr a n s m is e .

R essem blan ces [ 14]

— La bande 3400 cm"1correspond aux -OH lies constituant de longues series.

— Le pic 1640 cm"1 correspond aux doubles liaisons C = C (probablement benzeniques)et C= О
(cetonique et -COO"), avec l’epaulement П40 cm“1 des -COOH.

D ifferen ces

— La bande 1000—1100 cm"1 oil se trouve, vers 1035 cm“1, la bande C-O-C, est moins intense
chez les substances brunes provenant de cultures, et ой agissent les groupements substitues sur les
cycles. Mais il faut rappeier ä ce propos que les echantillons D et E sont formes ä partir d’acide
o-OH-benzolque, dcpourvu de tels groupements.
— Les acides humiques naturels absorbent vers 1400 cm“1, et non vers 1300 cm"1, ce qui
indique des - OH alcool III et/ou phenol. Par contre, les substances brunes absorbent aussi a
1300 cm"1, ce qui indique des - OH alcool I et II en plus des - OH alcool III et/ou phenol.
— Par la spectroscopie i.r., les substances brunes extraites des cultures ne different des acides
humiques naturels de «dimensions moyennes» que par une moindre teneur en -CH, -CH2- , -CH3,
 IAEA-SM-211/41 39

TABLEAU III. SOUCHES MICROBIENNES PRODUCTRICES DE SUBSTANCES

PARA-HUMIQUES

C om pos6 p h 6 n o liq u e su r
S o u ch e G enre
le q u el la so u ch e a 6te isolee

'B S 2 P seu d o m o n a s A cide o -O H -benzoique

■ BS02 P seudom onas A cide o -O H -benzoique

,G D 5 P seudom onas A cide o -O H -benzoique

BS4 P seu d o m o n a s A cide o -O H -benzoique

BS5 A c h r o m o b a c te r A cide o -O H -cinnam ique

JB S6 P seu d o m o n a s A cide o -O H -cinnam ique

1 b S06 P seu d o m o n a s A cide o -O H -cinnam ique

BS8 P seu d o m o n a s A cide 3,5-d i-O H -b en zo iq u e =

A cide a-r£ so rcy liq u e
JB S 1 0 P seudom onas T y ro sin e

I bsh P seudom onas A cide o -O H -cinnam ique

rG B 6 P seudom onas A cide o -O H -cinnam ique

GSB5 P seu d o m o n a s A cide o -O H -cinnam ique

G SB 05 P seu d o m o n a s A cide o -O H -cinnam ique

GC5 P seu d o m o n a s A cide o-O H -cin n am iq u e

G L3 P seudom onas A cide o -O H -cinnam ique
^G SC5 P seudom onas A cide o -O H -benzoique
G SA 5 P seu d o m o n a s A cide o -O H -cinnam ique
BS3 A c h r o m o b a c te r A cide o-O H -cinnam ique
■ BA4 A c h r o m o b a c te r A cide o -O H -benzoique
.BB5 A c h r o m o b a c te r A cide o -O H -benzoique
BSOl N o c a r d ia A cide o -O H -benzoique
BS7 F u n g i im p e r fe c ti A cide o -O H -benzoique

peut-etre due au choix des substances initiales, d’une part, et la presence des - OH alcooU et II
en plus des - OH alcool III et/ou phenol d’autre part.
Compte tenu des resultats de la spectroscopie i.r. et des hydrolyses et aussi d’autres resultats
en spectroscopie u.v. et en electrophorese [15], les substances brunes obtenues par culture paraissent
bien meriter l’appellation de substances para-hum iques.

3. ETUDE DES MICRO-ORGANISMES PRODUCTEURS DE SUBSTANCES PARA-HUMIQUES

A partir des cultures en milieu liquide produisant des substances para-humiques, on a isole, par
les techniques classiques (dilution, epuisement, etc.) un certain nombre de souches microbiennes
que Ton a cherche ä situer dans la classification [16].
Le tableau III presente les resultats obtenus. Les accolades regroupent les souches qui, quoique
provenant de travaux d’isolement distincts, ont paru semblables pour les caracteres examines.
Bien entendu, il s’agit lä uniquement de souches qui ont conserve, apres isolement, la
propriete de former des substances brunes ä partir de l’acide phenolique sur lequel elles s’etaient
d£velopp6es.
40 BAILLY et al.

De plus, on n’a ici que des souches pures, ce qui climine d’eventuelles associations micro-
biennes. Enfin, ce travail a ete mene uniquement sur agar-agar, et Гоп peut penser que 1’emploi
de silico-gels aurait permis l’isolement d’autres micro-organismes.
Un grand nombre de souches isolees peuvent etre attribuees aux genres P seudom onas et
A c h ro m o b a c te r; ces micro-organismes sont mobiles par des flagelles et ont un «temps de generation»
assez bref. II est done probable que Гоп a isole preferentiellement les germes les plus mobiles et
qui proliferaient le plus dans les milieux de culture. II reste cependant que de tels germes, presents
dans le sol, ä multiplication rapide, ä forte mobilitc, sont capables, ä partir de composes phenoliques
simples, de synthetiser des substances para-humiques.
Par ailleurs, le tableau III montre qu’un compose phenolique peut donner des substances
brunes sous Taction de souches nettement differentes. A l’inverse, des souches ayant les memes
caracteres (par exemple GL3 et GSC5) ont ete isolees sur deux composes differents. De plus les
souches BS5 et BS6, isolees sur acide o-OH-cinnamique et repiquees sur acide o-OH-benzoique,
continuent, quoique plus lentement, ä у produire des substances brunes.
Bien qu’on ne puisse encore generalise^ il semble done qu’il n’y a pas une grande specificite
des souches microbiennes pour les divers acides phenoliques.
Enfin, la presence dans la liste de Nocardia, actinomycetine, et de Fungi im p erfecti montre
que les eubacteries ne sont pas les seuls germes susceptibles de produire ces reactions. Des germes
appartenant ä d’autres groupes de la classification, peut-etre moins faciles ä mettre en evidence,
peuvent donner un resultat semblable.

CONCLUSION

Lorsqu’on cultive une microflore de sol avec comme seule source de carbone un acide
phenolique, dans des conditions ad equates et qui n’ont den d’exceptionnel, il у a, sous faction
des micro-organismes, formation de substances brunes meritant l’appellation de substances
para-humiques. Parmi les germes responsables il у a une certaine diversity, et on a mis particuliere-
ment en evidence des P seudom onas et des A ch rom obacter, germes particulierement mobiles et
qui se multiplient rapidement.
Cela nous parait etre un resultat important qui s’accorde, entre autres, avec le point de vue
de Mangenot [17] qui ecrit: «N ou s pen son s qu e la p o lym erisa tio n b iologiqu e e st un even em en t
p ro b a b le dans un so l a c tif ».

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P lan t S oil, sous presse.
[1 5 ] B A IL L Y , J.-R ., N K U N D IK IJE -D E S SE A U X , V ., S u r la fo rm a tio n de su b stan ce s n o ire s ä p a rtir de p h en o ls
sim ples p a r des m icro-organism es du sol, P la n t Soil 4 3 (1 9 7 5 ) 2 3 5 —57.
[1 6 ] B R E E D , S .R ., M U R R A Y , E .G .D ., SM ITH , R .N ., e t al., Bergey’s M anual o f D ete rm in ativ e B acterio lo g y ,
7 th E d ., W illiam s an d W ilkins C o m p a n y , B altim o re (1 9 5 7 ).
[1 7 ] M A N G EN O T , F ., «O n th e o x id a tio n o f p h en o lic co m p o u n d s by b ac teria and sy n th esis o f p a ra h u m ic
c o m p o u n d s» , T rans. In t. S ym p. H u m u s e t P lan ta V , S tudies a b o u t h u m u s, P rague, 13—17 S ep t. 1 9 7 1 , 3 7 —42.

DISCUSSION

F. ANDREUX: You have used spectra of salified compounds, which on the one hand causes
you to lose information on the intensity of the bands for the -COOH group, and on the other
obscures the peak at 1610-1640 cm"1 which is characteristic of the conjugate double bonds
C=C or C=0. Have you tried to make these spectroscopic comparisons more accurate by
decationizing beforehand, for instance, with an IF resin?
J.-R. BAILLY: I have been able to remove the ions from these brown substances only very
imperfectly by dialysis. Decationization on resin would require larger amounts of material. We
are working on the production of the brown substances with a fermentor. If we obtain 10 litres
of culture in that way instead of 200 ml I think we could try the treatment you suggested.
H.W. SCHARPENSEEL: We usually correlate the hue (spectral regions) of the Munsell colour
charts primarily with the presence of iron oxides (10 YR, 7.5 YR with goethite, 2.5 YR, 10 R
with haematite, etc.). The effect of the organic matter is more manifest in the categories of value
and chroma. Can your characterization of the humic compounds by spectral region be considered
sufficiently reproducible?
J.-R. BAILLY: Such a correlation would be significant only for a given concentration of
brown substances and for a known liquid thickness. That is impossible at present, as I do not
have enough brown substances. The Munsell colour plates shown on the slides beside the microbial
cultures were only intended to correct any distortion of the colours by the photograph.
W. FLAIG ( S cien tific S ecretary): You indicate in your paper that you isolated 22 strains
which form dark-coloured substances. Is it known whether any of these strains synthesize
phenolic compounds by metabolism?
J.-R. BAILLY: I was referring to 22 isolates, some of which may belong to the same strain.
I do not know whether brown substances are synthesized within the microbe cell or outside it
under the influence of exo-enzymes, but the browning is connected with the microbes’ development,
and phenolic acid is the only organic compound present.
 IAEA-SM-211/42

CARACTERISATION ET TRANSFORMATIONS EN
MILIEU MULL D’UN MODELE HUMIQUE ISSU DE
L’AUTOXYDATION DU SYSTEME CATECHOL-GLYCINE
ET MARQUE SELECTIVEMENT AU CARBONE-14

F. ANDREUX*, Dorota GOLEBIOWSKA**, Therese CHONE*,

F. JACQUIN+, M. METCHE*

* CNRS, Centre de pedologic biologique,

Vandoeuvre-les-Nancy,
France

** Akademia Rolnicza,
Zaklad Fizyki,
Szczecin,
Pologne

+ Institut national polytechnique de Lorraine,

Ecole nationale superieure d’agronomie
et des industries alimentaires,
Nancy,
France

Abstract-Е ёзи тё

D E T E R M IN A T IO N O F T H E C H A R A C T E R IST IC S A N D T R A N SF O R M A T IO N S IN A M U L L M ED IU M O F A
H U M IC M O D E L D E R IV E D F R O M A U T O -O X ID A T IO N O F T H E C A T E C H O L -G L Y C IN E SY STEM A N D
S E L E C T IV E L Y L A B E L L E D W ITH C A R B O N -14.
B ro w n n itro g e n -c o n ta in in g p o ly m ers are p rep are d b y a u to -o x id a tio n a t p H 7 .9 fro m a n eq u im o la r m ix tu re o f
c a te c h o l a n d glycine. S tric t c o n tro l o f th e e x p e rim e n ta l c o n d itio n s y ie ld s p ro d u c ts o f c o n s ta n t c o m p o sitio n , an d
a ffo rd s a basis fo r th e selective p re p a ra tio n o f th re e p o ly m ers labelled w ith 14C o n th e a to m s C , a n d C ; o f th e g ly cin e
a n d o n th e c a te c h o l ring. T h e se p o ly m e rs have a m e a n m olecu lar size sm aller th a n th a t o f h u m ic acid s e x tra c te d
fro m n e u tra l soils b u t th e ir s tru c tu re resem b les m o re th a t o f h y d ro ly tic resid u e s fro m e u tro p h ic h u m ic acid s an d
m elan in s. T h e ir s to ic h io m e tric ra tio is 2 m oles o f gly cin e t o 7 m o les o f ca te c h o l. T h e y are n o t c o m p le te ly h o m o ­
g en e o u s a n d c o n ta in tw o g ro u p s o f su b stan ce s: th e first a n d m o re a b u n d a n t g ro u p is larger an d p e rm its o p tim u m
sta b iliz a tio n o f th e g ly cin e in h ig h ly re s o n a n t s tru c tu re s ; th e seco n d g ro u p rem ain s m o re sensitive to h y d ro ly tic
tr e a tm e n t an d b io d e g ra d a tio n ; th e y are ch a ra cte rized b y p a rtia l fission o f th e a ro m a tic rin g s d u rin g sy n th esis, w h ich
is re sp o n sib le f o r th e fo rm a tio n o f th e ring-deriving -C O O H gro u p s.T h e b io d e g ra d a tio n o f th e se p o ly m ers in a n e u tra l m u ll
reveals tw o successive stages: (a) a n in itia l stage lasting five days, d u rin g w h ic h th e ring-deriving -C O O H g ro u p s are
a c tiv ely d e c a rb o x y la te d , w h ereas th e g ly cin e b o u n d to th e less stab le fra c tio n is d eg rad ed p re fe re n tia lly a n d th e n
m e tab o lized in th e u n e x tra c ta b le m icro b ia l su b stan ce s (m icro b ial h u m in ). T h e n o n -am in o resid u e s o f th e p o ly m ers
th e n h av e a d ep ressan t e ffe c t o n soil re s p ira tio n , w h ic h disappears o n ly w h en th e ir tra n s fo rm a tio n b y d e g ra d a tio n o r
re a c tio n w ith th e soil o rg an ic c o m p o u n d s o cc u rs; (b ) A second stage, ch a ra cte rized b y v ery slow m in eralizatio n and
affe c tin g a lm o s t exclusively th e m o re s tab le p o ly m ers in c o rp o ra te d in to th e h u m in b y p h y sico -c h em ic al in s o lu b iliz atio n .
T h e re is th e n n o f u r th e r p re fe re n tia l d e g ra d a tio n o f th e glycine, th e s ta b ility o f w h ich eq u als th a t o f th e ring-deriving
c a rb o n c o m p o u n d s o f a ro m a tic origin. T h e s tu d y show s th a t th e in c o rp o ra tio n o f am in o -acid resid u e s in to p o ly m ers
o rig in atin g fro m q u in o n e stru c tu re s re p re s e n ts a n im p o rta n t fo rm o f storag e f o r org an ic soil n itro g en .

C A R A C T E R IS A T IO N E T T R A N S F O R M A T IO N S EN M ILIEU M U L L D ’U N M O D E L E H U M IQ U E ISSU D E
L ’A U T O X Y D A T IO N D U S Y STE M E C A T E C H O L -G L Y C IN E E T M A R Q U E SE L E C T IV E M E N T A U C A R B O N E-14.
D es p o ly m eres b ru n s az o tes s o n t p re p a re s p a r a u to x y d a tio n ä p H 7 ,9 d ’u n m elange eq u im o le cu laire de ca tech o l
e t de g lycine. U n c o n trö le rig o u re u x des c o n d itio n s ex p e rim en tale s c o n d u it ä des p ro d u its d e c o m p o sitio n c o n s ta n te ,

43
44 ANDREUX et а].

e t au to rise la p re p a ra tio n de tro is p o ly m eres s£lectivem ent m a rq u es au 14C su r les ato m es d e ca rb o n e C i e t C 2 d e la

glycine e t su r le cycle d u ca tech o l. Ces p o ly m eres p re s e n te rs u n en c o m b re m e n t m o lecu laire m o y e n in fe rieu r ä celui
des acides h u m iq u e s e x tra its des sols e u tro p h e s , e t le u r s tru c tu re se rap p ro c h e davantage d e celle des residus
d’h y d ro ly se des acides h u m iq u es n a tu re ls e t des m elanines. L eur ra p p o rt sto ec h io m e triq u e est d e 2 m oles de glycine
p o u r 7 m oles de ca tech o l. Ils n e s o n t pas to ta le m e n t h o m o g e n es e t c o m p o rte n t d eu x g ro u p es de su b stan ces; les
p rem ieres, p lu s a b o n d a n te s , s o n t de plus g rande dim ension e t p e r m e tte n t u n e stab ilisatio n o p tim ale de la glycine dans
des s tru c tu re s h a u te m e n t re so n n an tes; les secondes d em e u re n t plu s sensibles au x tra ite m e n ts h y d ro ly tiq u e s e t ä la
b io d e g ra d a tio n ; elles se ca ra c te rise n t p a r u n e o u v e rtu re p artielle des n o y a u x a ro m atiq u es au co u rs de la s y n th ese, q ui
est resp o n sab le de la fo rm a tio n de g ro u p e m e n ts -C O O H m atriciels. L a b io d e g rad atio n d e ces p o ly m eres d an s u n
m u ll e u tro p h e m e t e n evidence d e u x S tapes successives: a) U ne e tap e p rim aire de 5 jo u rs , au co u rs de laq u elle les
g ro u p e m e n ts -C O O H m a tric iels s o n t a c tiv em en t d ec arb o x y les, ta n d is q ue la glycine lice ä la fra c tio n la m o in s stab le
est dcgradee p re fe re n tic lle m e n t, p u is m etabolisS e dans les co rp s m icro b ie n s in e x tra c tib le s (h u m in e m icro b ie n n e). Les
residue n o n am ines des p o ly m eres e x e rc e n t alors u n e ffe t d ep ressif su r la re sp ira tio n d u sol, q ui ne d isp ara ft q u ’avec
le u r tra n sfo rm a tio n p a r d eg rad a tio n o u re a c tio n avec les com poses o rg an iq u es d u sol. b ) U ne e ta p e seco n d aire,
caractcrisee p a r u n e m in eralisatio n tre s le n te q u i affe c te p resq u e ex clu siv em en t les p o ly m eres les p lu s stables
in c o rp o res dans l’h u m in e p a r in so lu b ilisatio n phy sico -ch im iq u e. II n ’y a p lu s alors de d eg rad a tio n p refere n tie lle de
la glycine, d o n t la sta b ilite est dgale a celle des com poses c a rb o n e s m atriciels d ’origine a ro m a tiq u e . C ette e tu d e
m o n tre q u e l’in c o rp o ra tio n des rdsidus am ino-acides dans des p o ly m eres d eriv an t de s tru c tu re s q u in o n iq u es c o n s titu e
u n e fo rm e im p o rta n te de sto ck ag e de l’az o te org an iq u e des sols.

1. INTRODUCTION

La stabilisation de l’azote organique dans les composes humiques des sols atteint des degres
divers en fonction du type de structure chimique dans lequel il est engage. Les composes
hydrolysables en milieu chlorhydrique ä chaud, les plus abondants, correspondent ä Tammoniaque,
aux amino-acides, aux amino-sucres, ainsi qu’ä des formes combinces solubles difficilement identi-
fiables [1—6]. L’azote non hydrolysable, abusivement appele parfois «azote heterocycle» correspond
le plus souvent ä des structures complexes provenant de l’interaction entre ces molecules simples et
d’autres composes. Maillard [7] fut l’un des premiers ä montrer que les amino-acides donnaient lieu
ä des condensations avec les sucres pour former des composes noirs, les mclanoi'dines, qu’il comparait
d£ja aux extraits de terreau et de furnier; de faqon generale, tous les aldehydes et les cetones peuvent
participer ä des reactions analogues [8]. Dans les sols la plus grande partie des polycondensats azotes
trouvent leur origine dans la polymerisation oxydative des polyphenols liberes par degradation de la
lignine, des tanins, ou des pigments vegetaux ou microbiens. Sur les sites electrophiles des noyaux
Ortho- ou Paraquinoniques ainsi form6s peuvent alors se fixer les groupements amines, en particulier
ceux des amino-acides N-terminaux des polypeptides [6, 9-11]. Ces reactions, apparentees aux
ph6nomenes de tanage, sont extremement gen6rales et sont egalement avancees pour expliquer la
biosynthese des melanines [12—18]. Lorsque de tels composes sont soumis ä une hydrolyse energique,
seule leur partie «peripherique» se trouve scindee au niveau des liaisons peptidiques, tandis que sub­
sisted les residus amino-acides directement lies ä la matrice polycondensee. La structure chimique
de ces combinaisons est souvent difficile ä elucider, en raison du nombre eleve de leurs precurseurs
phenoliques et des rearrangements profonds qu’elles subissent au cours de la pedogencse. De plus,
leur isolement est souvent malaise car elles peuvent etre modifiees par les reactifs d’extraction. C’est
pourquoi on fait souvent appel ä des systemes modeles: les modeles naturels, en particulier les
melanines fongiques ou les phytomelanines, possedent souvent une structure encore trop complexe,
et il semble qu’on puisse avantageusement leur substituer des modeles synthetiques obtenus en
conditions controlees. Le modele le plus courant correspond au Systeme catechol-glycine; les
mecanismes qui regissent sa preparation sont ä l’heure actuelle bien connus, tant en ce qui conceme
l’oxydation du catechol en quinone [19, 20], que la formation de produits d’addition glycine plus
catechol [21, 22]. Les reactions connexes de degradation oxydative de la glycine, avec formation
d’ammoniaque et d’acide glyoxylique, ont egalement ete etudiees et sont encore sujettes ä differentes
interpretations [21, 22]. Enfin, l’etude des polymeres noirs azotes, qui proviennent de la stabilisation
 IAEA-SM-211/42 45

finale des radicaux semi-quinoniques intermediaries, a fait l’objet de nombreux travaux mettant
indifferemment en jeu des techniques d’oxydation chimique ou enzymatique [23—28]. Les trans­
formations des polyphenols dans les sols ont ete etudiees, souvent par des methodes radioisotopiques,
et portent soit sur des composes libres (29, 30], soit sur leurs polycondensats syntltetiques [31,32].
Toutefois, peu d’auteurs ont jusqu’ä present etudie simultanement les relations structurales existant
entre les conditions de formation de tels polymeres et leur stabilite chimique et biologique. Nous
avons tente d’atteindre un tel resultat en recherchant des conditions moderement autoxydantes et
rigoureusement contrölees permettant une synthese reproductible de polymeres azotes ä partir du
Systeme catechol-glycine. Ces conditions etant fixees, il a ete possible de synthetiser des polymeres
marques selectivement au 14C et de les soumettre apres caracterisation ä differentes conditions de
degradation, soit par voie chimique, soit au contact de la microflore d’un sol ä mull.

2. METHODES EXPERIMENTALES

Un melange equimoldculaire 0,03M de catechol et de glycine dans un tampon phosphate

NaH2P04/Na2HP04 de pH 7,9 est soumis ä agitation pendant 5 jours dans un reacteur traverse par
un courant d’oxygene pur, a 20°C et ä l’obscurite. En cours de reaction, on effectue des preleve-
ments dont on enregistre le spectre d’absorption u.v.-visible (spectrophotometre «Beckman Acta 1»),
Une experience analogue est realisee avec 3 ml de melange, dans des fioles de Warburg de
15 ml montees sur un microrespirometre Gilson permettant de determiner la cinetique de
consommation d’oxygene par le milieu ä 20°C et sous pression normale.
Afin d’effectuer des mesures de chimiluminescence, 3 ml de melange sont preleves ä intervalles
reguliere et introduits dans une cuve en quartz placee contre la fenetre d’un photomultiplicateur
RCA 6655 A, dont la sensibilite maximale est atteinte pour 440 nm, et Ton enregistre le nombre
d’impulsions emises par unite de temps [33].
Trois polymeres sont prepares en reacteur avec 100 ml de solution equimoleculaire:0,03M,
en utilisant des precurseurs marques selectivement au 14C et de radioactivites specifiques connues:

0 catechol + glycine 14Ci

u) catechol + glycine 14C2
iii) catechol l4Cu‘ + glycine

A la sortie de chaque reacteur, le courant gazeux traverse deux pieges successifs contenant
15 ml de NaOH2N, destines ä mettre en Evidence les degagements eventuels de C02. En fin de
reaction, chaque solution est dialysee ä 5°C contre eau distilrie; les dialysats sont stockes au froid,
et les polymeres selectionnes par dialyse sont decationises sur resine Dowex 50 W X 8, 50—100 mesh
(H+), lyophilises et conserves sous vide.
La distribution des sites marques entre effluents gazeux, dialysats et polymeres est determinee
au moyen du spectrometre ä scintillation liquide Packard 3003 equipe d’un dispositif de standardisa­
tion externe [34]. Un bilan de l’azote est effectue par dosage dans les dialysats de l’azote ammoniacal
distillable [35] et de l’azote a-amine libre [36]. La presence de produits carbonyles de degradation
oxydative de la glycine est recherchee par preparation de dinitro-2,4-phenylhydrazones, Chromato­
graphie sur papier avec differents solvants [37] et autoradiographie des chromatogrammes.
Les polymeres sont ensuite soumis ä une etude physico-chimique: filtration sur gels de dextrane
Sephadex G 100 et G 50 superfine, en presence d’un tampon tris/HCl de pH 7,5; analyse elementaire
par combustion en four ferme (analyseur Carlo Erba 1104); spectre d’absorption infrarouge, au
moyen du spectrophotometre Beckman IR 10, apres pastillage dans KBr anhydre; determination des

M arquage u n ifo rm e d u n o y a u aro m atiq u e.

46 ANDREUX et al.

acidites COOH et OH par titration en continu dans l’eau distillee par NaOH 0,01N (Urectron 5,
Tacussel).
Vingt mg de polymeres sont soumis ä une hydrolyse acide par ebullition ä reflux dans 40 ml de
HCl 3N durant 3 heures. Les hydrolysats sont filtres sur membrane Millipore, concentres sous vide
jusqu’a elimination de l’acide, puis redisperses dans l’eau. La solution obtenue est alors percolee sur
une colonne de resine Dowex 50 W X 8, 50—100 mesh (H+), et les produits fixes sont elues ä l’aide
d'une solution de NH4OH 1,0N. Les dosages de C et N hydrolysables sont effectues, et l’on mesure
la distribution des differents sites marques 14C entre les fractions cationiques et anioniques.
Les polymeres selectivement marques 14C sont soumis ä une incubation d’un mois dans un mull
de sol brun lessive forestier dont les principales caractdristiques sont les suivantes:

pH dans l’eau = 5,6 Ferlibre = 2,3%

CEC = 16,2meq/100g C = 3,5%
Saturation en bases = 74,0% N = 0,25%
Argiles =22,6% C/N =14,0%

Des comparaisons sont realisees avec les precurseurs marqufis, glycine 14C!, glycine 14C2,
catechol 14CU; introduits ä l’etat libre dans le sol en quantites äquivalentes ä celles introduites sous
forme combinee dans les polymeres. Le sol seche ä l’air et tamise a 2 mm est reparti en lots de 20 g
dans des erlenmeyers de 500 ml. Chaque lot regoit respectivement 27 g de polymeres (1350 ppm),
3,4 mg de glycine (170 ppm) et 21,4 mg de catechol (1070 ppm), en solution dans un volume d’eau
calcule pour porter le sol ä Thumidite Äquivalente. Les erlenmeyers sont ensuite places dans un
bain thermostate ä 28°C et relies ä un Systeme clos muni d’un dispositif de circulation d’air [34].
Le C 02 dägage par les echantillons est regulierement entrafne par un courant d’air humide exempt
de C02, et piege ä la sortie des incubateurs dans une solution de NaOH 0,5N dont on determine la
radioactivity et que Ton peut titrer en retour par HC1 0,2N.
Apres incubation, les sols sont seches ä 40°C et leur radioactivity est determinee par combustion
dans un analyseur Wösthoff-Carmhograph 8 modifiy. Les essais de redissolution des produits
residuels sont effectuÄs en agitant 1 g de sol successivement avec 25 ml des reactifs suivants: H20,
tampon NaOH/tytraborate de sodium ä pH 9,7, pyrophosphate de sodium ä 1%, NaOH 0,1N [38].

3. RESULTATS ET DISCUSSION

3.1. CinÄtique de la rdaction d’oxydation

3 .1 .1 . S p ectres d ’ab so rp tio n u. v.-visible

Une pigmentation rouge se developpe des le depart de la reaction, se traduisant par une bande
d’absorption centräe ä 480 nm, dont l’intensite maximale est atteinte apres 25 min. Cette coloration
est indicatrice de la formation d’une combinaison entre glycine et catechol [21, 27]. On n’observe
par contre ä ce stade aucune modification des absorptions dans l’ultra-violet, caracterisees par les
maxima ä 215 nm (e max = 6300) et 275 nm (e max = 2300) propres au catechol.
Apres 5 heures, la preparation prend une teinte brun-verdätre coi'ncidant avec l’apparition d’un
maximum spectral ä 610— 620 nm, dü probablement ä la transition n -*• -n* du groupement 1C = 0 de
1'0. benzoquinone, tandis qu’on observe une augmentation sensible de l’extinction dans l’ultra-violet
(fig. 1). Un nouvel epaulement apparaft a 320— 330 nm, que Ton peut attribuer ä une combinaison
glycine-catychol, car on ne l’obtient pas avec le catechol seul. Apres 24 heures, la coloration brun-
verdätre s’accentue, puis la preparation prend une teinte brun-fonce, accompagnee d’un accroissement
general de Tabsorption, avec effacement parallele des maxima. L’extinction continue ainsi ä croftre,
parallelement au brunissement, jusqu’a l’obtention d’une valeur stable ä partir du quatrieme jour.
 IAEA-SM-211/42 47

F1G.1. V ariations au corns d u te m p s d u sp ectre u.v.-visible d e la p reparatio n ( C = 0 ,2 '1 0 ~ ъ m o le /l); P = p o ly m e re s

apres dialyse.

Le spectre est alors pratiquement monotone, avec decroissance progressive de l’extinction vers le
visible, comme e’est le cas pour les composes humiques stables extraits des sols [39, 40].

3 .1 .2 . O x yd a tio n du ca tech o l e t chim ilum in escence

La formation du pigment rouge apres 25 min n’affecte encore qu’une faible proportion de
molecules, comme en temoignent l’absence de modification du spectre u.v. et la faible intensity des
dchanges gazeux. La consommation d’oxygene s’etale sur 4 jours et sa cn^tique presente deux
etapes distinctes (fig. 2). La premiere etape, plus rapide, correspond aux 40 premieres heures de
reaction, et conduit ä la fixation d’environ 0,75 mole d’oxygene par mole de catechol introduit au
depart; la phase plus lente qui lui succede se termine par un palier correspondant ä la consommation
de 1,1 mole d’oxygene par mole de catechol.
La chimiluminescence, qui traduit le taux de desactivation des radicaux semi-quinoniques issus
de l’oxydation du catechol, montre d’abord une croissance pratiquement constante durant les
30 premieres heures, puis atteint un equilibre stationnaire durant 48 heures, avant de decroftre au
cours du quatrieme jour. II semble done que la desactivation des semi-quinones par polymerisation
n’atteint sa valeur optimale que lorsque la plus grande partie des molecules polyphdnoliques sont
oxyddes. Cette observation est parfaitement en accord avec les modifications du spectre u.v.-visible
apres 40 heures de reaction.
48 ANDREUX et al.

F IG .2. C in e tiq u e s com parees des echanges g a ze u x e t d e la c h im ilu m in escen c e au cours d e la syn th e se . A - C 0 2
p ro ven a n t d e la g ly c in e e t d u catechol; В = o x y g e n e c o n so m m e ; C = ch im ilu m in escen ce.

3 .1 .3 . D egradation e t p ro d u ctio n de СОг

La liberation de C02 par le milieu reactionnel interesse en premier lieu le groupement

carboxylique de la glycine, dont plus de 16% se trouvent decarboxyles (fig. 2). Elle est particuliere-
ment rapide en debut de synthese, puisque 13,5% des COOH sont d6ja degrades en 40 heures. Le
C2 de la glycine ne contribue pratiquement pas au degagement de C02, tandis que 3,8% de la radio­
activity du catechol у participent. Un tel degagement implique l’ouverture d’une proportion
 IAEA-SM-211/42 49

importante de noyaux aromatiques, qui peut atteindre 22,8% si Гоп admet qu’une molecule de
catechol libere une molecule de C02.
La desamination des amino-acides au cours de l’oxydation des polyphenols est liee ä l’oxydation
des combinaisons amino-phenoliques et au rearrangement des quinones ainsi formees [21, 22] ; eile
conduit ä la production d’un a-cetoacide, dont la decarboxylation peut etre spontanee en milieu
alcalin. La decarboxylation active de la glycine durant la premiere phase de reaction confirme que
les produits d’addition de la glycine sur le catechol se forment surtout au cours de cette phase et
qu’ils participent aux processus de polymerisation. L’ouverture des noyaux aromatiques se poursuit
durant la phase de polymerisation, et il n’est done pas exclu que des cycles ouverts participent ä la
structure des polymeres [41].

3.2. Identification des produits de reaction

3 .2 .1 . C o m p o sitio n des d ialysats

Dans les dialysats sont prdsents pres de 50% de la glycine initiale, et 17,2 ä 22,7% d’ions NH4+
(tableau I); l’acide glyoxylique provenant de la desamination de la glycine I4C2 est mis en evidence
par Chromatographie sur papier des DNP-hydrazones qui en derivent, suivie d’une autoradiographie.
On deduit du dosage des DNP hydrazones 14C2 que la quantite d’acide glyoxylique presente dans
le milieu correspondant ä moins de 2% des molecules de glycine de depart. L’acide glyoxylique a
done et€ decarboxyle ä mesure de sa formation en liberant du formaldehyde. Toutefois, il n’a pas
dte possible de mettre en evidence la DNP-hydrazone du formaldehyde, ce qui suggere que ce
compose s’est combine ä la glycine en donnant des bases de Schiff et des melanoidines [7, 8].

3.2.2. Caracterisation des p o ly m e re s .

Les polymeres lyophilises ont l’aspect d’une poudre brun-fonce, dont le poids atteint 16,4 ä
25,4% du poids total des composes de depart. Les bilans presentes au tableau II permettent d’observer
que ces polymeres incorporent 27,1% de la radioactivite du catechol, et moins de 10% de celle de
chacun des atomes Ci et C2 de la glycine.
Gräce au marquage selectif, on peut estimer que sur 23 atomes de carbone, 21 proviennent du
catechol et les deux autres respectivement des atomes Ct et C2 de la glycine. La coincidence entre
les nombres d’atomes d’azote et de carbone C! et C2 derivant de la glycine tend ä confirmer que des
moldcules entieres de cet amino-acide ont €t€ incorporees. En moyenne, 1 g de polymeres correspond
ä 1,5 millimole de glycine pour 5,4 millimoles de catechol, soit un rapport stoechiometrique de 1/3,5.
La comparaison entre la composition elementaire calculde et les resultats analytiques (tableau III)
montre une coincidence satisfaisante entre les teneurs en C, N et H des composes. Par contre, on
trouve par le calcul deux fois moins d’oxygene que par analyse. On en ddduit que les composes ont
incorpore de l’oxygene lors de la Synthese, soit par ouverture d’une partie des cycles, soit par hydroxy-
lation des quinones intermediates [20, 40—43].
La titration des acidites -COOH et -OH des polymeres, par comparaison avec celle des
equivalents glycine et catechol engages dans la structure des polycondensats, montre une diminution
significative des -OH initiaux, de 10,9 ä 2,9 meq/g, que Гоп peut imputer ä l’oxydation et ä la forma­
tion de structures hydroxyquinoniques [40, 42]; au contraire, l-’acidite carboxylique s’est elevee de
1,6 ä 4,8 meq/g par suite des ruptures de cycles. En admettant qu’un cycle est ouvert avec formation
de deux groupements -COOH [41 ], on peut estimer que l’acidite matricielle mesuree (3,2 meq/g)
est due ä l’ouverture de 29,6% des noyaux incorpores. Cette acidite est mise en evidence en
spectroscopie infrarouge par l’apparition d’un pic intense ä 1710 cm"1, deplace ä 1590 cm"1 par
salification [24].
En Chromatographie sur gel de dextrane Sephadex G 100, les polymeres montrent un pic
unique dont le maximum correspond а Кду = 0,55 et dont la branche descendante presente un
50 ANDREUX et al.

TABLEAU I. DISTRIBUTION DES FORMES DE L’AZOTE AU

TERME DE LA SYNTHESE (en % de N introduit au depart)

F o rm es de N D ialysats P o ly m eres P ertes

Exces

n n h Y 22,7
17,2 - -
N glycine libre 45,5 _
47 ,8 - -
N com bing 15,5 5,2
14,1 4,5
N to ta l 83,7 5,2 - 11,1
79,1 4,5 - 16,4

TABLEAU II. DISTRIBUTION DES DIFFERENTS SITES MARQUES

AU 14C AU TERME DE LA SYNTHESE (en % de la radioactivity de depart)

Sites co2 D ialysats P o ly m eres P ertes

m arqu6s Exces

G lycine 14C, 15,0 75,2 4,8 - 5,0

16,9 65,8 5,9 - 11,4
G lycine 14C2 0,1 72,8 4 ,7 - 2 2 ,4
0,2 9 6 ,4 7,3 + 3 ,9
0,2 9 3 ,9 7,7 + 1,8

C a te c h o l14Си 3,8 6 8 ,0 27,1 " 1.1

TABLEAU III. COMPARAISON DES COMPOSITIONS ELEMENTAIRES

CALCULEES ET MESUREES DES POLYMERES Resultats en %)

c H N 0

C atech o l 14C y 3 9 ,1 2 3,26 17,38

—COOH 1,86 0 ,1 6 - 4 ,9 6

D c h 2- 2 ,1 0 0,35 - -
nh 2- - 0,3 3 2,31 -

T o tal calcule 4 3 ,0 8 4 ,1 0 2,31 2 2 ,3 4

A nalyse 4 5 ,6 0 3,63 2 ,47 4 8 ,3 0

epaulement de Kav = 0,75. Par comparaison avec des protönes globulaires etalons [44], on peut
estimer que leurs masses molaires se situent entre 25 000 et 15 000 pour le constituant majeur, et
7000 et 4000 pour le second constituant. Ces valeurs, faibles par comparaison avec celles trouvees
pour les acides humiques des sols ä mull [45-48], peuvent s’expliquer par le taux 61eve des o v e r­
tures de cycles au cours de la synthese. En effet, dans les polycondensats naturels on observe souvent
une relation inverse entre le degrd de polycondensation et l’acidite matricielle [47, 49].
 IAEA-SM-211/42 51

TABLEAU IV. STABILITES COMPAREES DES SITES MARQUES

AU 14C ET DE L’AZOTE LORS DE L’HYDROLYSE ACIDE DES
POLYMERES (resultats en %)

Sites H ydrolysables C ations N on catio n s P ertes

14c , 56,7 30,6 22,9 3,2

,4C2 68,1 30,2 26,1 11,8

N -a am ine 30,0 - - -
N N H ,* 19,0 - - -
C atech o l 14Си 28 ,9 3,3 22,3 3,3

3 .2 .3 . D egradation a cid e d es p o lym eres

Lors de l’hydrolyse acide des composes humiques naturels et des modeles incluant des poly­
peptides, les amino-acides N-terminaux demeurent fix6s sur la matrice polycondensee [5, 10, 28, 47];
les polymeres obtenus ä partir du Systeme catechol-glycine devraient done opposer une resistance
61evde au traitement hydrolytique. Ainsi, leur spectre d’absorption presente les caracteristiques
habituelles des residus d’hydrolyse des acides humiques et des melanines [6], avec en particulier un
maximum tres net ä 1610 cm-1 attribue aux liaisons )C = О a - ß conjuguees [50].
L’hydrolyse acide ne modifie pas le spectre du residu solide; 28,9% de la radioactivite provenant
du catechol, 56,7% et 68,1% des radioactivites issues respectivement du C2 et du C2 de la glycine
sont retrouves dans les hydrolysats (tableau IV); 30% de la radioactivite du C2 et du C2 correspondent
ä de la glycine libre retenue sur resine cationique, la fraction non fix6e etant imputable ä des fragments
de polymeres et, ainsi que le suggerent Cranwell et Haworth [51], ä de l’acide glyoxylique. En effet,
la desamination de la glycine est probable, car 19% de l’azote sont liberes sous forme ammoniacale.
La rupture de la moitie de la glycine incorporee dans les polymeres suggere qu’il existe des liaisons de
stabilites differentes, en relation sans doute avec l’aromaticite des cycles matriciels.

3.3. Degradation des polymeres dans un sol ä mull

3 .3 .1 . D egagem en t d e C 0 2

Les courbes de mineralisation en C02 des diffdrents Substrats dans le mull actif sont presentees
a la figure 3. L’oxydation de la glycine libre est la plus active; le rapport i des atomes 14C! et 14C2
restant dans le sol apres 48 heures d’incubation laisse prevoir que la glycine non transformee emprunte
les voies metaboliques classiques de synthese proteique [52]. A l’dquilibre, apres quatre semaines,
78,5% du C2 et 48,6% du C2 sont oxydes en C02.
Dans le cas du catechol 14Cu libre, la vitesse d’oxydation, faible au depart, passe par un
maximum entre 48 et 72 heures et decroft ensuite; 9% seulement des atomes de carbone sont convertis
en C02. La resistance de ce compose ä la biodegradation peut etre attribute soit ä sa toxicite, soit
ä sa rapide humification par oxydation en quinones intermediaires, en accord avec les observations
faites sur des precurseurs polyphenoliques autoxydables, tels que le trihydroxy-1,4,5-naphtalene [6]
ou l’hydroquinone [29].
La mineralisation des polymeres est active et atteint sa vitesse maximale au cours des premieres
24 heures d’incubation, puis decroit et devient pratiquement constante ä partir du cinquieme jour.
Les taux de mineralisation en C02 s’elevent ä 39,5% et 25,9% respectivement pour le Q et le C2 de
la glycine, et 17,7% pour le carbone du catechol. L’incorporation de la glycine et du catechol dans
les polymeres se traduit done par une stabilisation de l’amino-acide, et par une fragilisation du
catechol en raison de l’existence des groupements -COOH matriciels.
52 ANDREUX et al.

F I G .3 . C o u r b e s c u m u l a tiv e s d e la m in e r a lis a ti o n e n C 0 2 d e s S u b s tr a t s m a r q u e s i n c u b e s d a n s le s o l. 1 = g ly c in e 14C 1;

2 = g l y c i n e 14C2; 3 = c a te c h o l 14C u ; 4 - p o l y m e r e s g l y c i n e 14C j; 5 ~ p o ly m e r e s g ly c in e l i C 2 ; 6 = p o ly m e r e s c a te c h o l

14C u,‘ 7 = p o l y m e r e s g ly c in e 14C u + c a t e c h o l 14C u .

3 .3 .2 . M o d ifica tion s de la stru ctu re des p o ly m ir e s

Les essais de reextraction des residus d’incubation avec l’eau et les solvants alcalins [38]
montrent qu’une faible proportion seulement de la radioactivite initiale est remise en solution
(tableau V). Dans le cas des polymeres, cette insolubilisation suggere l’existence de modifications
structurales profondes. Les rapports atomiques C1/C2/Ccatechol sont determines dans le C02 degage
et dans les differents extraits (tableau VI). Par comparaison avec les rapports atomiques de depart,
et en choisissant C2 constant et egal ä 1, on observe qu’au debut de la mineralisation, l’oxydation en
C02 affecte preferentiellement le C, de la glycine. Apres 4 jours d’incubation, les atomes Cj et C2
sont degrades ä la т ё т е vitesse, tandis que l’oxydation des atomes matriciels provenant du catechol
commence ä croftre, jusqu’ä un rapport final proche de celui des produits de depart. A l’equilibre,
les polymeres se trouvent done degrades selon un processus congruent tres lent et continu. La parti­
cipation de processus physico-chimiques ä ces degradations est tres possible dans ce sol neutre
particulierement riche en hydroxydes de fer amorphes.
Les produits de degradation les plus appauvris en radioactivite due au catechol et au Ci sont
reextraits respectivement ä l’eau et dans NaOH ä pH 9,7. Les rapports atomiques des autres extraits
alcalins et des residus se rapprochent davantage de celui des polymeres initiaux avec toutefois une
augmentation sensible du carbone matriciel (tableau VI). Comme dans le cas de l’hydrolyse acide,
l’action de la microflore semble s’etre exercee preferentiellement sur des structures presentant un
moindre degre de stabilisation de la glycine, ainsi que sur des groupements -COOH matriciels
 IAEA-SM-211/42 53

TABLEAU V. DISTRIBUTION DE LA RADIOACTIVITE RESIDUELLE DANS LES

EXTRAITS AQUEUX ET ALCALINS DES SOLS INCUBES

Substrats H2Oa NaOH Pyrophosphate NaOH Total extrait

pH 9 ,7 a l%a 0 ,lN a a b

G lycine 14Ci 2,5 0,6 0,4 0,6 4,1 19,1

G lycine 14 C2 5,5 1,8 1,2 1,9 10,4 20,2

Catechol 14Си 2,7 5,3 6,3 8 ,0 22,3 2 4,5

Polym eres G lycine 14Ci 6 ,i 3,7 3,7 2,5 16,0 2 5 ,6
Polym eres G lycine 14C2 7,7 7,4 4,1 3,4 2 2 ,6 , 29,4
Polym eres C atechol 14Q j 5,5 7,3 4,8 3,8 2 1 ,4 '2 5 ,8

a En % de la radioactivity introduite.
b En % de la radioactivite residuelle apres incubation.

TABLEAU VI. COMPARAISON DE LA DISTRIBUTION DES TROIS SITES MARQUES ET

DE LEURS RAPPORTS ATOMIQUES ENTRE LES DEGAGEMENTS GAZEUX ET LES
COMPOSES RESIDUELS

C 0 2 degage dans M icroatom es C /20 g de sol Rapports atom iques

les periodes
(jours) C, C2 0-catechol C, C2 ^catechol

0 -1 7,2 2,8 2 2 ,6 2,6 1 8,1

1 -2 2,3 1,5 22,6 1,5 1 15,0

2 -3 0,8 1,0 13,2 0,8 1 13,2

3 -4 0,7 0,7 9,6 1 1 13,7
4 -7 1,2 1,0 17,0 1,2 1 17,0
7 -9 0,6 0,6 9,7 1 1 i6 ,2
9 -1 1 0,6 0,6 9,1 1 1 15,2
1 1 -1 5 0,9 0,9 14,5 1 1 16,1
1 5 -1 8 0,4 0,45 8,0 0,9 1 17,8
1 8 -2 2 0,5 0,55 9,6 0,9 1 17,5
2 2 -2 7 0,7 0,7 13,0 1 18,6
1
C 0 2 total 16,1 10,8 148,9 1,5 1 13,8
Extrait H2 0 2,5 3,2 4 6 ,6 0,8 1 14,6
Extrait NaOH pH 9,7 1,5 3,1 61,5 0,5 1 19,8
Extrait Na4P20 7 1,5 1,7 40 ,3 0,9 1 2 3,7
Extrait NaOH 0,1 N 1,0 1,4 3 2 ,0 0,7 1 2 2,8
N on extrait 18,2 21,5 51 1 ,9 0,8 1 23,8

Produit de depart - - - 1 1 2 1 ,0
54 ANDREUX et al.

provenant de noyaux ddjä ouverts. II est probable qu’en cours d’incubation, d’autres noyaux se
trouvent rompus par la microflore, conduisant ä des structures aliphatiques riches en groupements
-COOH, analogues ä l’acide muconique et ä ses derives [53-55]. De tels composes se trouveraient
stabilises sous forme de complexes pseudosolubles avec les cations polyvalents du sol [38, 56, 57].
Les composes les moins degrades correspondraient au contraire ä des macromolecules ä structure
resonnante assurant la stabilisation de la glycine, et susceptibles d’etre insolubilisees par voie physico-
chimique.

3 .3 .3 . In flu en ce su r la m ineralisation des com poses organiques p reex ista n ts

Le dcgagement de C02 total par les sols enrichis est compare avec celui d’un sol non enrichi
incube dans les mcmes conditions. L’apport de glycine exerce une action stim u la n te [58] pratique-
ment immediate: apres 4 jours d’incubation, le degagement de C02 a augmente d’environ 250% par
rapport au temoin. Cette action est au contraire depressive dans le cas du catechol libre et des poly­
meres, au moins durant les premiers jours de l’incubation. Ces composes provoquent done une
perturbation dans l’equilibre biologique du sol, qui ne disparait qu’apres adaptation de la microflore.
Dans le cas des polymeres, il ne semble pas que cette perturbation soit düe ä une toxicitd directe car,
contrairement au catechol seul, leur vitesse initiale de mineralisation est tres elevee; il semble plutot
que leur degradation conduise ä la formation de composes intermediaires presentant une toxicite
temporaire. On a cherche ä confirmer cette hypothese en etudiant revolution au cours de l’incubation
du rapport C2/C] des atomes de carbone degage sous forme de C02. Dans le cas des polymeres, се
rapport passe de 0,38 ä 0,46 entre le premier et le second jour, puis ä une valeur constante comprise
entre 0,50 et 0,60. Dans le cas de la glycine libre, cette valeur est atteinte des le premier jour. On
peut done conclure que durant les premiers jours, les amino-acides presentant les liaisons les plus
fragiles sont d’abord decarboxyles avant d’etre detaches de la matrice des polymeres et de participer
au metabolisme microbien [52, 59]. La fraction matricielle residuelle manifesterait alors un effet
toxique, avant d’etre transformee ä son tour.

4. CONCLUSION

L’autoxydation d’une solution equimoleculaire de catechol et de glycine ä pH 7,9 et 20°C

conduit en 4 jours ä un etat d’equilibre caracteris6 par la formation de polymeres azotes de couleur
foncee, saliflables grace ä une teneur elevee en groupements carboxyliques. Leur elaboration met
en jeu l’oxydation du catechol avec formation intermediaire de radicaux semi-quinoniques et de
quinones, qui reagissent avec la glycine [21,22]. Les produits d’addition ainsi formes s’oxydent ä
leur tour en quinones actives qui subissent des rearrangements provoquant la desamination d’une
Partie de la glycine libre, avec formation d’aeide glyoxylique CHOCOOH, dont la decarboxylation
est tres rapide.
La premiere phase —qui dure entre 30 et 40 heures —de la reaction est caracterisee par
l’oxydation du catechol, et la production de C02 par degradation de la glycine (15 ä 18% du C,)
et des noyaux aromatiques (3,8% du carbone du catechol). A la fin de cette phase, les molecules
oxydees predominent et la polymerisation des radicaux semi-quinoniques peut alors se d6velopper.
A l’equilibre, presque tous les radicaux libres ont ete desactives en conduisant ä des polymeres dont
les quantites et les dimensions dependent etroitement des conditions chimiques de l’equilibre.
Les produits ainsi stabilises contiennent peu de radicaux libres [60], mais leur traitement
alcalin conduit comme dans le cas des acides humiques naturels ä une nouvelle activation des groupe­
ments quinoniques et ä l’accroissement des ouvertures de cycles avec production de C02 [49]. Les
polymeres isol6s presentent des caracteristiques analytiques comparables ä celles des acides humiques
extraits des sols eutrophes; nöanmoins, leur encombrement moleculaire est moindre, en raison du
maintien des conditions d’autoxydation durant toute la duree de la Synthese. Celles-ci, contrairement
 IAEA-SM-211/42 55

ä l’oxydation enzymatique ä pH faiblement acide [19, 26], tendent en effet ä ralentir la polymerisa­
tion au profit de la degradation des noyaux aromatiques [49, 61]. Toutefois, il est bon de preciser
que dans la nature l’autoxydation accompagne toujours l’oxydation enzymatique des precurseurs
polyphenoliques.
Parmi les polymeres isotes, peuvent etre distinguds deux types de composes; les uns, les plus
abondants, se caracterisent par des structures hydroxyquinoniques hautement resonnantes qui
permettent une stabilisation dlevee des substituants glycine; les seconds, de plus petite taille,
contiennent davantage de cycles ouverts porteurs de groupements -COOH [61 ] et presentent un
etat de conjugaison plus faible, qui explique la stabilisation moindre de la glycine. En effet,
l’hydrolyse chlorhydrique ä chaud parvient ä detacher la moitie des molecules de glycine tandis que
l’autre moitie demeure incorporee ä un ptecipite noir tres stable representant environ 70% du poids
total des polymeres. Lors de l’incubation dans le sol, 17,5% du carbone des polymeres sont degages
sous forme de C02. Par rapport ä la glycine et au catechol libres, la combinaison dans les polymeres
se traduit par une stabilisation de la glycine, dont la biodegradation diminue de 50%, et au contraire
par une fragilisation du catechol, dont l’oxydation en C02 augmente de 100%. La biodegradation
parait affecter pteferentiellement les motecules les plus petites, en assurant ä la fois la rupture des
amino-acides et la decarboxylation des groupements -COOH matriciels. Contrairement ä la glycine
libre, les polymeres exercent, vis-ä-vis de la biodegradation des composes organiques preexistants
dans le sol, un effet depressif qui ne prend fin qu’apres 4 jours, avec la fin de la phase primaire de
biodegradation de la glycine liee. Cet effet est etroitement lie ä l’dlimination de l’amino-acide, avec
liberation de composes toxiques d’origine matricielle qui pourraient se fixer aux parois cellulaires
des micro-organismes ou bien etre partiellement biodegrades.
La «phase secondaire» de l’incubation, ainsi que la qualifient de nombreux auteurs [62—64],
est caracterisee par une mineralisation tres lente et stoechiontetrique des polymeres, ce qui suggere
leur incorporation aux composes humiques les plus stables. Les composes reextractibles ä l’eau ou
par NaOH ä pH 9,7 correspondent ä des structures aliphatiques riches en groupements -COOH issus
de la biodegradation des noyaux aromatiques et susceptibles de donner lieu ä la formation de
complexes pseudo-solubles. Les extraits alcalins et l’humine sont au contraire enrichis en noyaux
aromatiques et ne mettent pas en evidence de decarboxylation preferentielle de la glycine. L’activite
biologique tend done ä epargner une fraction homogene et hautement polymerisfe des macromole-
cules introduites, dont la stabilisation dans 1’humine semble faire intervenir des processus physico-
chimiques. Cette stabilisation est ä rapprocher des insolubilisations obtenues dans les sols apres
degradation microbienne des chafnes polypeptidiques rattacltees aux phytomelanines [56]; eile
s’avere etre une forme importante de stockage de l’azote organique dans l’humine des sols.

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[2 7 ] SCH ANEL, L., “ Studies about hum u s” , Trans. Int. Sym p. Hum us et Planta IV , Prague (1 9 6 7 ) 73.
[28] L A D D , J.N ., BU TLER, J.H .R ., A ust. J. SoU Res. 4 (1 9 6 6 ) 41.
[2 9 ] SCHVARTZ, C., «E volution des hydrosolubles de litieres de calluna e t de hetre au cours du processus
d’hum ification», These, N ancy (1 9 7 5 ) 81 p.
[3 0 ] WANG, T.S.C ., CHANG, S.Y ., YEH, K .H ., J. Agric. Ass. China 7 2 (1 9 7 0 ) 30.
[3 1 ] V ERM A, L„ M ARTIN, J.P., H A ID ER , K., SoU Sei. Soc. A m ., Proc. 3 9 2 (1 9 7 5 ) 2 7 9 .
[3 2 ] H A IDER , K „ M ARTIN, J.P., SoU Sei. Soc. A m ., Proc. 39 4 (1 9 7 5 ) 657.
[3 3 ] GOLEBIOWSKA, D ., “ Studies about hum us”, Trans. Int. Sym p. Humus et Planta V , Prague (1 9 7 1 ) 3 69.
[3 4 ] GUCKERT, A „ ROGER, P„ JA CQ UIN, F ., Bull. E N SA N 10 2 (1 9 6 8 ) 69.
[3 5 ] STEVENSO N , F.J., K ID DER , G., TILO, S.N ., SoU Sei. Soc. A m ., Proc. 31 1 (1 9 6 7 ) 71.
[3 6 ] MOORE, S„ STEIN, W .N., J. Biol. Chem. 211 (1 9 5 4 ) 907.
[3 7 ] SMITH, I., in C hromatographie and E lectrophoretic T echniques, V ol. I, William Heinem ann Medical B ooks,
Interscience Publishers, London (1 9 6 0 ) 261.
[3 8 ] BRUCKERT, S„ HETIER, J.M ., GU TIERR EZ, F „ Sei. Sol - Bull. A FES 4 (1 9 7 4 ) 2 25.
[3 9 ] FLA IG , W„ Sei. Sol - BuU. A FES 2 (1 9 7 0 ) 39.
[4 0 ] FLA IG , W., Proc. Int. M eet. H um ic Substances, N ieuw ersluis, Pudoc, W ageningen (1 9 7 2 ) 19.
[4 1 ] CZUCHAJOWSKI, L„ KRECZEK, J., R ocz. G leboz. 16 1 (1 9 6 6 ) 3.
[4 2 ] M ARTIN, J.P., H A IDER , K., BO NDIETTI, E., Proc. Int. Meet. Hum ic Substances, N ieuw ersluis, Pudoc,
W ageningen (1 9 7 2 ) 171.
[4 3 ] PIERPOINT, W .S., B iochem . J. 9 8 (1 9 6 6 ) 567.
[4 4 ] ANDREW S, P„ B iochem . J. 91 (1 9 6 4 ) 22 2 .
[45] BA ILLY , J.R ., M ARGULIS, H., Plant SoU 2 4 (1 9 6 8 ) 34 3 .
[46] BA ILLY , J.R ., Plant Soil 4 4 1 (1 9 7 6 ) 4 9 .
[4 7 ] A N D R E U X , F ., METCHE, M., Rapp. 1er Coli. Int. Biodegradation e t H um ification, N ancy, 1 9 7 4 , PIERRON Ed.
(1 9 7 4 ) 47 9 .
[4 8 ] POSPISIL, F „ R ostl. V yroba 2 0 8 (1 9 7 4 ) 795.
[4 9 ] SWIFT, R .S., PO SNER, A.M ., J. SoU Sei. 23 4 (1 9 7 2 ) 381.
[5 0 ] THENG, B.K .G ., PO SN ER , A .M ., SoU Sei. 104 (1 9 6 7 ) 191.
[5 1 ]
CRANWELL, P .A ., HAWORTH, R .D ., Proc. Int. Meet. H um ic Substances, N ieuw ersluis, Pudoc, Wageningen
(1 9 7 2 ) 13.
[52] M A Y AU D O N , J„ SIM ONARD, P., Ann. Inst. Pasteur 109 (1 9 6 5 ) 2 24.
[5 3 ] H A Y AISH I, O., K A TA G IRI, M„ ROTHBERG, S ., J. Am . Chem. Soc. 7 7 (1 9 5 5 ) 5 4 5 0 .
[54] R U N G , F ., Folia M icrobiol. 16 1 (1 9 7 1 ) 41.
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[5 6 ] C A R BA LLAS, T „ A N D R E U X , F „ JA CQ UIN, F „ Sei. S ol - Bull. A FES 3 (1 9 7 1 ) 29.
[5 7 ] LESPINAT, P .A ., H ETIER, J.M., THOM ANN, C., CHONE, T ., Sei. Sol - BuU. A FES 1 (1 9 7 6 ) 53.
[5 8 ] JENKINSON, D .S ., “ The turnover o f organic m atter in soU” , Rep. FA O /IA E A Technical M eeting on the U se o f
Isotop es in SoU Organic Matter Studies, Pergamon Press, Oxford (1 9 6 6 ) 187.
[5 9 ] THIM ANN, V ., in The Life o f Bacteria, 2nd Ed., McMUlan C om pany, N ew York (1 9 6 3 ) 9 0 9 p.
[6 0 ] A TH ERTON, N .M ., CRANW ELL, P .A ., FLO YD , A .J., HAWORTH, R .D ., Tetrahedron 2 3 (1 9 6 7 ) 1653.
[61] N G U Y EN , Q .H ., METCHE, M„ U RIO N , E„ Bull. Soc. Chim. Fr. 7 (1 9 6 6 ) 2 2 3 2 .
[62] D A N N EBER G , O., B odenkultur 2 2 3 (1 9 7 1 ) 264.
[6 3 ] H A N R IO N , M., TOUT A IN , F ., BRUCKERT, S„ JA CQ UIN, F ., O ecologia Plantarum, 1 0 2 (1 9 7 5 ) 169.
[6 4 ] McGILL, W .B., SHIELDS, J.A ., PA U L, E.A ., SoU B iol. B iochem . 7 (1 9 7 5 ) 57.
 IAEA-SM-211/42 57

DISCUSSION

R.W. ALDAG: In your model experiments, when you observed loss of I4C from the carboxyl
group of glycine, did you also notice an increase in ammonia in the reaction solution or was the
ammonia that may have evolved simultaneously bonded to the newly formed polymer?
F. ANDREUX: Analysis of the dialysable compounds reveals a quantity of free ammonia
corresponding almost exactly to the amount of C02 released. This proves that almost all the
glyoxylic acid formed by de-amination of the glycine is decarboxylated. It seems, therefore, that
all the nitrogen incorporated in the polymers comes from the glycine and not from the ammonia.
 IAEA-SM-211/43

EFFECT OF NITROGEN ON THE FORMATION OF

PYROCATECHIN-HUMIC ACID AND THE
NITROGEN LINKAGE CHARACTERISTIC OF THIS ACID

H. ÖZBEK
Faculty of Agriculture,
University of (Jukurova,
Adana,
Turkey

Abstract

EFFECT O F NITROGEN ON THE FORM ATION OF PYROCATECHIN-HUMIC ACID A N D THE NITROGEN

LINK AG E CHARACTERISTIC O F THIS ACID.
An evaluation o f syn th etic hum ic acids as fertilizers has been undertaken. T he effects o f nitrogen on
h um ification, and th e w ay nitrogen is linked to th e main structure, were studied. T he use o f sy n th etic procedures
to resolve the com p lex problem s o f hum ic acid form ation is n ot a particularly new m ethod but researchers believe
that m od el com p ou nd investigations can m ake valuable contributions to this subject. In th e present stud y,
pyrocatechin was polym erized b y oxid ative coupling reaction w ith nitrogenous com pounds arranged in five
groups including th e con trol group. The polym erization was achieved using air o x y g en as th e oxidizing agent
in alkaline m edium at pH 8.6. To ascertain the h um ification rate, aliquot sam ples were taken after 13, 2 8 , 6 3 ,
8 4 and 102 days o f th e reaction. The samples were precipitated w ith 1N NCI. T h e am ounts o f to ta l hum ic
acids form ed in th e reaction and the nitrogen linked to hum ic acids, as w ell as hydrolysable nitrogen, were
determ ined. As a result, it is suggested that th e form o f nitrogen affects the rate o f hum ification as w ell as the
am ount o f nitrogen linkage to hum ic acids. The longer the h um ification period, th e greater th e am ount o f
nitrogen linked to hum ic acids in the h ydrolysable form . When the am ounts o f hum ic acids, nitrogen con ten t
and th e hydrolysable nitrogen w ere taken in to consideration, th e m ost appropriate form o f nitrogen for
hum ification was found to be nitrogen in th e form o f ammonia.

INTRODUCTION

It is well known that humic acids which do not contain nitrogen do not occur in the nature [1 ].
Humic acids are not only a degradation product, but are also formed by polymerizations in the soil.
Thus, nitrogen is linked to the structure during polymerization [1,2]. It is evident that the linkage
of nitrogen to humic acids can be explained by theoretical chemical principles; however, the
amount of nitrogen linkage to humic acids is particularly important from the point of view of the
soil scientist. Nitrogen also plays an important role in humification. It is almost impossible in
nature to observe how this reaction occurs. All researchers agree that model compound studies
can make a valuable contribution to interpretation of the results.
In recent years, research workers have been united in their efforts to find a source that will
supply the essential need of nitrogen when the plant requires it [3]. This is only possible when
nitrogen is given as organic fertilizer to the soil. Synthetic humic acids containing nitrogen may
serve as a source. On this basis, this study was carried out to elucidate the effects of nitrogen
on humification and the manner in which nitrogen is linked to the main structure.

MATERIALS AND METHODS

Lignin has the most important role in the humification process in the soil [1,4]. Lignin
molecules or degradation products of lignin may directly polymerize to yield humic acids. It is

59
60 ÖZBEK

widely accepted that the basic monomeric units of humic acids are oxidation products of lignin [5].
It is known that oxidative degradation of lignin yields mostly polyphenols. For this reason,
pyrocatechin has been taken as a model compound in the present communication [6]. To provide
nitrogen, several nitrogen-containing compounds were studied, and five different groups were
arranged [7].
The groups are as follows:

Group I Pyrocatechin
” II ” +(NH4)2S04
” III ” + KN03
” IV ” + H2N-CO-NH2
” V ” + H2N-CH2-COOH

A reaction mixture of 5 g pyrocatechin and the nitrogenous compound corresponding

to 1 g nitrogen was placed in a reaction flask equipped with an oxygen inlet tube. The reaction
was established in an alkaline medium at pH 8.6. The alkaline medium is necessary in the oxidative
polymerization to enable the hydrogen in the phenolic hydroxide group to dissociate for coupling
reactions [8]. The pH values lower than 8.6 are not a good medium for the reaction, since only
a few pyrocatechin molecules take place in the polymerization. The pH values over 10 or 11 are
also not suitable, because humic acids formed may have been degraded to yield degradation
products under very alkaline conditions [9].
Oxygen that was used as the oxidizing agent was passed through the stirred solution, and the
reaction mixture was left for humification with continuous bubbling of oxygen [ 10, 11 ]. In order

Sample
( reaction mixture)

centrifuge

I i
centrifugate precipitate
(discarded) washed w ith

acidified H 0
4; ^
centrifuge

l
precipitate centrifugate
(discarded)
drTe^under vacuum
at 105° C
determination of humic
acid formed
___ ±_____

K jeldahl Hydrolysis
Analysis 6 N HCl
I . . Kjeldahl Analysis
Total N determination

Hydrolysable N
determination

F I G .l. S e p a r a tio n o f th e p o ly m e r iz a tio n m ix tu r e .

 IAEA-SM-211/43 61

to follow the humification rate, aliquot samples were taken after 13, 28, 63, 84 and 102 days
of the reaction. They were precipitated with 1N HC1 and were separated according to the scheme
shown in Fig. 1. The amounts of total humic acids formed and the nitrogen linked to humic
acids (total N) were determined.
Determination of hydrolysable nitrogen was carried out, after hydrolysing the samples
with 6N HC1 [9]. The difference between the total and the hydrolysable nitrogen gives,us the
amount of humic acid nitrogen linked as heterocyclic units [2]. The hydrolysable nitrogen is
the nitrogen that can be mineralized during a vegetation period to provide the essential require­
ment of the plant [9].

DISCUSSION

In the five groups, the humification rate and the amount of humic acids formed increased with
time (Table I). Nitrogen speeds up humification, and the rate of this reaction also changes
according to the form of nitrogen used (Fig.2).
As seen in Table 11, in the five groups, at the end of 102 days, the amount of humic acid formed
in the reaction varies from one to group to another.
In the group without nitrogen, about 20% of pyrocatechin is polymerized by 102 days.
However, this ratio is greater in percentage in the other groups.

TABLE I. AMOUNT OF HUMIC ACIDS FORMED DURING

102 DAYS OF HUMIFICATION »

A m ount o f hum ic acids (m g)

Days 13 28 63 84 102

Group I 3 4 0 .0 503.5 65 0 .0 8 5 0 .2 1 0 00.0

Group II 9 9 8 .0 1 0 29.0 1438.0 1 7 6 8 .0 2 0 2 9 .8

Group III 6 2 0 .0 6 2 0 .0 81 0 .0 1 3 5 4 .0 1 3 1 8 .0
Group IV 6 9 4 .0 1 0 24.0 1400.0 2 0 2 8 .0 1 9 9 8 .0
Group V 9 0 0 .0 9 2 0 .0 1106.0 1 1 6 8 .0 1 3 5 5 .0

F IG . 2. F o r m a tio n o f h u m ic a c id s r e la te d to tim e a n d fo r m o f n itr o g e n u se d .

62 ÖZBEK

TABLE II. RATIO OF HUMIC ACIDS FORMED TO THE

PYROCATECHIN USED DURING 102 DAYS (%)

R atio o f hum ic acids to th e p yrocatechin used (% )

Days 13 28 63 84 102

Group I 6 .8 10.07 13.0 17.05 2 0 .0

Group II 19.9 2 0 .4 28.8 3 5 .4 4 0 .6 .
Group III 12.4 12.4 16.1 2 7 .0 2 6 .3 7
Group IV 15.3 20.2 28 .0 40.5 4 0 .0
Group V 18.0 18.4 22.3 22.3 27.1

As humification proceeds, the linkage capability of nitrogen in the reaction mixture to the
humic acid structure has increased, but there are still noticeable differences among them (Fig.3).
These differences are not parallel with the amount of humic acids formed in the reaction. In other
words, the ratio of increase in the amount of humification to the increase in the amount of
nitrogen linked to humic acids is not similar. If this ratio had been equal, the curves in Fig.4 would
have been parallel to the x-axis. Therefore, it is evident that the form of nitrogen used in humifi­
cation mostly affects the linkage capability of nitrogen to humic acid structure.
The amounts of total and hydrolysable nitrogen linked to humic acids during humification
reactions can be seen in Table III. The linkage ratio of nitrogen is indicated in Table IV. The
total nitrogen contents of the humic acids formed and the hydrolysable and unhydrolysable
nitrogen are given in Table V.
 IAEA-SM-211/43 63

F I G .4 . C h a n g e in n itr o g e n c o n t e n t o f h u m ic a c id s d u r in g h u m ific a tio n p e r io d .

TABLE III. AMOUNT OF TOTAL (tot-N) AND HYDROLYSABLE NITROGEN (hyd-N)

LINKED TO HUMIC ACIDS

N itrogen con ten t (m g)

Days 13 28 63 84 102

T ot-N Hyd-N Tot-N Hyd-N Tot-N Hyd-N Tot-N Hyd-N Tot-N Hyd-N

Group II 15.8 5.8 16.0 7.7 25.2 11.5 3 6 .2 15.4 4 4 .8 2 4 .7

Group III 1.1 0.6 1.4 0.7 2.4 1.0 2.3 1.0 12.5 5.2
Group IV 1.2 0 .8 3.5 2 .2 5.6 3.4 2 6 .6 15.9 4 3 .4 2 3 .4
Group V 2 7 .0 10.2 3 3 .0 10.0 42 .2 10.5 4 1 .4 10.4 51.3 12.0

In group II, the humic acids formed in the reaction contain 1.5, 1.74, 2.2% nitrogen after
13, 63, 102 days respectively (Table V). In the other groups, a similar increase can also be
observed. Although humic acid yields at the end of 102 days in group IV are more than in group V,
the amount of nitrogen linkage is higher in group V. In groups III and V, equal amounts of humic
acid are formed. However the amounts of nitrogen linked to the structure vary.
It can be concluded that the longer the humification period, the greater is the amount
of nitrogen linked to humic acids in the hydrolysable form. As seen in Fig. 5, an increase in the
humic acid rate is almost parallel with nitrogen linkage, but the longer the humification, the
smaller is the percentage of hydrolysable nitrogen in the humic acid structure.
64 ÖZBEK

TABLE IV. LINKAGE RATIO OF NITROGEN USED IN THE EXPERIMENT TO THE

HUMIC ACIDS AS TOTAL AND HYDROLYSABLE NITROGEN

Linkage ratio (%)

Days 13 28 63 84 102

Tot-N Hyd-N Tot-N Hyd-N Tot-N Hyd-N Tot-N Hyd-N Tot-N Hyd-N

Group II 1.58 0.58 1.6 0.77 2.52 1.15 3 .6 2 1.54 4 .4 8 2.47

Group III 0.11 0.0 6 0 .1 4 0 .0 1 0 .2 4 0 .1 0 0 .2 3 0 .1 0 1.25 0 .5 2
Group IV 0 .1 2 0.0 8 0.35 0.2 2 0.56 0 .3 4 2 .66 1.59 4 .3 4 2.34
Group V 2 .7 0 1.02 3.3 0 1.03 4 .2 2 1.05 4 .1 4 1.04 5.13 1.20

TABLE V. AMOUNT OF TOTAL, HUMIC AND HYDROLYSABLE NITROGEN (AS

PERCENTAGE OF HUMIC ACID) IN THE HUMIC ACIDS FORMED IN GROUPS II, III, IV
AND V (AS PERCENTAGE OF TOTAL NITROGEN)

Group II Group III

(A s percentage o f total nitrogen) (A s percentage o f to ta l nitrogen)

Total-N
Days D ays
(%) H um ic—N Hydrolysable-N
<.%)
Humic-N Hydrolysable-N

13 1.5 3 1 .0 69 .0 13 0 .17 54.7 54.3

28 1.58 31.3 6 8 .0 28 0.22 5 0.0 5 0.0

63 1.74 34.2 6 5 .8 63 0.29 58.3 4 1.7

84 2.0 0 37.5 62.5 84 0 .1 7 58.3 4 1 .7

102 2 .2 0 4 4 .7 55.3 102 0 .9 4 58.5 4 1.5

Group IV Group V

(A s percentage o f total nitrogen) (A s percentage o f to ta l nitrogen)

Total-N Total-N
Days Days
(%) (%)
Humic-N Hydrolysable-N Humic-N Hydrolysable-N

13 0.1 8 3 3 .4 66.6 13 3.00 6 2.2 37.7

28 0 .3 4 35.7 64.3 28 3.59 6 8.6 3 1 .4

63 0.4 0 3 8 .8 6 1 .2 63 3.81 7 5.2 2 4 .8

84 1.31 4 0 .0 6 0 .0 84 3 .5 4 74.8 2 5.2

102 2.17 46 .2 53.8 102 3 .78 76.7 2 3.3

In group II, the amount of hydrolysable nitrogen in humic acids is 69% after 13 days, and
it decreases to 55.3% at the end of 102 days. Similar variations are also observed in other groups
(Table V). It is obvious that when the humification rate increases, nitrogen is linked to the humic
acids in the form of heterocyclic units. The form of nitrogen here plays the most important role.
For example, the humic acid that links higher amounts of nitrogen is formed in group V which
contains the nitrogen as NH2 group (Fig.4). However, there is less hydrolysable nitrogen in
group V in comparison with other groups (Fig.6).
 IAEA-SM-211/43 65

x---------- G ro u p П
3 * ---------- Group Ш
h----------- Group ЕГ
0.......... G rou p 21

0 5

13 28 63 84 102 T im e
(d a ys)

F IG . 5. L in k a g e o f n itr o g e n to h u m ic a c id s in h y d r o ly s a b le fo r m a c c o r d in g to th e d iffe r e n t n itr o g e n s a m p le s

u s e d in th e r e a c tio n .

100 х--------- Group П

•--------- Group Щ
+ ---------- Group Ш"
0..... Group 2
z
Ъ 70 *-------- --------------
о ----------------- к-

50' ч-
О
er)
о

c 30
.о
о
СП
>»
о Ю
тэ
>»
X
I3 28 63 84 I0 2 ^ т е ,
(d a y s )

F IG . 6. C h a n g e in a m o u n t o f h y d r o ly s a b le n itr o g e n r e la te d to h u m ific a tio n r a te .

CONCLUSION

Pyrocatechin has been successfully polymerized with certain nitrogen-containing compounds

by oxidative coupling. After calculating the amount of humic acids formed, the total and
hydrolysable nitrogen was determined according to the Kjeldahl technique. The form of nitrogen
that affects the rate of humification has been investigated.
According to the results evaluated in this work, it is suggested that the form of nitrogen
affects the rate of humification as well as the amount of nitrogen linked to humic acid structures.
When the following three factors —amount of humic acids, nitrogen content and the
hydrolysable nitrogen —were taken into consideration, the most appropriate form of nitrogen
for humification was found to be nitrogen in the form of ammonia.
66 ÖZBEK

REFERENCES

[1] FLAIG, W. SALFELD, J. Chr., H A IDER , K., Z w ischenstufen bei der Bildung von natürlichen Huminsäuren
und synth etischen V ergleichssubstanzen, Landwirtsch. Forsch. 16 2 (1 9 6 3 ) 85.
[2] FLAIG, W., C hem ische Untersuchungen an H um instoffen, Z. Chem. 4 7 (1 9 6 4 ) 262.
[3 ] FLAIG, W., SÖCHT1G, H., Grundlagenforschung zur Strohdüngung, Kali-Briefe Fachgebiet 8 2 (1 9 6 6 ) 5.
[4] FLAIG, W., BR EYH AN , Th., Über das Vorkom m en von Indolverbindungen in Schwarzerde-Hum insäuren,
Z. Pflanzenernaehr. Bodenkd. 7 5 ( 1 9 5 6 ) 132.
[5 ] K U BIENA, W.L., Entw icklungslehre des Bodens, Springer Verlag, Wien (1 9 4 8 ).
[6] SCHARPENSEEL, H.W., K R A USE, R., Am inosäureuntersuchungen an verschiedenen organischen Sedim enten,
besonders Grau- u. Braunhuminsäurefraktionen verschiedener B od en typen (einsch ließ lich 14C-markierter
Huminsäuren; Z. Pflanzenernaehr. Bodenkd. 9 6 ( 1 9 6 2 ) 11.
[7] IWASCHKEWITSCH, T.M., KUPREWITSCH, W .F., TSCHERBAKOW A, T ., D ie freien Am inosäuren im
B oden, Ber. Akad. WissBelouruss. S.S.R . V I (1 9 6 2 ) 523; Z. Pflanzenernaehr. Bodenkd. 1 0 5 ( 1 9 6 4 ) 56.
[8] ZIECHMANN, W,, KICKUTH, R., D ie Struktur der Huminsäuren, Kolloid Z. 1 7 4 1 (1 9 6 1 ) 39.
[9 ] SCH EFFER , F ., ULRICH, B ., Humus und Humusdüngung I , Stuttgart (1 9 6 0 ) 1 —4 9 , 7 1 —214.
[1 0 ] HOLLEM AN, A .F ., WIEBERG, E., Lehrbuch der anorganischen Chem ie, Walter de Gruyter, Berlin (1 9 6 4 ) 35.
[1 1 ] KICKUTH, R., C hem ische A bbauversuche an einer natürlichen Hum insäure, D issertation, U niversity o f
G öttingen (1 9 5 9 ) 24.
 IA E A -SM -211 /4 4

C H E M IC A L A L T E R A T IO N S O F N A T U R A L L IG N IN B Y j
IN T E R A C T IO N S W IT H H U M IC -L IK E A U T O X ID A T IO N
P R O D U C T S O F P Y R O G A L L O L ( 1 ,2 ,3 -T R IH Y D R O X Y B E N Z E N E )
I
T. WEICHELT j
Interfakultatives Lehrgebiet Chemie, |
University of Göttingen, J
Göttingen, !
Federal Republic of Germany |

Abstraa I
I
CHEMICAL ALTERATIONS OF N A T U R A L LIGNIN BY INTERACTIONS WITH HUMIC-LIKE A UTO XID ATIO N
PRODUCTS OF PYROGALLOL (1 ,2 ,3-TR IH YD R O X YBEN ZEN E). j
Investigations on chem ical interactions b etw een natural lignin and au toxid ation products o f pyrogallol are
described. Pyrogallol after au toxid ation catalysed by Si0 2 under con d ition s similar to nature (soil, p eat) was used
as a m od el substance for a group o f phenols reacting in alm ost th e same w ay. T he m easurem ents w ere carried ou t
b y m eans o f absorption spectroscop y in the regions o f electron excitation as w ell as m olecular vibration and
rotation, and later b y a Warburg apparatus to register th e gas dynam ics, and b y electrical con d uctivity, thin-layer
chrom atography, special colour reactions and various oth er analytical m ethods. Apart from th e fact that interactions
b etw een lignin and autoxidizing pyrogallol could be tested exactly, other im portant results o f the investigations were
th e adsorption o f O 2 and th e release o f CO 2 in the reaction m ixture, and th e structure o f lignin was altered in m any
respects. T he electron rr-orbitals decreased, m ainly because o f a m ore or less drastic dim inution o f fu nction al groups
such as aldehyde and a - and 0 -k eto groups and structures such as stilb en oid e and phenylcoum arone elem ents in th e
aliphatic parts o f lignin, w here also alcoholic and p henolic h yd roxy groups were lost. On the other hand, carboxyl
or ester lactone groups increased as w ell as m eth oxyl. B y chrom atography m eth od s th e form ation o f a com plex
b etw een lignin and parts o f th e hum ic-like au toxid ation products o f pyrogallol could be identified. In general,
lignin was degraded by the indicated chem ical interactions m ainly follow ing o xid ative reaction m echanism s. The
release o f CO 2 even show s th e total d ecom p osition o f som e o f th e material.

1. INTRODUCTION ;

As no reaction between natural lignin and already system-stabilized humic substances by

the methods described here could be proved, the lignin was added to phenols, which during
their autoxidation reacted with lignin to form humic-like substances. This means that the humic
substances can only react with lignin when they are still nascent.
The following experiments, for which pyrogallol was chosen as the model substance to form
humic-like substances, are divided into two groups of special interest: first the experiments to show
the chemical effects of the reaction mixture on the whole, and secondly the group of analytical
measurements that investigate in detail the altered lignin.

2. EXPERIMENTS

2.1. Test conditions

Pyrogallol was the basic material used to produce humic-like autoxidation products catalysed
by finely divided Si02, whereas lignin itself was not altered by Si02.
The experiments were so arranged that all single components (lignin, phenol, catalyst, solvent)
as well as their combinations (reaction mixtures) could be measured and interpreted separately.

67
68 WEICHELT

TABLE I. INTERPRETATION OF ULTRA-VIOLET SPECTRA TO INDICATE CHEMICAL

INTERACTIONS BETWEEN NATURAL LIGNIN AND AUTOXIDIZING PYROGALLOL
0.1 cm cells, E = absorbance (log I/log I0)

N o. C om ponents Meas ured Interpretation (E )

cone. me X.
W ithout Sum o f lignin D iff. betw een
(see Section 2 .2 )
nm E solvents and pyrogallol sum and 4
and S i 0 2 (2 + 3) (effec t o f reaction)
Де

Solvent m ixture 283 0.36

1 plus catalyst 277.5 0.395 - - -
( S i 0 2) 279 0 .3 9

2 Lignin in 1 283 1.24 0 .8 8

2 .27 -
3 Pyrogallol in 1 277.5 1.785 1.39

C om bination o f -0 .2 9
4 279 2.37 1.98 -
2 and 3 in 1 (dim inution)

2.2. Reaction mixture

The reaction mixture consisted of 500 mg lignin, 63 gpyrogallol and 26 g Si02' in 1 litre
7-butyrolacton/H20 (1:1 vol.%). In relation to the whole solution the concentration of pyrogallol
was 0.5M and that of Si02 0.4M. The single components measured in parallel had the same
concentration. Continuous shaking guaranteed a supply of oxygen. Care was taken to ensure that
the concentration was not altered and no evaporation occurred during the reaction procedures so
that exact measurements of all components investigated in parallel could be obtained.

2.3. Investigation of the reaction mixture

2.3.1. E le ctro n spectroscopy

To indicate the chemical interactions between lignin and autoxidizing pyrogallol, first the
absorption spectra in the ultra-violet and visible region were measured after a reaction time of
four weeks. The spectra registered were interpreted at their maxima or at any other suitable
wavelength.

2.3.1.1. Absorption in the ultra-violet region

The registered and interpreted measured values of the ultra-violet spectra from 210—340 nm
are summarized in Table I.

2.3.1.2. Absorption in the visible region

The interpretation of the spectra measured from 340—700 nm show again a diminution of
absorption in the reaction mixture (Table II).
In Tables I and II the absorption was interpreted only at a certain wavelength. But whereas
the spectra in the visible region of all single components (lignin, autoxidizing pyrogallol and solvent)

Merck.
 IA E A -SM -211/44 69

TABLE II. INDICATION OF CHEMICAL INTERACTIONS BETWEEN NATURAL LIGNIN

AND AUTOXIDIZING PYROGALLOL BY THE ABSORPTION OF LIGHT IN THE
VISIBLE REGION
0.1 cm cells, E = absorbance (log I/log I0)

N o. C om ponents Measure d values Interpretation (E )

cone.: ma X. W ithout Sum o f lignin D iff. betw een
(see S ection 2 .2 ) E
nm solvents and pyrogallol sum and 4
and S i0 2 (2 + 3) (effec t o f reaction)
Дб !

Solvent m ixture
1 plus catalyst 400 0 .1

( S i0 2)

2 Lignin in 1 400 0 .375 0.2 7 5

1.575 -
A utoxidizing
3 400 1.4 1.3
pyrogallol in 1

C om bination - 0 .3 5 5
4 400 1.32 1 .2 2
o f 2 + 3 in 1 (dim inution)

TABLE III. EXPERIMENTAL ARRANGEMENTS TO MEASURE THE GAS DYNAMICS

DURING THE REACTIONS BETWEEN LIGNIN AND AUTOXIDIZING PYROGALLOL

N o. Variants Main chamber Side arm*

1 Therm obarom eter 2 .0 ml 7 -butyrolacton: —

H20 ( 1 : 1 vol.%)
2 Solvent m ixture SM = 1.8 ml 7 -butyrolacton: 0 .2 ml 0 .4 S i0 2-
(SM) plus S i0 2 H 2 0 ( 1 :1 vol.%) suspension
3 Lignin in 2 1 . 8 ml lignin solu tion in Suspension
SM, cone.: as above
5 m g/m l = 9 g lignin
4 Pyrogallol in 2 1 . 8 ml pyrogallol solu tion in Suspension
SM, cone.: as above
1/2 m = 63 mg pyrogallol
5 C om bination o f 1 . 8 ml solu tion o f the Suspension
lignin and com bination in SM, cone.: as above
pyrogallol in 2 5 mg lignin in 63 mg pyrogallol

a Start o f the reaction w hen th e S i0 2 in th e side arms has been converted to th e m ain chambers.

show a continual trend of diminishing absorption without special maxima, minima and shoulders,
the ultra-violet spectra (see Section 2.5.1) had rather different shapes depending on the substance
and wavelength.

2.3.2. Gas dyn a m ic in a Warburg apparatus

The chemical interactions between lignin and autoxydation products of pyrogallol were also
investigated by measuring the gas dynamics (Table III).
70 WEICHELT

O j- uptake

F I G .l. 0 2 - u p ta k e o f lig n in ( 1 ), a u to x id iz i n g p y r o g a llo l ( 2 ) a n d th e c o m b i n a tio n (3 ) o f b o th , w ith o u t th e a b ility

o f a b s o r b in g C 0 2 b y N a O H a n d a fte r th e e f f e c t o f th e c h o s e n s o lv e n t m ix tu r e w i th S i 0 2 is s u b tr a c te d . C u rve 4

is t h e c a l c u l a t e d s u m o f o x yg e n u p ta k e o f t h e s e p a r a te ly r e g is te r e d s in g le c o m p o n e n ts lig n in a n d a u to x id iz i n g

p y r o g a llo l (1 + 2 ). T h e a r r o w s in d ic a te th e d iff e r e n c e in 0 2 ~ u p ta k e b e tw e e n th e s u m (4 ) a n d th e c o m b in a tio n (3 ).

D im in u tio n o f 0 2 - u p ta k e in d ic a te s r e le a s e o f g a s.

0 2- uptake

F I G .2 . 0 2 - u p ta k e o f lig n in (1 ), a u to x id iz in g p y r o g a llo l (2 ) a n d th e c o m b in a tio n (4 ) o f b o th w ith th e p o s s ib ility

o f a b s o r b in g C 0 2 b y N a O H . T h e e ffe c t o f th e m e d iu m (s o lv e n t, S i 0 2 ) w a s s u b tr a c te d . T h e a r r o w s in d ic a te th e

0 2-u p ta k e a s th e d iffe r e n c e b e tw e e n c u r v e s 4 a n d 3 , th e la s t b e in g th e c a lc u la te d s u m o f th e s e p a r a te ly m e a s u r e d

s in g le c o m p o n e n ts 1 + 2 .

2.3.2.1. Warburg experiments without C02-adsorption

The gas dynamics were measured at first without NaOH (no C 02-absorption). Some of these
results are shown in Fig.l.
As a result of the reaction between lignin and autoxidizing pyrogallol, the combination of
these two components took up less oxygen than the separately measured single components.
This effect can be explained by a release of gas. A further Warburg experiment was undertaken to
investigate the kind of gas released during the reaction process.
 IAEA -SM -211/44 71

TABLE IV. CHROMATOGRAPHICAL INVESTIGATIONS ON THE REACTION

PRODUCTS OF LIGNIN AND PYROGALLOL

Experim ental D escription o f the chromatogram Remarks

arrangements
Lignin A utoxidizing C om bination
pyrogallol o f lignin
and pyrogallol

Thin layer plates: Rf = 0.89 Rf = 0.75 R f = 0 .6 9 The reaction

cellu lose ( 0 - 0 .2 5 mm) Red spot Brow n spot Very weak red m ixture and
M obile solvent : parts o f it had
n-butanol/dioxan ( 2 : 1 ) Red trace the smallest
Tem p.: 23°C m obility
E xposure tim e: 3 1 /2 h Red coloured Red sp o t at
Colour test: spot at th e the beginning
phloroglucin/H C l beginning

2.3.2.2. Warburg experiment with C02-absorption

It was believed that C02 was the gas released. In order to absorb it, NaOH was placed in the
central chambers of each Warburg vessel. The results are summarized in Fig.2.

2.3.3. Chrom atography

Chromatographic investigations should give further information on the interactions between

lignin and autoxidizing pyrogallol after a reaction time of two months. The summarized results in
Table IV clearly demonstrate a bond between parts of lignin and some of the humic-like aut-
oxidation products of pyrogallol.
The reduced mobility of the new complex between lignin and humic-like autoxidation
products of pyrogallol may be caused by the formation of greater particles (larger molecular mass)
or by a reduction of functional groups because of chemical bonds.

2.3.4. Electrical c o n du ctivity

By measuring the electrical conductivity in the fluid medium of dioxan/water, the interactions
between lignin and autoxidizing pyrogallol could be tested also. Whereas lignin did not change its
conductivity over the whole reaction time, pyrogallol increased its conductivity during its humi­
fication. However, this increase in electrical conductivity was significantly higher when lignin
was also added.

2.3.5. pH-values

The concentration of hydrogen ions in the solutions used in these experiments was very
similar to those in soil. This is shown in Table V.

2.4. Separation of the reaction mixture

To investigate the reacted lignin separately it had to be isolated from the reaction mixture
without further alterations, which was possible by the method of separation outlined in Fig.3.
72 WEICHELT

TABLE V. pH-VALUES DURING THE INTERACTIONS BETWEEN LIGNIN AND

AUTOXIDIZING PYROGALLOL

No. C om ponents pH-v dues

A t the beginning After 2 \ m onths

o f experim ents

7 -b u tyrolacton /H 2 0
1 ( 1 : 1 vol.% ) 5.2 5.2
plus S i0 2

2 Lignin in 1 5.1 5.1

A utoxidizing
3 4.5 3.5
pyrogallol in 1

C om bination
4 (lignin + pyrogallol) 4.5 3.5
in 1

Reaction mixture

(filtration of SiO^)

Coagulation of the lignin in the thirtyfold

amount of Na2S04-solution (1%)

Separation of lignin by filtration Filtrate encases the autoxidation

(membrane ~ 2 jum) or products of pyrogallol
centrifugation. The coagulate
is cleaned by repeated washing
with dist. H20 and n-butanol

The complex between lignin The above-mentioned products

and humic-like autoxidation coagulate with HCI (pH < 3) and
products of pyrogallol is are separated by filtration or
probably present in the centrifugation
butanol fraction

F I G .3 . S e p a r a tio n o f r e a c tio n m ix tu r e .

2.5. Investigations on the reacted lignin

2.5.1. Ultra-violet spectroscopy

The comparison of the u.v. spectra between unaltered lignin and lignin already reacted and
isolated from the reaction mixture shows significant differences (Fig.4). With the help of the so-
called Де-method [1—3], where one spectrum is subtracted from'the other, the difference is made
more evident (Fig.5).
The diminution of absorption of the u.v. light by the reacted lignin can be caused by the loss
of carbonyl groups of various kinds, as well as by the transformation of phenylcoumarone in
phenylcoumaran elements. Further possibilities are the decay of the aliphatic cis- and trans-double
bonds or of such bonds in stilbenoide structures. Some more details are revealed by i.r. spectroscopy.
 IA E A -SM -211/44 73

F I G .4 . U ltr a -v io le t s p e c tr a : ( 1 ) u n r e a c te d lig n in ; ( 2 ) r e a c te d lig n in . C o n c e n tr a tio n : 0 .5 m g fm l; 0 .1 cm c e lls ;

s o lv e n t « n -d io x a n -H 2 0 f9 :1 v o l.% ) , d o u b le - b e a m s p e c tr o p h o to m e te r .

FJG.5. R e s u lt o f th e tie - m e th o d ( th e r e d u c e d a b s o r p tio n o f th e r e a c te d lig n in is d e m o n s t r a te d ) .

F I G .6 . In fr a -r e d s p e c tr a : ( 1 ) u n r e a c te d lig n in , ( 2 ) r e a c te d lig n in . C o n c e n tr a tio n : 3 m g lig n in p lu s 3 0 0 m g K B r.

 -J

TABLE VI. INTERPRETATION OF SOME IMPORTANT INFRA-RED ABSORPTION BANDS (COMPARISON BETWEEN UNREACTED
LIGNIN AND LIGNIN REACTED WITH AUTOXIDIZED PYROGALLOL
D = Density in %; E = Extinction

D ifferen ce b etw een I + II

Reason for absorption + = increase o f absorpt.
Sarkanen (1 9 7 1 ) Unreacted lignin I R eacted lignin II - = decrease o f absorpt.
cm 1 o f th e reacted lignin

2935 OH-stretching, m eth yl, D 7 0 -1 9 6 .9 8 - 14 -

m ethylene E 0 .1 5 5 - 0 .721 = 0 .5 6 6 0 .1 5 5 - 0 .8 5 4 = 0 .6 9 9 - 0 .1 3 3

1920- Carbonyl valence = U nconjugated D 6 2 - 3 5 .9 59 - 37.1 -

10 k eto and carboxyl groups E 0 .2 0 8 - 0 .4 4 5 = 0 .2 3 7 0 .2 2 4 - 0 . 4 3 1 = 0 .2 0 7 - 0 .0 3 0

WEICHELT
1660- Carbonyl valence = para- D 5 7 .2 - 25 5 3 .0 - 2 4 .2 -
55 substituted aryl k eton e E 0 .2 4 2 - 0 .6 0 2 = 0 .3 6 0 .2 7 6 - 0 . 6 1 6 = 0 .3 4 - 0 .0 2 0

1595 A rom atic skeletal vibrations D 5 1 - 9 .3 4 5 .6 - 9 .8

E 0 .2 9 2 - 1 .032 = 0 .7 4 0 .3 4 1 - 1 .0 0 9 = 0 .6 6 8 - 0 .0 7 2

1270 Guaiacyl ring breathing D 4 3 .2 - 0 .1 3 6 .5 - 0.5 -

w ith CO-valence E 0 .3 6 5 - 3 .0 0 0 = 2 .6 3 5 0 .4 3 8 - 2 .3 0 0 = 1 .8 6 2 - 0 .7 7 3

1220 Syringyl ring breathing D 4 2 .7 - 2.2 3 5 .7 - 2 -

w ith CO-valence E 0 .3 7 - 1 .6 5 8 = 1.288 0 .4 4 7 - 1 .6 9 9 = 1 .252 - 0 .0 3 6

1085 0 = 0 deform ation sec. D 41 - 6.2 3 3 .3 - 4 -

alcohol and aliphatic ether E 0 .3 8 7 - 1 .2 0 8 = 0.821 0 .4 7 9 - 1 .3 9 8 = 0 .9 1 9 + 0 .0 9 8

1035- A rom atic C-H in plane D 4 0 .3 -1 .9 3 2 .4 - 1.5 -

30 deform ation, Guaiacyl typ e and E 0 .3 9 5 - 1 .7 2 2 = 1.32 0 .4 9 0 - 1 .8 2 4 = 1 .3 3 4 + 0 .0 0 7
C = 0 d eform ation , prim, alcohol

975 > CH o u t o f plane deform ation D 3 9 .5 - 2 5 .8 3 0 .3 - 2 0 .9 -

(trans) F. 0 .4 0 3 - 0 . 5 8 8 = 0 .1 8 5 0 . 5 1 9 - 0 . 6 8 = 0 .161 -0 .0 2 4
TABLE VII. COMPARISON OF THE EFFECT O F THE PHLOROGLUCIN/HC1 COLOUR TEST ON THE UNREACTED LIGNIN
1 cm cells in th e reference beam : n-dioxan/HCl

N o. Variants Lignin Phloroglucin/H Cl- HClc End cone, E 555 E ^555 E rel. D ecrease o f
solu tion 3 solu tion b o f lignin max. max. aldehyde
colour groups
(m l) (m l) (m l) (m g/m l) effect (%) (%)

IA E A -SM -211 /4 4
a C ontrol
Unreacted solution 2.5 0 .2 5 0 .2 4
1
b lignin Colour
solution 2.5 0.25 - 1.2 0 .9 6 100 -

a C ontrol
solution 2.5 0 .2 5 0 .2 3 _ _
R eacted
b lignin C olour
solu tion 2.5 0.25 - 0 .9 0 .6 7 6 9 .8 3 0.2

Cone. = 5 .0 m g/m l solvent.

b
Cone. = 8 .3 3 m g/m l; H C l:d = 1.125.
D ensity o f HC1= 1.125.
TABLE VIII. INTERPRETATION O F u.v. SPECTRA A FTE R REDUCTION O F THE UNREACTED AND REACTED LIGNIN BY SODIUM On
BOROHYDRIDE.
Solvent: dioxan/H 20 (9 :1 vol. %); 0.1 cm cells; reaction tim e: 11 h; for concentrations see A ppendix.

Measured values in E = ex tin c tio n parts Interpretation

(A ) Unreacted lignin (B ) R eacted lignin

и II j he iv 4 Vs V l6 V II 7 V III 8 IX 9 X,o
X lignin lignin in lignin in Reacted Reacted Reacted D iff. betw een D iff. b etw een D iff. betw een D iff. b etw een V III 8 + IX 9
nm alone NaOH NaOH + lignin alone lignin in lignin in E + 1V 4 n 2 + H i3 v 5 + V I6 (decreased absorption o f
NaBH„ NaOH NaOH + NaBH4 (decreased (e ffe c t o f (e ffe c t o f reacted lignin = higher
absorption o f redu ction o f redu ction o f co n ten ts o f carbonyl groups)
the reacted the unreacted the reacted
lignin) lignin) lignin)

275 1.27 1.38 1.36 1 .1 6 1.135 1.21 - 0 .1 1 - 0 .0 2 - 0 .0 2 5 - 0 .0 0 5

WEICHELT
280 1.49 1.52 1.5 1.31 1.335 1.31 - 0 .1 8 - 0 .0 2 - 0 .0 2 5 - 0 .0 0 5
285 1.585 1.61 1.577 1.37 1.38 1 .3 5 - 0 .2 1 5 - 0 .3 3 -0 .0 3 0 - 0 .0 0 3
2 8 5 .5 1.55 1.613 1.573 1 .3 3 1.376 1.345 -0 .2 2 -0 .0 4 0 - 0 .0 3 1 - 0 .0 0 9
290 1.43 1.57 1.52 1.24 1.335 1.300 - 0 .1 4 - 0 .0 5 - 0 .0 3 5 - 0 .0 1 5
295 1.13 1.41 1.365 0 .9 9 1.2 1 .162 - 0 .1 4 - 0 .0 4 5 - 0 .0 3 8 - 0 .0 0 7
300 0.95 1.29 1.24 0 .8 3 1.1 1.051 - 0 .1 2 - 0 .0 5 - 0 .0 4 9 - 0 .0 0 1
305 0.8 8 5 1.18 1.13 0 .7 6 0 .9 9 2 0 .9 4 5 - 0 .1 2 5 - 0 .0 5 - 0 .0 4 7 - 0 .0 0 3
310 0 .8 4 6 1.050 0 .985 0 .7 2 0 .8 9 0 .8 4 4 - 0 .1 2 6 - 0 .0 6 5 - 0 .0 4 6 -0 .0 1 9
315 0.8 0.93 0 .878 0.6 8 7 0 .8 0 0.75 - 0 .1 1 3 - 0 .0 5 2 -0 .0 5 -0 .0 0 2
320 0 .778 0 .856 0 .8 0.65 0 .7 3 5 0 .6 8 7 - 0 .1 2 8 -0 .0 5 6 - 0 .0 4 0 -0 .0 1 6
325 0.7.4 0 .8 0 4 0 .7 5 0.61 0 .6 9 5 0 .6 3 8 - 0 .1 3 -0 .0 5 4 - 0 .0 5 7 + 0 .0 0 3
330 0.71 0 .778 0 .7 2 0 .5 7 0 .6 6 7 0 .6 0 7 - 0 .1 4 -0 .0 5 8 -0 .0 6 0 + 0 .0 0 2
335 0.6 7 5 0 .755 0 .6 9 7 0 .5 2 8 0 .6 4 0 .5 8 - 0 .1 4 7 -0 .0 5 8 -0 .0 6 0 + 0 .0 0 2
340 0 .6 2 0 .7 3 4 0 .6 7 2 0 .4 8 0 .6 1 5 0 .5 5 - 0 .1 4 -0 .0 6 2 -0 .0 6 0 -0 .0 0 2
345 0.5 4 3 0.71 0.6 4 2 0 .4 0 .5 8 7 0 .5 2 3 - 0 .1 4 3 -0 .0 6 8 -0 .0 6 4 -0 .0 0 4
350 0.4 7 7 0 .687 0.6 1 8 0 .3 7 4 0 .5 5 7 0 .4 9 8 - 0 .1 0 3 -0 .0 7 0 - 0 .0 5 9 - 0 .0 1 1
355 0 .4 0 8 0 .6 5 0 0.4 8 3 0 .3 2 0 .5 2 3 0 .4 7 0 - 0 .0 8 8 -0 .0 6 7 - 0 .0 5 3 -0 .0 1 4
360 0 .3 2 0 .6 0 2 0 .5 4 0 .2 6 5 0 .4 8 3 0 .4 3 4 - 0 .0 5 5 -0 .0 6 2 - 0 .0 4 9 - 0 .0 1 3
 IA E A -SM -211/44 77

2 .5 .2 . In fra -r e d s p e c tr o s c o p y

Com parison o f the i.r. spectra o f the unreacted and th at o f the reacted lignin shows also m any
differences (Fig.6). An exact interpretation o f the spectra was carried o u t in Table VI.

2 .5 .3 . P h lo r o g lu c in /H Q c o lo u r r e a c tio n

The determ ination o f aldehyde groups, which are conjugated w ith the arom atic system o f
lignin, chiefly coniferyl, but also p-coum ar and sinapin aldehyde structures, was carried out by
quantitatively measured colour reactions w ith phloroglucin and HC1 on the unreacted lignin as
well as on th a t reacted w ith autoxidizing pyrogallol. In Table VII the experim ental arrangements,
the measured values and the in terpretation are summarized.
The difference betw een the content in aldehyde structures o f the unreacted lignin and o f
the (reacted) lignin isolated from the reaction m ixture, as show n in Table VII, is very great.

2 .5 .4 . R e d u c tio n w ith s o d iu m b o r o h y d r id e

The purpose o f the NaBH4-reduction was to eliminate all keto groups in the a - and /3-position
o f th e aliphatic side chains as well as all aldehyde groups, in order to show w hether the chemical
interactions betw een lignin and autoxidizing pyrogallol inducted new carbonyl groups as in
carboxyl, lacton o r ester groups. The effect o f the reduction was investigated by u.v. and i.r.
spectroscopy.

2.5.4.1. NaBH4-reduction followed by ultra-violet spectroscopy

The m ethod o f interpretation sum m arized in Table VIII was partly carried out as described
in Refs [1 -3 ].
The results indicate th a t during the interactions betw een lignin and autoxidizing pyrogallol,
except for a decrease in carbonyl groups (as described in Section 2.5.1), new groups were created,
such as carboxyl, ester and lacton groups.

2 .5 A .2 . NaBH4-reduction follow ed by infra-red spectroscopy

The interpretation o f the NaBH4-reduction by i.r. spectroscopy (Table IX) gave some m ore
detailed results on the newly form ed carbonyl groups in the reacted lignin.

2 .5 .5 . P h e n o lic h y d r o x y l g r o u p s

D eterm ination o f the free phenolic hydroxyl groups (Table X) was carried o u t by a
spectroscopically measured colour reaction using l-nitroso-2-naphthol. The experim ents were
controlled by eugenol (1-propane, 2-hydroxy-, 3-m ethoxy-benzene) as a m odel substance for lignin.
More details are given in th e A ppendix.

2 .5 .6 . M e th o x y l g r o u p s

The m ethoxyl groups o f lignin were determ ined as described in Ref. [4]. The measured values
and their in terpretation are sum m arized in Table XI. As show n by th e control substance o-m ethoxy-
benzoic acid, the m easurem ent error was about 3% , so th a t the relatively high increase in m ethoxyl
caused by the interactions betw een lignin and autoxidation products o f pyrogallol is trustw orthy.
 -J
00

TABLE IX. INTERPRETATION O F SOME IMPORTANT FREQUENCIES IN i.r. SPECTRA O F UNREACTED AND REACTED LIGNIN A FTER
ITS REDUCTION WITH NaBH4
For m ore details see Appendix

cm ”1 Reason for Unreacted lignin R eacted lignin D iff. b etw een I + II Remarks
absorption reduced w ith NaBH 4 reduced w ith NaBHU (values unrelated to
Ia IIa reacted lignin)b

OH-valence, 1) 6 1 . 4 - 3 5 4 .7 - 3 .6 —
3420 H bonded 2) 0 .2 1 1 - 1 .5 2 3 = 1 .312 0 .2 6 2 - 1 .0 1 8 = 0 .7 5 6 - 0 .5 5 6
3) 5 8 .4 51.1 - 7 .5

OH-stretching 1) 5 8 . 6 - 9 5 0 .8 - 1 3 _
2935 m ethyl and 2) 0 . 2 3 2 - 1 .0 4 6 = 0 .8 1 4 0 .2 9 - 0 .8 8 6 = 0 .5 9 6 -0 .2 1 8

WEICHELT
m ethylene 3) 4 9 .6 3 7.8 - 1 1 .8
T he reacted lignin
Carbonyl valence 1) 3 8 .7 - 3 6 .5 3 2 .2 - 2 9 .6 - show s an increase in
1 7 5 0 -1 0 unconjugated carbonyl 2) 0 . 4 1 2 - 0 .4 3 8 = 0 .0 2 6 0 .4 9 2 - 0 .5 2 9 = 0 .0 3 7 + 0.0 1 1 carboxyl, ester or
carboxyl groups 3) 2.2 2.6 + 0 .4 lactone groups

1) 2 9 .4 - 1 1 2 4 .6 - 9 .2 _
Carbonyl valence
1 6 6 0 -5 5 substituted 2) 0 . 5 3 2 - 0 .9 5 9 = 0 .4 2 7 0 . 6 0 9 - 1 .0 3 6 = 0 .4 2 7 ±0
aryl k eton e 3) 1 8.4 1 5.4 - 3 .0

Arom atic 1) 1 9 .7 - 3 .3 1 6 .7 - 3 .4 _
1595 skeletal 2) 0 . 7 0 6 - 1 .4 8 2 = 0 .7 7 6 0 . 7 7 7 - 1 .4 6 9 = 0 .6 9 2 -0 .0 8 4
swinging 3) 16.4 13.3 - 3 .1

a 1 = O ptical d en sity in percentage; 2 = E xtinction (log I/log I0); 3 = Scale parts.

b + = increased absorption; — = decreased absorption.
TABLE X. COMPARISON O F THE FREE PHENOLIC HYDROXYL GROUPS IN UNREACTED LIGNIN AND IN LIGNIN REACTED WITH
AUTOXIDATION PRODUCTS O F PYROGALLOL

I = control Lignin 1- Nitrose-

Dioxan H N 0 3b HC1 End. D ilution d = cm E % 385 R eal colour Proportion o f th e C ontent o f Decrease
Substances

sam ple solu tion 3 2- naphthol- cone. (cells) max. effec t OH -groups per phenolic in
II = colour solu tion b (effec t o f w eight unit and OH-groups phenolic
reaction colour fin al absorbance OH-groups
(m l) (m l) (m l) (m l) (m g/m l) reaction) valueb

о I 5 - 1 0.2 5 1.5 3.7 - 0.1 - - „ - -

IA E A -SM -211/44
.00 и 5 1 - 0.25 1.5 3.7 1/3 0.1 0 .3 9 3 1 0 0 .3 9 1.1 100% -
3
= 11.7 = 10.6 4

тз
о I 5 - 1 0.25 1.5 3.7 - 2 - - - - -
Unrea
lignin

II 5 I 0.25 1.5 3 .7 2 1.035 3 - 0 . 5 - 1.035 1 14.6%

= 1.55 1.55 -

•a I 5 - 1 0.2 5 1.5 3.7 - 2 - 8 - -

React
lignin

II 5 1 0.25 1.5 3.7 2 0 .9 7 3 - 0 .5 - 0 .9 7 1 13.6% -6 .8 %

= 1.45 1.45 (reacted
lignin)

3 C oncentration: 0 .5 m g/m l.
b See A ppendix.

-j
vo
80 WEICHELT

TABLE XI. COMPARISON OF THE METHOXYL GROUPS IN THE UNREACTED LIGNIN

AND IN LIGNIN REACTED WITH AUTOXIDATION PRODUCTS OF PYROGALLOL

N o. Sample Weight o f th e V olum e o f OCH3-con ten ta Increase o f

investigated thiosulphate m eth oxy groups
substances
(mg) (m l) (m g) (%) (%)

o-m eth oxy-b en zoic 4 .1 5 -

1 21 6.8
acid 4 .2 8 b —

2 Lignin 22 .0 6 5.23 3 .1 9 14.5 -

Lignin reacted w ith

3 22 .4 7 7 .1 2 4 .3 4 19.3 4.8 ± 0 .1 4 4
au toxy. pyrogallol

a C ontent o f m eth oxyl: volum e o f thiosulphate X 0 .5 1 7 0 6 X factor o f th e thiosulphate solution = mg OCH3 ;

1 ml thiosulp hate = 0 .5 1 7 0 6 m gO C H 3. T h e factor o f th e thiosulphate solution was 1 .1 7 9 , so that the
factor necessary to calculate th e con ten t o f m eth oxy is 0 .6 0 9 6 .
b Calculated value.

F IG . 7. R e la tio n s b e tw e e n lig n in a n d a u to x id a tio n p r o d u c ts o f p h e n o ls a s w e ll a s f u r t h e r fa c to r s c a u s in g th e

d e c o m p o s itio n o f lig n i n .

3. INVESTIGATION ON THE AUTOXIDATION PRODUCTS OF PYROGALLOL

The formation of humic-like substances during the autoxidation process of pyrogallol, which
is parallel with the creation of other substances under the chosen conditions, was demonstrated by
electrical conductivity, absorption spectra in the u.v., visible and i.r. region, also by the solution
and coagulation in various solvents and at different pH-values. However, a detailed description
is not given here.
 IA E A -SM -211/44 81

4. DISCUSSION

With the help of a variety of methods including electron spectroscopy in the u.v. and visible
region, i.r. spectroscopy, and the Warburg technique, and also thin-layer chromatography, the use of
electrical conductivity and the application of special colour reaction, as well as several other
analytical methods, it was not only possible to indicate chemical interactions between natural
lignin and autoxidation products of pyrogallol under test conditions very similar to nature (soil,
peat, etc.), but the many alterations that occurred in the structure of lignin could be measured
analytically also. So apart from the uptake of oxygen, a release of C02 and a diminution of
light absorption in the u.v. and visible region, registered on the reaction mixture of lignin and
pyrogallol, dissolved in 7-butyrolacton/H20 (1:1 vol.%) in the presence of Si02 as catalyst, the in­
vestigations indicated that the тг-orbitals of the lignin had decreased, particularly in cis- and trans­
double bonds and a- and /З-keto as well as aldehyde groups in the aliphatic side chains of lignin and
in phenylcoumarone and stilbenoide structure elements. On the other hand, some ^-electronic
systems were increased by the reaction procedures (carboxyl, ester and/or lacton groups). The
esterification of carboxyl with alcoholic hydroxyl groups, besides radical mechanisms, possibly
forms complexes between lignin and humic-like substances of autoxidized pyrogallol. The
reduction in phenolic hydroxyl groups by the interactions seemed to proceed at least partly parallel
with the formation of oxygen bridges, as could be shown by i.r. spectra, and the increasing content
of methoxyl groups indicated a relative diminution of aliphatic structures.
The reactions and structural alterations of lignin by the interactions with autoxidation
products of pyrogallol are typical for the decay of lignin, where condensation and, above all,
oxidative processes play a large part. The total decay is chiefly related to the aliphatic structures
of lignin.
Because of the fact that lignin in soil can produce phenols by the activity of microbes,
autoxidation of at least part of these phenols leads to the (abiological) decay of lignin, as described
here. These aspects and some further relations are expressed in the scheme shown in Fig.7.
In order to guarantee a better decomposition of straw in soil, where lignin is the limiting
factor of the turn-over in this material, the supply of lignin in soils should be as high as possible,
and burning of straw or other methods of total removal should be kept to a minimum.

5. SUMMARY

Chemical interactions between natural lignin and humic-like autoxidation products of

pyrogallol (1,2,3-tri-hydroxybenzene) produced many effects on the reaction mixture
( 0 2-uptake, C02-release, decrease of absorption in the u.v. and visible region) and altered the
structure of lignin to a large extent (decrease of rr-electron orbitals, loss of phenolic hydroxyl,
alcoholic, aldehyde and keto groups, whereas the methoxyl, carboxyl, ester and/or lacton groups
increased). On the whole the lignin was degraded abiologically, mainly in its aliphatic structure
elements. The results of the experiments described are in many respects similar to the effects which
were obtained in consequence of the chemical interactions between lignin and hydrochinon
(p-di-hydroxybenzene) under soil conditions, although there were also some remarkable
differences [5].

Appendix
DETAILS OF EXPERIMENTS

Apparatus Perkin-Elmer, E T S—3T (u.v.; visible region; near i.r.)

Perkin-Elmer, Grating 4 5 7 (i.r. 4 0 0 0 — 4 0 0 cm -1 ).
82 WEICHELT

M ethods

R ed uction w ith NaBH4 :

(a) 4 m l lignin solu tion (unreacted or from the reaction-m ixture-separated lignin), concentration: 1.0 m g/m l
in n-dioxan; 2 m l distilled H20 w ere added.

(b ) 4 ml lignin solu tion (see above) plus 2 m l 0 .0 3 N NaOH.

(c) 4 m l lignin solu tion (see above) plus 2 ml NaBH4-solution in 0 .0 3 N NaOH, w hich had a concentration o f
0.1 mg NaBH4 per m l 0 .0 3 N NaOH.

T he u.v. spectra was measured on th e dissolved substances, and th e i.r. spectra were measured after
evaporation and preparation o f KBr-pellets.

Phenolic h yd roxy groups

C oncentrations: lignin = 0 .5 m g/m l dioxan

l-nitrose-2-naphthol: 0.01%
H N 0 3 = 2.5N ; H C l:d = 1.125.

The solu tion s were added as described in Table X; reaction tim e: 6 hours; m easurem ent at Xmax 3 8 5 nm.

To calculate the ratios o f th e w hole con ten t o f phenolic OH groups per unit o f w eight, th e m olecular mass
o f a phenylpropane unit in lignin w as assumed as 180. Therefore o n e gram o f lignin has
6.02
-------X 1023 = 3 3 .3 X Ю20 phenylpropane units, whereas eu gen ol has 3 6 .7 X Ю20 m olecules, so that th e ratio
180
o f phenol-bearing units is 1 :1 .1 per unit o f w eight.

Lignin preparation

The lignin was isolated from P i c e a e x c e l s a b y th e slightly m od ified m ethods o f Björkman [6 ] and Pepper,
Baylis and Adler [7]. T hese m eth od s gave lignins very like th ose described in R efs [ 8 ,9 ], and w hich were tested
b y further experim ents [1 0 —12].

REFERENCES

[1] A ULIN -ERD TM A N , G ., U ltraviolet spectroscopy o f lignins and lignin derivates, T echn. A ssoc. Pulp. Paper
Ind. 3 2 ( 1 9 4 9 ) 260.
[2] A ULIN -ERD TM A N , G., Sven. Papperstidn. 56 (1 9 6 8 ) 1187 .
[3] A ULIN -ERD TM A N , G., S A N D E N , R ., A cta Chem. Scand. 2 2 (1 9 6 8 ) 1187.
[4] GATTERM ANN-W IELAND, D ie Praxis des organischen Chem ikers, 41st E dn, Walter DeGruyter, Berlin,
(1 9 6 2 )7 4 .
[5] WEICHELT, T h ., “C hem ical interactions betw een lignin and autoxidizing h ydroch in on ” , Int. Peat Sym posium ,
D anzig, 1 9 74, 88.
[6] BJÖRKM AN, A ., Studies o n fin ely divided w ood: I. Extraction o f lignin w ith neutral solvents, Sven.
Papperstidn. 59 (1 9 5 6 ) 4 7 7 .
[7] PEPPER, J.M ., et al., The isolation and properties o f lignin obtained b y th e acidolysis o f spruce and aspen
w ood in dioxan-water m edium , Can. J. Chem. 37 (1 9 5 9 ) 1241.
[8] ZIECHMANN, W., WEICHELT, Th., Chem ische Veränderungen bei der Isolierung von Lignin,
T eil I: A usbeuten, M ethoxylgehalte, U V-Spektren, Z. Pflanzenernaehr. Bodenkd. (in press).
[9] ZIECHMANN, W., WEICHELT, T h ., Chem ische Veränderungen bei der Isolierung von Lignin,
Teil II: IR-Spektren, Phloroglucin-HCl-Nachweis, Z. Pflanzenernaehr. Bodenkd. (in press).
[10] WEICHELT, Th., M ÜLLER-WEGENER, U ., Fluorescence o f lignin (unpublished).
[11] KRESS, B.M., WEICHELT, Th., ZIECHMANN, W., Phosphorescence o f lignin (unpublished).
[12] WEICHELT, Th., T h e use o f fluorescence and p hosphorescence to investigate biological material,
Int. Peat S ym posiu m , Posen, Sept. 1976 (in press).
 IAE A-SM -211 /4 4 83

DISCUSSION

M. METCHE: In view of your results one question arises whether the autoxidation catalyst
of the pyrogallol'was not copper. Several observations mentioned in the literature seem toindicate
that this autoxidation does not occur when the reaction takes place in a silica vessel and in a
solution of triple-distilled water.
Your paper seems to me very interesting because the model you studied includes processes
involving radicals and it is therefore conceivable that the autoxidation of lignin is promoted by
coupling with a reaction leading to oxidation of a simple compound (pyrogallol). We now have
some results which tend to confirm your observations.
Moreover, such models are made considerably more interesting by observations made on the
organic matter of soils. T. Carballas and F. Andreux have recently demonstrated the primary role
of the process of walnut leaf a-hydrojuglone oxidation in the development of the organic matter
of a soil covered by mixed vegetation (Graminaceae and walnut). 1
T. WEICHEÜT: From the literature and from my own investigations I know that several
copper and various silicium oxide compounds can initiate the so-called autoxidation of a variety
of phenols. Although the research I described was not primarily directed towards studying the
effects of various catalysts that cause the autoxidation of phenols, I did study the effect of Si02,
Si02 •2H20 , Si02 •nH20 , Cun S04, KOH and NaOH in parallel experiments using a variety of
phenols, such as pyrogallol, hydroquinone, p-quinone and pyrocatechin. I first took as a medium
distilled H20 alone and then added one of the catalysts mentioned above. All these gave a clearly
positive effect; I selected Si02 because it had a rather long reaction time, and because it presents
conditions very similar to those of soil (pH, clay minerals). The effect of the various catalysts I
measured by using electron spin resonance (decrease after the starting period), u.v. spectra
(decrease ~ 270—295 nm), absorption spectra in the visible region (increase) and also gravimetrical
determination of the humic substances.
In my investigations, pyrogallol was used as a model substance to form humic substances
(autoxidation products) that altered the lignin by chemical interactions in many ways. But during
these interactions new phenols such as pyrogallol can be produced by decomposition of lignin,
too, as I indicated in Fig.7.
Many thanks for your interest and the reference to additional literature.
W. FLAIG (S cien tific S ecreta ry): How do you explain the increase in ester groups?
T. WEICHELT: The increase in ester groups is due to oxidative processes on various
functional groups of lignin, such as coniferyl and unconjugated and conjugated aldehyde groups,
the latter can be of a syringyl, coniferyl and a p-coumaric nature. All these groups form carboxyl
groups that react with aromatic as well as aliphatic hydroxyl groups of the humic substances
produced by pyrogallol, thus producing the corresponding ester groups. Some of the humic
substances were bound by such ester linkages, whereas another part reacted with lignin by radical
mechanisms.
W. FLAIG ( S cien tific S ecreta ry): Did you consider the possibility of tropolone systems being
formed during oxidation of pyrogallol in your explanation of reactions? The formation of this
type of compound is very rapid.
T. WEICHELT: I regarded the aromatic tropolone systems that have many electron я-orbitals
as very important in the chemical interactions between lignin and autoxidizing pyrogallol, mainly
because of their ability to form bi-radicals and the corresponding mesomeric formations. As the
investigation of the chemical alteration of lignin was the main purpose, the tropolones (or very
similar systems) were estimated only by using u.v. spectra, as was the case for purpurogallols,
gallic acid, etc. Other measurements using fluorescence and phosphorescence (emission) spectro­
scopy also indicated the importance of tropolone systems but the results are not published here.
 IAEA -SM -211 /4 5

P O S S IB IL IT IE S O F A N E C O N O M IC A N D
N O N -P O L L U T IN G U T IL IZ A T IO N O F L IG N IN

W.H.M. SCHWEERS,
Bundesforschungsanstalt für
Forst- und Holzwirtschaft,
Hamburg

W. VORHER
Ordinariat für Holztechnologie der
• Universität Hamburg,
Hamburg,
Federal Republic of Germany

Abstract

POSSIBILITIES OF A N ECONOMIC A N D NON-POLLUTING UTILIZATION OF LIGNIN.

The problem o f lignin u tilization , in spite o f m any years o f effo rt, has n o t been solved satisfactorily. One
o f the m any reasons for th e difficulties encountered in the prod uction o f useful products, either polym eric or
m onom eric, is th e fact that in technical processes, such as w ood pulping or w ood hydrolysis in w hich lignin is
obtained as a by-product, the natural lignin undergoes condensation reactions and/or is transform ed to sulphur-
containing derivatives. These alterations o f the natural lignin m olecu le make it less reactive, and influence
strongly its degradability to m on om eric products. If lignin is isolated from w o o d y plants or oth er lignocellulose-
containing m aterials b y phenolysis, lignin is obtained in a less condensed state and h en ce can easüy be degraded
w ith good yield to m onom eric phenols. A lso its chem ical reactivity, particularly because o f its high con ten t in
phenolic OH-groups, is m uch higher than the reactivity o f th e usual technical lignins. For exam p le, it can be
reacted easüy w ith dnsocyanates to obtain polyurethanes. It could be show n that the phenolysis o f lignin also
is an alternative to existing pulping procedures. H ence it should be possible to obtain also the by-product lignin
from these processes w hich n ow am ount to approxim ately 4 0 m illion ton s annually in a less condensed and higher
reactive state.

Lignocellulosic material, such as wood and straw, is one of the largest renewable resources of
carbon-containing raw material in the world. The annual net primary production of carbon fixed
by various major land ecosystems amounts to approximately 54 X 109 t [1 ].
Since the carbon content of lignocellulosic material is approximately 50%, the production of
total dry mass amounts to 108 X 109 t annually. If one works on an average lignin content of
20% of this material, the annual production of lignin in nature is 21.6 X 109 t, representing
12 X 109 t of organic bound carbon.
So far this vast amount of organic bound carbon has been almost unused as a source of the
production of chemicals or chemical products, since we do not have at our disposal technically
and economically feasible processes to isolate the lignin in such a structural state that allows easy
chemical degradation to useful products.
Technical processes by which lignin is separated from the carbohydrate portion of the ligno­
cellulosic material, such as the different pulping processes and processes for wood hydrolysis,
yield lignin in a drastically altered state. It is more condensed than in its natural form, and further­
more in many of these processes such as the sulphate- and sulphite pulping it is obtained as a
sulphur-containing derivative. The amount of lignin which at present is obtained in such an altered
state as a by-product of these pulping processes is approximately 40 X 106 t annually. Hence
the ‘technical lignins’, äs they are obtained at present, are difficult to degrade and less reactive than
natural lignin, and because of the sulphur content are not suitable for a series of further chemical

85
86 SCHWEERS and VORHER

сн, он сн,он
I 2 I 2
н-с н-с-
H'-С-0— / ~ Л — С-С- СН20Н Н-С+ + НО— S— С-С- СН2ОН

а осн, а осн,
ь 5
I
он

СН2ОН
о
Н-с-
H.-?—О - он
осн,
0 i
1 F I G .l. R e a c tio n o f p h e n o l w ith lig n in .

TABLE I. PULPING OF PINE WOOD WITH PHENOL; PULPING

CONDITIONS

Tim e Tem perature

(h) (°C )

0.5 2 0 to 110

1 110

2 110 to 170

3 170
0-25 170 to 50

Hydrom odul: 1:10

A cid catalyst: HC1 or HOOC-COOH 0.05 — 2%

TABLE II. PULPING OF PINE WOOD WITH PHENOL;

MATERIAL BALANCE

Wood 2 0 0 0 g (o .d .)

Phenol 12364g

Lignin in w ood 28% = 5 6 0 g

Screened pulp 5 3 .3 % = 1066 g

Kappa N o. 6 3 .6
Lignin in pulp 9 .5 4 % = 101.7 g

Screenings 3.5% = 7 0 g

Lignin in screenings 2 8 % = 19.6 g

Lignin extracted 4 3 8 .7 g

Phenol recovered by distillation 12.2 5 6 g

Phenol condensed to lignin 90 g

Crude phenol lignin obtained 1024 g

 IA E A -SM -211/45 87

TABLE III. PULPING OF PINE WOOD WITH PHENOL; PULP QUALITY

Pulp yield

U nscreened 56.8%

Screened 53.3%

Screenings 4.5%

Kappa N o. 6 3.3

T ensile strength 9 .2 0 0 m

Tear factor 53

Burst factor 60

Folding resistance (all at 50° SR) 1.300

Behaviour in bleaching C /E /H /D /E /D 99% GE

TABLE IV. PULPING OF PINE WOOD WITH PHENOL; PYROLYSIS OF PHENOL LIGNIN

P y r o ly s is c o n d itio n s

Temperature 550°C

Sw eeping gas (con tin u ou s) Water vapour

P r o d u c ts o b ta in e d

% based on % based on
crude lignin lignin removed from w ood

Phenol 2 0 .0 4 6 .7 !

o-Cresol 3.9 6.7

p-Cresol 2.0 4.7

E thyl phenol 1.2 2.8

T otal phenols 26.1 6 0 .9

Surplus o f
Phenol 26.2

Phenol m ixture 4 0 .4

Charcoal 29.8

Gases (CO, CH4 , C 0 2) 16.0

Neutrals 14.0

A cids 0 .4

L osses (w ater) 13.7

reactions unless they are desulphurized before use. If one takes into account that the chemical
industry today uses approximately 90 X 106 t annually of organic bound carbon for the production
of its organic products, and is mainly based on crude oil as raw material — a limited and
unrenewable resource which will be available only for the next 30—50 years —it is obvious that
in future it will not be tolerated to waste such large amounts of lignin which contain 22 X 106 t
of organic bound carbon, and hence represent 24% of the amount of carbon that is consumed by
the organic chemical industry.
88 SCHWEERS and VORHER

For an economic use of lignin as a raw material for the production of chemicals it is necessary
to develop processes that allow the separation of lignin from the lignocellulosic raw material in a
less altered form. On the other hand, such a process should also be suitable to obtain the carbo­
hydrate portion of the raw material as pulp in good quality and good yield.
In this connection we have studied the possibilities of pulping wood, and other ligno­
cellulosic matter such as straw, using phenol in the presence of small amounts of an acid catalyst
as pulping agent [2-8]. Phenol reacts with lignin in the presence of acids, as shown schematically
in Fig.l.
The lignin macromolecule is degraded by splitting the ether-bonds, and phenol is condensed
to reactive centres in the а-position of the propane side chains of the phenylpropane units, avoiding
internal condensation of the lignin molecule [9—12].
The phenol lignin formed in this reaction dissolves in the phenol and can be finally extracted
from the crude pulp by isopropanol and 1% NaOH.
The crude phenol lignin is isolated together with the dissolved portion of carbohydrates as
a brown powder by distilling off unreacted phenol and the solvent. This crude material can easily
be utilized for the production of simple monomeric phenols by pyrolysis. The analytical
characteristics of the purified phenol lignin show that it is high in OH-content, and has a low
molecular weight compared with other technical lignins. Recent experiments have shown that
it can be easily reacted with diisocyanates to form polyurethanes.
The pulp obtained by the phenolysis of lignocellulosic materials has also been investigated.
It could be obtained in good yield and it is easily bleachable. Tensile strength, burst factor and
folding resistance are comparable with results obtained by the classical pulping processes. Only
the tear factor is lower, but it is hoped that by changing the pulping condition a better tear
resistance can be reached.
In Tables I—IV details about pulping conditions, the results of pyrolysis of phenol lignin
and data showing pulp quality are presented.
From the results shown in Table IV it can be seen that phenolysis of lignocellulosic material
offers the possibility of converting the lignin extracted to a simple mixture of phenols with a
yield of 40%. This yield is far higher than that obtained in pyrolysis or hydrogenolysis of sulphite
or sulphate lignins as reported in the literature [13, 14].

REFERENCES

[1 ] O LSON, J.S., “Carbon cycles and tem perate w oodlands”, E cological Studies (REICHLE, D .E ., E d.),
Chapman & H all, L on d on (1 9 7 0 ) 226.
[2 ] SCHWEERS, W., PER EIRA , H .N ., Über den Holzaufschluss m it Phenolen, 1. Mitt.: Tränkung verschiedener
H ölzer m it Phenol, H olzforschung 2 6 (1 9 7 2 ) 51.
[3 ] SCHWEERS, W., BEH LER , H ., B E IN H O FF, O ., Über den H olzaufschluss m it Phenolen, 2 . M itt.: V or­
läufige U ntersuchungen über die Phenolbilanz, H olzforschung 2 6 (1 9 7 2 ) 103.
[4] SCHWEERS, W., RECHY, M., Über den H olzaufschluss m it Phenolen, 3. Mitt.: Über den A ufschluss von
Kiefern- und B u ch en h olz, Papier (D arm stadt) 27 (1 9 7 3 ) 6 3 6 .
[5] SCHWEERS, W., RECHY, M., BEINH O FF, 0 ., Phenol pulping, Chem. T ech. (A m sterdam ) (1 9 7 4 ) 4 9 0 .
[6] RECHY, M., U ntersuchungen über die M öglichkeiten des A ufschlusses von H olz m it P h en olen, D octorate
Thesis, Hamburg (1 9 7 3 ).
[7] VORHER, W., E ntw icklung eines Kontinuierlichen Pyrolyseverfahrens zum Abbau von Phenollignin m it
dem Ziel der Ligninverwertung unter besonderer Berücksichtigung der Rückgewinnung von Phenol aus
Ablaugen eines P h en olzellstoffp rozesses, D octorate Thesis, Hamburg (1 9 7 6 ).
[8] V ORHER, W., SCHWEERS, W.H.M., U tilization o f phenol lignin, Appl. P olym . Sym p. 2 8 (1 9 7 5 ) 2 77.
[9] B R A U N S, F .E ., The Chem istry o f Lignin, A cadem ic Press, N ew York (1 9 5 2 ) 4 79.
[1 0 ] B R A U N S, F .E ., B R A U N S, D .A ., The Chemistry o f Lignin, Suppl. V ol., A cadem ic Press, N ew York (1 9 6 0 ) 4 7 7 .
[1 1 ] KRATZL, K., Z A U N ER , L , C LAUS, P., R eaktionen m it m arkierten Ligninen und M odellen, 1. Mitt.:
K ondensation m it Phenol, H olzforschung 18 (1 9 6 4 ) 47.
 IA EA -SM -211/45 89

[12] NIMZ, H., Kondensationsreaktionen des Lignins, Holzforschung 23 (1969) 84.

[13] GOHEEN, D.W., MARTIN, J.B., Preparation o f guajacol from spent pulping liquors, Canadian Patent
791.772, 1968.
[14] GOHEEN, D.W., “ Low molecular weight chemicals” , Lignins —Occurrence, Formation, Structure, and
Reactions (SARKANEN, K.V., LUDWIG, C.H., Eds), Wiley Interscience, New York, London, Sidney,
Toronto (1971) 575.

DISCUSSION

M.H.B. HAYES: Your phenolysis mechanism suggests cleavage of an ether linkage, release
of an alkoxide, formation of a carbonium ion and finally condensation with the 4-position of
the phenol. Such reactions can have very considerable implications in humic acid structure deter­
minations (Jackson, Swift, Posner, S oil Sei, and Piper and Posner, S o il Sei, so I would appreciate
a more detailed description of this mechanism.
W.H.M. SCHWEERS: We do not add acidic phenol, but hydrochloric or oxalic acid. The
reaction mechanism has not been studied in our laboratory, but there are publications on it by
Kratzl and co-workers [11] and Nimz [12]. They studied reactions of phenols with lignin or
lignin model compounds, and also looked at degradation products, in particular those obtained
from the oxidation of the condensation products with nitrobenzene. They succeeded in showing
that condensation takes place between the first carbon of the side chain, i.e. the one next to the
aromatic ring, and the ortho or para position of the phenol. Cations react easily with a phenol in
either position; this is a well-known substitution reaction. When the cation is formed by splitting
of the ether, a proton, H+, is added to the ether bridge, which is split, and the resulting cation
reacts with the phenol.
B.R. NAGAR: What type of pyrolyser did you use, and what was the pyrolysis time?
W.H.M. SCHWEERS: We used hydrochloric or oxalic acid as catalysts for phenolysis; for
some reason sulphuric and acetic acid did not work in this way. We did not use catalysts at all
for pyrolysis; we simply heated the material. The pyrolysis time was rather short; we did not
measure it accurately, since the raw material was introduced into a tube heated to 550°C with a
device that moved the raw material along this tube. However, it was approximately 30—60 s.
The main problem is that one must use a sweeping gas, water vapour or nitrogen, because the
monomeric phenols —the degeneration products —have to be removed from the hot zone promptly,
before they are further degraded. Thus the monomeric material remains in the hot tube for
seconds only, whereas the phenol lignin is heated for 30—60 s.
 IA E A -SM -211 /4 6

O X ID A T IO N O F S O M E P H E N O L IC S U B S T A N C E S
A S IN F L U E N C E D B Y C L A Y M IN E R A L S

Z. FILIP
Institut für Landwirtschaftliche
Mikrobiologie,
J ustus-Liebig-Universität,
Giessen

W. FLAIG, E. RIETZ
Institut für Biochemie des Bodens
der Forschungsanstalt für Landwirtschaft,
Braunschweig-Völkenrode,
Federal Republic of Germany

Abstract

O X IDA TIO N OF SOME PHENOLIC SUBSTA N CES A S INFLUENCED B Y CLAY M INERALS.

In m od el experim ents, the oxygen uptake o f 5-m ethylpyrogallol, 2 ,3,4-trihyd roxyb en zoic acid, 2 ,3 ,5 -
trihyd roxytoluene, as w ell as o f the m ixtures o f these p henols w ith protocatechu ic acid, caffeic acid and orcine,
was n o t at all or to a small ex ten t influenced by the presence o f m ontm orillon ite in the water suspension buffered
at pH 7 or 8. Furtherm ore, m ontm orillon ite and kaolinite w hen saturated w ith 1 Г , K +, Ba2 + , Ca2 +, A l3 + or
F e3 + ion s, and added to the pyrogallol solu tion buffered at pH 6 .0 , influenced neither the speed o f the colour
changes, nor the in ten sity o f the brow n colour, evaluated as an extin ction at 3 0 0 nm . H ow ever, b oth phenom ena
were influenced b y m uscovite. N o direct relationship has been found betw een the valence o f the cations in the
exchange p osition s o f m uscovite used and the catalytic effect o f this clay mineral.

INTRODUCTION

Soil humic acids consist of more than 45% humus-like fungal melanins and more than 20%
of different phenolic substances [if. According to Haider and co-workers [2], the oxidative
reactions of phenolic substances, sometimes without any participation by microbial phenoloxidases,
play an important role in the humification processes. As the production of fungal melanins is
markedly accelerated and enhanced after the addition of clays to the culture media [3], tests were
made to establish the possible effect of some clay minerals on the oxygen uptake of phenols and
their mixtures, and also on the browning reaction of pyrogallol.

MATERIAL AND METHODS

For the respirometric tests, 20 /nmol of protocatechuic acid, caffeic acid or orcinol were dis­
solved in phosphate buffer at pH 7.0 or 8.0 and were placed in the main container of the Warburg
vessels with or without addition of 0.5% (wt/vol.) of montmorillonite (Wyoming). In the side arm
of the Warburg vessels, 5.0 pmol of 2,3,5-trihydroxytoluene, 2,3,4-trihydroxybenzoic acid or
5-methylpyrogallol dissolved in 0.5 ml H20 were placed. In the central container of the vessels,
0.2 ml of 10% NaOH solution was present. After heating to 28°C both reagents were mixed together
and the oxygen uptake of the mixtures was measured for 3 to 4 h. In some experiments 10 ppm
of Fe2+ ions were also added to the reaction mixtures.

91
92 FILIP e t al.

TABLE I. OXYGEN UPTAKE FROM REACTION MIXTURES OF PHENOLS WITH AND

WITHOUT MONTMORILLONITE

R eaction m ixtures д т о 1 0 2 at pH 7 д т о 1 0 2 at pH 8
Conrol M ontm. Control M ontm .

5 д т о 1 5-MPG a 5 .6 5.5 6.1 6.2

+ 20 Mmol PCA 6 .0 5 .8 6.4 6.2
+ 20 jUmol CA 6 .5 6 .5 9.3 9.2
4- 20 jUmol Ore, 6 .0 5.8 6.3 6.5

5 Mmol 2,3,4-TH BA 3 .3 2.3 6.0 6.4

+ 20 Mmol PCA 3 .5 3 .6 6.9 7.0
+ 20 д т о 1 CA 5 .0 5 .6 13.0 11.3
+ 20 д т ol Ore, 3 .6 3 .5 11.5 11.4

5 Mmol 2,3,5-TH T 5.5 5 .6 5.9 6.4

+ 2 0 Mmol PCA 6 .4 6.8 8:0 7.5
+ 2 0 д т о 1 CA 8 .0 7.0 10.4 10.0
+ 20 д т о 1 Ore. 6 .5 6.2 6.8 6.2

5 д т о 1 5-MPG +
10 ppm F e3 + 8.6 7.8 11.2 12.4
+ 20 Mmol PCA 8.6 6.6 12.6 13.5
+ 20 д т о 1 CA 10.4 8.1 15.8 16.2
+ 20 д т о ! Ore. 9.7 8.6 13.9 14.0

a 5-MPG — 5-m ethylpyrogallol; PCA = protocatechu ic acid; CA = caffeic acid; Ore. = orcinol;
2,3,4-T H B A 2 ,3,4-trihyd roxy ben zoic acid; 2,3,5-T H T = 2,3,5-trihyd roxytoluene; F eJ + = FeSC).-,.

For the spectrophotometric tests, 100.0 mg of pyrogallol were dissolved in 40 ml of 0.1N

natrium acetate solution buffered at pH 6.0. With the exception of control variants, 40.0 mg of
fine ground quartz, or the same quantity of clay minerals (a fraction < 2 pm) were added. The clay
minerals used (kaolinite, muscovite, montmorillonite) were saturated with H+, K+, Ba2+, Ca2+,
Al3+ or Fe3+ ions before use. The flasks with the reaction mixtures were shaken at 100 rev/min
under a light intensity of 600 W/l m2 at a constant temperature of 22°C. After 4, 24 and 48 hours
respectively, parallels were examined. First the mixtures were centrifuged at 12 000 g for 20 min
and then the pyrogallol solution free from minerals was measured in the range of 250—600 nm using
a SP 1800 Phillips/Unicam spectrophotometer. As well as recording the extinction curves, the
extinction values at 300 nm were determined for the evaluation of colour intensity.

RESULTS AND DISCUSSION

The data in Table I show that the oxygen uptake of phenol mixtures has in fact not been
positively influenced by the presence of montmorillonite. In some cases it was lower than in the
controls. As the sorption of the phenols used can be omitted at the given pH values according to
previous tests [4], the suitable conditions for a heterogenic catalysis at the clay surface are
perhaps neglected. After the addition of Fe2+ ions, the oxygen uptake increased a little at pH 8.0,
but this effect disappeared at pH 7.0, probably because the cations became strongly fixed by clay.
 IA E A -SM -211/46 93

F I G .l. A b s o r p tio n s p e c tr a o f r e a c tio n m ix tu r e s : F I G .2 . A b s o r p tio n s p e c tr a o f r e a c tio n m ix tu r e s :

p y r o g a llo l. p y r o g a llo l + k a o lin ite .

F I G .3 . A b s o r p tio n s p e c tr a o f r e a c tio n m ix tu r e s : F I G .4 . A b s o r p tio n s p e c tr a o f r e a c tio n m ix tu r e s :

p y r o g a llo l + m o n tm o r illo n ite . p y r o g a llo l + m u s c o v ite .

94 FILIP e t al.

These results indicate that montmorillonite alone does not act as a catalytic factor by auto-
oxidative changes of phenolic mixtures which are common in the young cultures of soil fungi
producing dark-coloured melanins.
As the humification must be regarded as to some extent a browning phenomenon of organic
substances, further experiments were carried out using pyrogallol, a trihydric phenol, which can
be oxidized by molecular oxygen to a dark-coloured polymer product. It is also known that
pyrogallol can be formed during oxidative changes of more simple phenols of biological origin [2].
The oxidative changes of pyrogallol were estimated spectrophotometrically in the u.v. range,
where a newly prepared colourless solution very strongly absorbs at 266 nm only. With the lapse
of time the pyrogallol solution became yellow, reddish brown and finally brown. In Fig.l one
can see a strong absorption shoulder at 300 nm and a weak one at 370 nm after 4 h. The first
appeared as a strong absorption band after 24 hours and became a little weaker after 48 hours but
the 370 nm shoulder disappeared almost completely at the same time.
Whereas the molecular oxygen only caused the oxidation and the respective spectral changes
in the pure pyrogallol solution, a secondary oxidation by electron transfer could additionally be
taken into consideration when clay minerals are present in the reaction mixtures. However, the
absorption spectra were only very little changed by kaolinite and montmorillonite. The absorption
at 300 nm and at 370 nm is a little weaker in Figs 2 and 3, but an additional absorption band at
340 nm appeared, particularly when Fe3+ or Al3+ were the exchangeable cations. A blue-colour
reaction, probably caused by reduction of trivalent cations, was responsible for these spectral
changes, which are no longer detectable after 24 hours.
Among the mineral substances used, the muscovite influenced most extensively the absorption
spectra of the pyrogallol solution. In Fig.4 the spectral curve of the 48-hour reaction solution
shows only a very low absorption shoulder at 266 nm instead of a strong band, which is characteristic
for pyrogallol. Below the 300-nm absorption shoulder, the spectral curve shows no longer any
clear-cut absorption bands, similar to soil humus and humus-like substances [5].
Considering that the absorption band or shoulder at 300 nm is very characteristic of oxidative
changes of pyrogallol under the experimental conditions, the optical density of pyrogallol solutions
measured at that wavelength has been chosen for some comparative evaluation of the degree of
pyrogallol oxidation. Other authors also [6,7] recommend the use of the optical density at 300 nm
for estimating the amount of oxidized material formed by a polymerization of polyhydric phenols.
The data in Table II show a general increase in the optical density at 300 nm in dependence on a
reaction time from 4 to 48 h. Compared with the pure pyrogallol solution, the extinction values
are only very little influenced by kaolinite and montmorillonite, but they are distinctly influenced
by muscovite. Therefore o n ly the muscovite, a mica clay, enhanced the formation of a brown
polymer substance in a buffered pyrogallol solution, whereas both kaolinite and montmorillonite
were only a little or not at all active. Measured according to the end effect, i.e. after 48 h of the
reaction time, even the pure quartz (Merck) exerted more influence than either kaolinite or
montmorillonite. It is also of interest to note that mono- and divalent cations in exchangeable
positions of clay minerals were more active than Fe3+ and Al3+. Therefore trivalent metal ions,
when in exchange sites, do not need to increase the oxidation, which can be brought about much
more by the activation of oxygen through chemisorption on or polarization by a solid surface.
Similarly Goodman and Siegel [8] have demonstrated the effect of solids on the oxidation of pyro­
gallol when cellulose was present in the reaction mixture. Other authors observed the oxidation of
hydroquinone to p-benzoquinone even in a nitrogen atmosphere, when Fe3+ or Cu2+ were
exchangeable cations [6], but the opposite reaction, namely the reduction of Prussian brown, was
also observed to be enhanced in the presence of di- and trivalent cations in exchange sites of
bentonite [9]. One can also assume that the possible oxidation power of trivalent metal ions,
which is based on electron transfer reactions, could be made ineffective with regard to the
experimental substance when some oxidizable impurities (e.g. organic materials) are present in the
clays added. In some accordance with our results Kumada and Kato [10], using different natural
 IA EA -SM -211/46 95

TABLE II. INFLUENCE OF QUARTZ AND CLAY MINERALS ON THE BROWNING

REACTIONS OF PYROGALLOL IN A NATRIUM ACETATE SOLUTION (pH 6.0)

E xtin ction values at 3 0 0 п т

R eaction m ixtures after 4 h after 2 4 h after 4 8 h

Pyrogallol (P) 0 .0 6 3 0 .5 0 0 0 ,5 9 2

P + Quartz 0 .075 0 .6 2 0 0 .7 9 5

P + K aolinite-H 1+ 0 .055 0 .5 8 7 0 .6 8 5

P + K aolinite-K 1+ 0 .0 5 8 0 .4 1 7 0 ,6 2 2

P + K aolinite-Ba2+ 0.051 0 .6 4 5 0 .6 4 5

P + Kaolinite-Ca2+ 0 .0 6 6 0 .5 8 7 0 .7 1 5

P + K aolinite-A l3+ 0 .0 5 6 0 .5 5 0 0 ,5 6 5

P + K aolinite-F e3+ 0.0 5 6 0 .4 1 7 0 .6 3 0

P + M uscovite-H1+ 0 .0 7 6 0 .7 5 0 1.2 6 0

P + M uscovite-K 1+ 0 .0 8 6 0.7 5 5 1.1 3 0

P + M uscovite-Ba2+ 0.0 7 3 0 .6 1 2 0 .6 7 0

P + M uscovite-Ca2+ 0 .0 7 0 0 .7 4 0 1.2 5 0
P + M uscovite-A l3+ 0 .0 7 0 0 .4 8 0 1 .2 2 0

P + M uscovite-Fe3+ 0.0 6 3 0 .5 8 0 1.0 4 0

P + M ontm orill.-H 1+ 0 .0 5 0 0.3 5 5 0 .7 2 0

P + M ontm orill.-K l+ 0 .0 6 0 0 .5 2 2 0 .7 2 7

P + M ontm orill.-Ba2"1" 0 .0 6 0 0 .5 2 0 0 .7 1 0

P + M ontm orill.-Ca2+ 0.0 5 5 0 .4 3 2 0 .5 4 0

P + M ontm orill.-A l3+ 0.0 6 5 0 .4 4 0 0 .4 4 0

P + M ontm orill.-Fe3+ 0.2 8 5 0 .4 6 0 0 .6 1 0

and ore clays, refer to their influence on the speed of browning reactions but not to spectral
changes in the pyrogallol solution.
From the results achieved in our experiments, and also from the results of other authors,
it can be concluded that both clays and other solid particles can act as catalytic factors in the
oxidation of йоте phenolic substances important in humification processes. However, a positive
or negative clay effect depends on specific conditions that are not yet exactly determined.
Because the effects mentioned are not only important for the theory of humus-forming processes,
but could also be of practical importance for the alteration of phenolic substances in waste water,
they will be further studied in detail.

REFERENCES

[1 ] SCHNITZER, M ., N E Y R O U D , J.A ., Soil B iol. B iochem . 7 (1 9 7 5 ) 365.

[2 ] H A ID ER , K „ M ARTIN, J.P., FILIP, Z., “Hum us biochem istry”, Soil B iochem istry IV (PA U L , E.A .,
McL a r e n , А Л ., Eds), M. D ekker, N ew York ( 1 9 7 5 ) 196.
[3 ] FILIP, Z ., H A ID ER , K ., M ARTIN, J.P., Soil B iol. B iochem . 4 (1 9 7 2 ) 147.
[4 ] FILIP, Z., “W echselbeziehungen zw ischen Mikroorganismen und Tonm ineralen und ihre Auswirkung auf
die B odendynam ik”, H abilitationsschrift, University o f Giessen (1 9 7 5 ).
96 FILIP e t al.

[5 ] FILIP, Z., SEM OTÄN, J., KUTILEK, M ., Geoderm a 15 (1 9 7 6 ) 131.

[6 ] THOMPSON, T .D ., MOLL, W .F., Jr., Clays Clay Miner. 21 (1 9 7 3 ) 337.
[7] THOMPSON, T .D ., T SU N A SH IN A , A ., Clays Clay Miner. 21 (1 9 7 3 ) 3 51.
[8] GOODM AN, N .S ., SIEGEL, S.M., N ature (L ondon) 184 (1 9 5 9 ) 53.
[9 ] Y A R IV , S., Israel J. Chem. 7 (1 9 6 9 ) 4 5 3 .
[1 0 ] K U M A D A, К ., К А Т О , H ., Soü Sei. Plant Nutr. 16 (1 9 7 0 ) 1 95.
 B IT U M E N S IN
S O IL O R G A N IC M A T T E R
(S e ssio n 7a)
 IAEA-SM-211/47

LIPIDS OF MICROBIAL ORIGIN

IN SOIL ORGANIC MATTER*
, G.H. WAGNER, E.I. MUZOREWA
University of Missouri,
Columbia,
Missouri,
United States of America

Abstract

LIPIDS OF MICROBIAL ORIGIN IN SOIL ORGANIC MATTER.

Fats, waxes and resins occur in soil organic matter, and may arise from microbial synthesis or accuihulate
as resistant remnants of plant residues undergoing humification. Lipids have been reported to account for about
5% of the organic matter in mineral soils. The significance of micro-organisms in contributing to soil lipids was
investigated by determining the amount and distribution of 14C in lipid fractions after incubating various labelled
substrates in the soil. Amendments were glucose, dextran, and cell wall and cytoplasmic fractions of fungal
sclerotia. After humification of glucose, the l4C recovered using benzene-ethanol to extract lipids equalled 7%
of the residual 14C in soil from a fertilized corn plot, and nearly 8% in soil from an untreated timothy plot.
Lipid 14C extracted from soil in which dextran had been incubated was only about one-half that recovered from
the soil incubated with glucose. Production of l4C microbial tissue was probably less in dextran-treated soil,
and an alternate quantity of 14C may have occurred as polysaccharide. The lipid 14C was distributed among
waxes, resins and asphalt in a similar pattern for all substrates investigated. Cell wall and cytoplasmic components
of sclerotia showed considerable resistance to decomposition; however, recovery of 14C in lipids following
incubation of these substrates was very much less than the amount in parallel extractions of the materials before
incubation. The discrepancy may indicate that added microbial lipids became incorporated into soil humus
without undergoing major degradation, and this condensation rendered them inaccessible to extraction.

INTRODUCTION

Lipids are among the least studied components of soil organic matter. These materials
comprise 1 to 5% of the organic matter in mineral soils, and up to 20% of that in organic soils [ 1].
Lipids are found in fossils of soil-forming rocks, in sediments, and in residues of plants, animals
and micro-organisms. Soil lipids can exercise a considerable effect on soil properties. Waxy
substances may waterproof soil particles to prevent movement of nutrients from mineral surfaces
into solution for plant uptake. They may similarly retard mineralization of organic nitrogen.
Low molecular weight components may behave as organic nutrients, and are likely to produce
harmful effects on plant growth. Numerous lipid components could exert stabilizing forces upon
soil aggregates by cementing mineral particles together, and by imparting water repellency to
loosely held aggregates.
The object of this investigation was to evaluate the significance of micro-organisms in
contributing to soil lipids. It was proposed to determine the amount and distribution of I4C in
several lipid fractions after incubating various labelled substrates in the soil.

* Contribution from the Department of Agronomy, Missouri Agricultural Experiment Station, Journal
Series No.7599.

99
100 WAGNER and MUZOREWA

TABLE I. ANALYTICAL DATA FOR SOILS AND RECOVERY OF CARBON-14 IN

EXTRACTED LIPIDS

pCi of 14C/100 g soil

Soil plota,
Organic C Extracted by
incubation period, Lipid
(%) p Initially After
and amendment incubation ^ Benzene- 14C
added
HCI ethanol (% of total)

Timothy 1.47 5.0

5 months
Glucose 3.98 0.959 0.195 0.074 7.7

Corn 1.33 6.0

5 months
Glucose 3.98 0.616 0.134 0.044 7.1

Corn 1.24 6.0

3 months
Dextran 3.27 0.851 0.092 0.030 3.5

Sclerotial
cytoplasm 7.44 4.82 0.156 0.186 3.8
Sclerotial
cell wall 7.32 6.54 0.096 0.067 1.0

Plot of Sanborn Field [4].

EXPERIMENTAL

The soil under study was a Mexico silt loam (Alfisol) from the upper 15 cm of Sanborn
Field. One plot cultivated continuously to com with adequate fertilization had an organic carbon
content of 1.3% and a pH of 6.0. A contrasting plot for study was cultivated continuously to
timothy with no fertilizer treatment, and had an organic carbon content of 1.5% and a pH of 5.0.
The soils were labelled with 14C because of previous incubations for periods up to 5 months with
labelled amendments including glucose [2], dextran, cell wall and cytoplasmic fractions of
sclerotia of M acroph om in a phaseoli [3]. Analytical data for the soils under study and activity
associated with the amendments are summarized in Table I.
Soils were first leached with 0.1N HC1 to remove basic cations and improve extractability
of lipid components [5]. An extraction-fractionation scheme as shown in Fig. 1, and similar to
that suggested by Morrison and Bick [6], was followed. Lipids were extracted from 20 g units
of soil using 130 ml of 2:1 benzene-ethanol in a Soxhlet apparatus for 20 to 21 h. The extract
was concentrated in a rotary evaporator under reduced pressure at 40°C, and an aliquot taken
for 14C determination. The remainder of the lipid-containing solution was taken to dryness,
re-suspended in 20 ml of light petroleum (b.p.63° - 75°C), and extracted in a micro-Soxhlet for
20 to 21 h to yield a crude wax fraction in solution and an insoluble asphalt. The soluble crude
wax fraction was reduced to a fixed volume, an aliquot taken for 14C counting, and the remainder
dried in vacuo. Crude waxes were further fractionated by refluxing for 1 h in 1:1 methanol-iso­
propanol after which any insoluble materials in the hot solvent were added to the previously
separated asphalt. Upon cooling the methanol-iso-propanol, the extracted matter separated into
 IAEA-SM-211/47 101

SOIL

Pre-Treatment
0.1N HCI

Soxhlet
Extraction
2:1 Benzene:Ethanol

Soxhlet
Extraction

F I G .l. S c h e m a tic d ia g r a m o f p r o c e d u r e f o r e x tr a c tio n a n d fr a c tio n a t io n o f s o il lip id s .

a refined wax precipitate and a soluble resin fraction. Aliquots were taken before and after
cooling for 14C determination.
Aliquots for 14C measurement were added to a PPO-POPOP scintillation cocktail [7], and
radioactivity was measured using a Packard Tri-Carb liquid scintillation counter. A standard
14C benzoic acid solution was used to determine counting efficiency.
Total carbon was determined by combusting samples in an electric furnace, trapping the
evolved C0 2 in a scrubbing tower containing NaOH, precipitating the C0 2 with BaCl2 and
titrating with HCI. Radioactivity in the precipitated BaC03 was counted by the liquid scintillation
method [ 8].

RESULTS AND DISCUSSION

The incubation of 14C glucose in soil for a period of 5 months to label soil organic matter
by microbial synthesis yielded residual 14C in the soil, as reported in Table I. The major portion
of the activity initially added to soil had been liberated as C02, and 24% and 15% remained in
the timothy and com soils respectively. Previous investigations have shown that glucose is very
rapidly utilized in these soils [ 8, 9]. During the prolonged incubation it must be assumed that
a number of successive generations of micro-organisms had metabolized the labelled carbon.
102 WAGNER and MUZOREWA

TABLE II. ACTIVITY IN LIPID FRACTION OF M acroph om in a phaseoli

SCLEROTIA

Fraction Total 14C Lipid I4C Waxes Resins Asphalt

(pCi/200 mg) (Percentage of lipid l4C)

Cytoplasm 7.44 3.85 2.2 65.1 15.3

Cell wall 7.32 0.68 1.5 68.2 11.4

F IG . 2. D is tr ib u tio n o f lip id 14C a m o n g r e s in s , w a x e s a n d a s p h a lt f o r s o ils in c u b a te d w ith d iff e r e n t s u b s tr a te s .

At the end of the incubation, glucose carbon had become humified to the extent that the
decomposition rate for 14C was approaching that of much of the native organic matter present
in the soil. A number of investigations have dealt with the distribution of 14C in these soils, and
have included classical fractions of soil organic matter [ 10], amino components [ 11] and
carbohydrates [ 12].
The preliminary leaching of each soil sample with dilute mineral acid removed from 15 to
20% of the residual 14C. This leachate and associated 14C was not investigated further, but it
would include a variety of simple organic components.
The quantity of 14C in the lipid fraction extracted using benzene-ethanol is reported in the
last two columns of Table I. Lipid I4C relative to total residual 14C after incubating glucose was
 IAEA-SM-211/47 103

slightly but significantly higher for the timothy soil than for the corn soil. The timothy soil has
a higher total organic matter content and a lower pH, and the latter parameter would indicate
that fungi had been relatively more significant than bacteria in the humification of the glucose.
Results for extraction of lipids after incubation of dextran in the corn soil are also reported
in Table 1. The dextran-treated soil yielded less lipid 14C than soil incubated with glucose. The
shorter incubation period was not assumed to be the major cause, and rate of decomposition
of dextran at the end of the incubation had declined similarly to that for glucose in a parallel
incubation [12]. A study that compared glucose and dextran during an incubation extending
for three months showed some resistance to decomposition for the polysaccharide [12]. The
lower proportion of 14C in extracted lipid from the dextran-treated soil may reflect a different
population or less 14C microbial biomass with an amount of 14C alternately occurring as
polysaccharide.
The 14C in lipids extracted from soil incubated with cell wall and cytoplasmic components
of sclerotia is shown in Table I. These amendments displayed considerable resistance to
decomposition [3], and the 14C in the extracted lipid must be considered to have arisen from
unaltered or only partially altered amendment as well as from newly synthesized microbial
materials. Extraction of the cell wall and cytoplasmic material before their incorporation into
the soil showed lipid 14C to equal 9% and 52% of the respective totals. Activity associated with
these materials and the distribution of this activity among the lipid fractions is reported in
Table II. After incubating the sclerotial materials in soil, the proportion of residual 14C that
could be extracted as lipid was greatly reduced. Even though decomposition of the amendments
was limited, a marked resistance to extraction with benzene-ethanol developed.
The distribution of the 14C among the several fractions obtained from the extracted lipid
material is reported in Fig.2. Soils incubated with diverse substrates give a similar distribution
pattern for waxes, resins and asphalt. Results for glucose which had been completely utilized
to yield in situ microbial products and tissues were found to be similar to results for highly
resistant sclerotial tissues incubated in soil. Some difference was observed in refined waxes which
were relatively higher for the glucose-treated soils at the expense of asphalts. The latter were
higher for the sclerotial treatments and for dextran also.
The fractionation procedure was not highly refined, and more elaborate procedures
apparently will be needed to determine lipid differences associated with differing microbial
populations in the soil.
The resin fraction constituted the largest proportion of lipid material for all treatments
under study. Some materials were lost during the fractionation procedure, and it is not known
if this loss was equally distributed among the fractions obtained. More likely the loss occurred
during concentration of the initial extract by rotary-vacuum evaporation after which material
adhered tightly to the walls of the flask. Total recovery in the fractions ranged from 75 to 85%
of the crude lipid fraction.
Results of this investigation show that lipids extracted from soil organic matter in mineral
soils may be of microbial origin. Microbially synthesized products of a lipid nature in soil may
become incorporated into soil humus without undergoing major degradative modification.
This presumed condensation imparts significant resistance to extraction by organic solvents to
the lipid materials.

REFERENCES

[1] BRAIDS, O.C., MILLER, R.H., “ Fats, waxes, and resins in soil” , Ch. 6, Soil Components I, Organic
Components (GIESEKING, J.E., Ed.), Springer Verlag, New York (1975).
[2] CHAHAL, K.S., WAGNER, G.H., Decomposition of organic matter in Sanborn Field soils amended with
C-14glucose, Soil Sei. 100(1965) 96.
104 WAGNER and MUZOREWA

[3] WAGNER, G.H., MEDYNSKA-RABINSKA, H., The contribution of melanic fungal tissues to soil organic
matter formation, Humus Planta V (1971) 27.
[4] WAGNER, G.H., “ Significance of microbial tissues to soil organic matter” , Isotopes and Radiation in Soil
Organic-Matter Studies (Proc. Symp. Vienna, 1968), IAEA, Vienna (1968) 197.
[5] HANCE, R.J., ANDERSON, G., Extraction and estimation of soil phospholipids, Soil Sei. 96 (1963) 94.
[6] MORRISON, R.I., BICK, W., The wax fraction of soils: Separation and determination of some components,
J. Sei. Food Agric. 18(1967) 351.
[7] DOBBS, H.E., Oxygen flask method for the assay of tritium, carbon-14 and sulfur-35 labeled compounds,
Anal. Chem. 35(1963) 783.
[8] WAGNER, G.H., “ Microbial growth and carbon turnover” , Ch.6, Soil Biochemistry 3 (PAUL, E.A.,
McCLAREN, A.D., Eds), Dekker, New York (1975).
[9] BEHERA, B., WAGNER, G.H., Microbial growth rate in glucose-amended soil, Soil Sei. Soc. Am., Proc.
38(1974) 591.
[10] MUTATKAR, V.K., WAGNER, G.H., Humification of carbon-14 labeled glucose in soils of Sanborn Field,
Soil Sei. Soc. Am., Proc. 31 (1967) 66.
[11] WAGNER, G.H., MUTATKAR, V.K., Amino components of soil organic matter formed during humification
of 14C glucose, Soil Sei. Soc. Am., Proc. 32 (1968) 683.
[12] OADES, J.M., WAGNER, G.H., Biosynthesis of sugars in soils incubated with 14C glucose and 14C dextran,
Soil Sei. Soc. Am., Proc. 35(1971) 914.
 IAEA-SM-211/48

ANALYSE ET ROLE DES BITUMES

DANS LES SOLS SABLEUX ACIDES

E. FUSTEC-MATHON*, P. JAMBU*,
G. JOLY**, R. JACQUESY**
Universite de Poitiers,
Poitiers,
France

АЫггаа-Яёвитё

ANALYSIS AND ROLE OF BITUMENS IN ACID SANDY SOILS.

In the acid sandy soils of the M6doc Landes (France), the podzols are observed to have a much higher bitumen
content than the hydromorphic type. Study of the acid and neutral fractions of the bitumens extracted from
surface vegetation and various horizons of these soils shows that the acid fractions and the hydrocarbons undergo
less transformation and less degradation in the Ai horizons of the podzols than in the corresponding hydromorphic
soil horizons. The neutral fractions are for the most part only slightly transformed, but are more rapidly degraded,
in the soils. The incubation of soil samples in the presence of 14C-labelled maize straw indicates that the soil
bitumens originate mainly from plants, but that microbial synthesis products are incorporated into this fraction,
more especially in the podzols. The bitumens have a toxic effect on the soil microflora as a whole. The intensity
of this effect varies with the concentration of the extracts, pH of the soil and origin of the microflora. The acid
fraction and the hydrocarbons are the strongest inhibiting agents. The depressant action depends to some extent
on the length of the chains and the nature of the functions. Acidity seems to play the main part in accumulation
of bitumens in the podzols studied. Generally speaking, the bitumens appear to take part in the formation of mor
type humus.

ANALYSE ET ROLE DES BITUMES DANS LES SOLS SABLEUX ACIDES.

Dans les sols sableux acides des Landes du Medoc, on note un taux de bitumes beaucoup plus 61ev6 dans les
podzols que dans les sols hydromorphes. L’etude des fractions acides et neutres des bitumes extraits de la v6getation
superficielle et de divers horizons de ces sols montre que les fractions acides et les hydrocarbures sont moins
transform ^ et moins d6grad6s dans les horizons A | des podzols que dans les horizons correspondants des sols
hydromorphes. La plupart des fractions neutres sont peu tra n sfo rm ^ mais plus rapidement degraddes dans les
sols. L’incubation d’6chantillons de sol en presence de paille de mais marquee au l4C montre que les bitumes
du sol proviennent pour la plus grande part des vegetaux, mais des produits de synthese microbienne s’incorporent
ä cette fraction, en particulier dans les podzols. Les bitumes exercent une action toxique ä l’egard de la microflore
globale des sols. L’intensite de cette action varie en fonction de la concentration des extraits, du pH du sol et de
l’origine de la microflore. La fraction acide et les hydrocarbures sont les plus inhibiteurs. L’action depressive
depend pour une part de la longueur des chaines et de la nature des fonctions. L’acidite semble jouer le role
principal dans Гaccumulation de bitumes au niveau des podzols 6tudies. D’une maniere generale, les bitumes
interviendraient dans la formation des humus de type mor.

1 - INTRODUCTION
Sous le terme de ’‘bitumes" ou simplement de " lip id es du s o l ” on d^signe un
ensemble de produits directem ent e x tr a its du so l par des so lv en ts organiques et
notamment par un melange §thanol - benzene. Cette fra ctio n heterogene (1 ), essen-
tiellem en t lip id iq u e , en e f f e t , n’a ete que peu etu d iee. Cette d esa ffe ctio n est
probablement due au f a i t que le s bitumes ne representent qu'une fra c tio n n e g li-
geable de la m atiere organique dans de nombreux types de s o ls (21. Ces c o n s ti­
tuents sont cependant tr e s abondants dans le s s o ls pauvres, biologiquement peu

* Laboratoire de pedologie — P6dologie des pays atlantiques.

** Laboratoire de chimie organique (XII).

105
106 FUSTEC-MATHON et al.

a c t if s t e l s que le s s o ls acldes e t le s tourbes (1, 3) ou leur ro le e s t certa in e-

ment Important. I ls agira ien t sur le s proprietes physiques du so l et exerceraient
une action toxique sur la germination et la croissan ce des Organes v6getaux sou-
terra in s ; i l s in tervien d raien t egalement sur l ' a c t i v i t e microbienne des s o ls
(1, 4, 5 ).
Au cours de notre etude sur 1 'evolution des s o ls sableux acides de la re­
gion des Landes du M§doc [France), nous avons ete frappes par le s d ifferen ces
importantes des taux de bitumes le long des sequences d ' hydromorphie cro issa n -
te (6 ). Les s o ls de o e tte region sont a ffe c te s par une nappe phreatique peu
profonde e t se rep a rtissen t en fonction du degre d’hydromorphie : des s o ls hy-
dromorphes peu humiferes se developpent dans le s zones b asses, humides alors
que des podzols humiques se d iffer en cien t dans le s zones le s plus e le v e e s. Dans
le s horizons A des s o ls hydromorphes, le taux de bitume represente 0,4 % du sol
e t 6 %du carbone t o t a l. Ce taux augmente tandis que 1 'hydromorphie diminue et
a t te in t dans le s horizons A des podzols 1,7 %du so l e t de 25 ä 30 %du carbo­
ne t o t a l. Les horizons В des podzols en sont depourvus.
Nous avons done essaye de p reciser le s conditions de c e tte accumulation de
bitumes dans certa in s horizons en analysant leur com position, leur evolution et
leur action sur le s p rop rietes b iologiq ues des s o ls .

2 - NETHODES 0 ’ETUDE
Les bitumes ont ete e x tr a its des horizons Aj e t A2 d'un podzol e t de 1 'ho­
rizon A d'un so l hydromorphe au soxh let pendant 0 heures, s o it par un melange
ethanol - benzene, s o it par un melange ether de p etro le - a ceta te d ’eth y le 4 /1 ,
ce dernier permettant d 'e v ite r 1 'e s te r if ic a tio n des acides lib r e s au cours de
1 ’extraction [7 ). Les bitumes ont e te egalement e x tr a its de la vegetation pre-
lev ee en t o t a l i t e ä la surface de ces s o ls , broyee e t homogSneisee La zo­
ne etu diee n 'e st pas actuellem ent p lantee en p ins, mais e i l e l ' e t a i t ä une P e ­
riode relativem ent recente.
Les e x tr a its ont 6te sgpares en une fra ctio n neutre e t une fra c tio n acide.
Dans chaque fr a c tio n , p lu sieu rs fa m ilie s de composes ont ete is o le e s selon le
protocole u t i l i s e par Albrecht (0 ). Les co n stitu en ts de chaque fa m ille ont ete
id e n t if ie s par Chromatographie en phase gazeuse avec programmation de tempera­
tu re. L 'estim ation q u an tita tiv e de chaque substance separee a ete deduite de la
surface du pic correspondent sur le s chromatogrammes. Nous avons e ta b li le s rap­
ports correspondant au "Carbon P re feren tia l Index" [9) : le C .P .I. qui trad uit
la dominance des composes a nombre impair d'atomes de carbones dans le s hydro-
carbures e t le C .P .I.a qui trad u it c e lle des chaines paires dans le s acides et
le s a lc o o ls . Pour 'les auteurs qui ont etudie le s co n stitu a n ts organiques de se ­
diments anciens, le s v ariation s de ces in d ices indiquent une evolution des
con stitu a n ts sous l ' e f f e t de la diagenese (0, 10).
Afin de p reciser l'im portance r e la tiv e des produits d 'o rig in e v eg eta le ou
microbienne dans le s bitumes, des ech a n tillo n s d 1horizon Aj de podzol e t de sol
hydromorphe maintenus ä la cap acite au champ ont ete en rich is en p a ille d e m a is
marquee uniformement au carbone 14 ä raison de 100 mg de p a ille broyee pour 100
g de so l se c . Des ech a n tillo n s de s o l identiques ont ete en rich is en glucose 1**C
ä raison de 2 %par rapport au carbone t o ta l du s o l. Les ec h a n tillo n s ont ete in ­
cubes 3 semaines ä 28°C. L'atmosphere e t a it renouvelee par balayage d ’a ir humi-
d if i § . Apres une extraction ргёа1аЫе des co n stitu a n ts hydrosolubles, le s b itu ­
mes ont ete e x tr a its comme precedemment. La r a d io a c tiv ite de ces fra c tio n s a e t e

La vegetation e s t con stitu ee essen tiellem en t de M o t i n i a e a e r u l e a , E r i c a s c o -

p a r ia , E. t e t r a l i x , E. a i l i a r i s , C a llu n a v u l g a r i s , U le x e u r o p e n s , U. n a n u s
en proportions variab les selon le degre d ’hydromorphie.
 IAEA-SM-211/48 107

TABLEAU I. DISTRIBUTION DES DIFFERENTS TYPES DE CONSTITUANTS DES BITUMES

DANS LES HORIZONS A, ET A2 DE PODZOL ET A! DE SOL HYDROMORPHE [2]
(en mg/100 g de sol sec) I

Podzol Sol hydro-

C onstituants des bitumes morphe
Horizon Aj Horizon A2 Horizon Aj
F r a c tio n a c id e (l )
Honoacides 10,6 3,7 10,4
□ iacides 11,5 2,1 5,2
Acides plus p ola ires 53,0 n.d. 21,4
F r a c tio n n e u tr e
Hydrocarbures 6,1 1,2 2,3
Cetones 10,6 2,1 4,5
A lcools 30,3 1,4 11,0
Esters longs 4,5 0,3 1,0
Glycerides 15,9 2,9 6,5

tl) A c id e s l i b r e s

mesuree en s c in t illa t io n liq u id e dans un melange Toluene - PPO - Р0Р0Р. Les b itu ­
mes ont ete egalement e x tr a its de la p a ille de mais non incubee apres ex tra c­
tio n des composes hydrosolubles.
L 'influ en ce des bitumes sur la m icroflore du so l a ete estim£e ä l'a id e
d'un t e s t de croissan ce de micropopulations t o t a le s . C e lle s -c i provenaient d’un
so l brun le s s iv e [pH 7 ,3 ), des horizons d'un podzol [pH 3,5) e t d ’un so l hy-
dromorphe [pH 5 ,0 ) . Aux m ilieux de cu lture t£moins, 0 ,5 ml d’ethanol-benzene
e ta ie n t ajoutes pour 25 ml de m ilieu , aux m ilieux t e s t s , 0,5 ml de so lu tio n s de
bitumes ä d iffe r e n te s con centration s. 5 a 10 b o ite s ont ete in ocu lees pour cha-
que e s s a i. Les r e s u lta ts ont ёЬ ё exprimes sous forme d'un in d ice de croissan ce :
jc = nombre de colo n ies sur m ilieu t e s t x -|qq
nombre de colo n ies sur m ilieu tSmoin
Un in d ice superieur a 100 trad u it un e f f e t stim ulant de la substance etu -
d iee , un in d ice in fe r ie u r a 100 indique un phenomene d 'in h ib itio n (11, 5 ).

3 - RESULTATS
3.1 - Etude des con stitu an ts
L’ importance r e la tiv e des d iffe r e n ts types de co n stitu a n ts des bitumes e s t
donnee dans le tableau I . Dans le s t r o is horizons, nous retrouvons le s memes f a ­
m ilie s de composes organiques separables en une fra ctio n acide et une fra ctio n
neutre. La fra c tio n acide represente de 55 ä 60 % de 1 ’e x tr a it t o t a l. Bien que
le taux global de bitumes varie d'un horizon a 1 'autre, le s proportions r e l a t i ­
ves des d iffe r e n te s fa m ilie s de composes sont a peu pres id en tiqu es dans chaque
horizon.
3.1.1 - F r a c t i o n a c i.d e
La fra c tio n acide lib r e con tien t des mono et des d ia cid es lin £ a ir e s satures
dans le sq u els dominent le s composes ä nombre pair d'atomes de carbone. E lle ren-
ferme Sgalement des hydroxyacides qui ne sont pas examines dans ce tr a v a il.
1 08 FUSTEC-MATHON et al.

A Plantes

B-C Podzol Ai

D-E Podzol ^2

F -G Sol hyd romorphe A

25 30 nb.de С

M O N O A C I DES DI A C I D E S
F I G .l. H is to g r a m m e s d e r e p a r titio n d e s m o n o a c i d e s ( A , B , D , F ) e t d e s d ia c id e s (С , E , G ).

La fig u re 1 permet de comparer le s histogrammes de re p a r titio n des monoaci­

des et des d iacid es en fon ction du nombre d’atomes de carbone dans la vegetation
e t dans le s t r o is horizons de s o l. Le tableau II indique quels sont le s composes
le s plus abondants dans ces fra c tio n s e t le s valeurs du CPIa.
On con state que pour le s monoacides la longueur des chaines diminue dans le
so l par rapport ä la vägetation de meme que le CRIa. La diminution du CPIa e st
n ette entre le s deux horizons de podzol. Les d ia cid es ne sont pas rencontrSs
dans le s p lantes et leur evolution dans le s divers horizons de so l e s t v o isin e
de c e lle des monoacides.
 IAEA-SM-211/48 109

TABLEAU 11. LONGUEURS DE CHAINES DOMINANTES ET VALEURS DU

C A R B O N P R E F E R E N T IA L IN D E X DANS LES MONO- ET LES DIACIDES

Monoacides D iacides

Maximum CPI. a Maximum CPI.a

»
Vegetation c 30 4,60 Neant Neant

Podzol horizon Aj C2t 3,44 c26 _ c28 2,40

Podzol horizon A2 C20 ' C28 2,22 c24 1,75

Sol hydromorphe c24 ” C28 3,20 c24 2,34

3.1.2 - F r a c tio n n e u tr e
Cette fra c tio n e s t con stitu ee de fa m ilie s de composes lin e a ir e s sa tu res, a
1 'exception des a lc o o ls lib r e s e t des cetones, d erives terpeniques dont la com­
p o sitio n e st pratiquement identique dans le s plantes et dans le s bitumes des
d iffe r e n ts horizons.
La re p a r titio n des hydrocarbures dans le s p lantes e t le s horizons de sol
e s t representee sur la figu re 2 , le tableau III indiquant quels sont le s com­
poses le s plus abondants e t le s valeurs du CPI. Pour ces composes anombre im­
pair d'atomes de carbone dominant, la longueur des chaines tend egaiement ä d i-
minuer dans le s o l. Le CPI, Sieve dans le s vegetaux, diminue tr e s nettement de
1 ’horizon Aj ä 1 'horizon A2 du podzol, sa valeur etant interm ediaire dans le
so l hydromorphe. II convient de noter que le s hydrocarbures du pin, ä chaine
relativem ent courte [in fer ie u r e ä C2o) ne se retrouvent pas en quantite nota­
b le dans le s bitumes des s o ls .
Les g ly cerid es co n stitu en t une part importante de la fra ctio n neutre des
c ir e s e x tr a ite s de la vegetation et egaiement des bitumes des d iffe r e n ts h o ri­
zons a lo r s q u 'ils son t, en general, consideres comme peu abondants dans le s s o ls .
On trouve des mono-, d i- e t tr ig ly c e r id e s , ces derniers en proportion r e la t iv e ­
ment f a ib le . L1hydrolyse basique de ces gly cerid es conduit ä des monoacides
dont le s histogrammes sont tr e s d iff6 r e n ts de ceux des monoacides lib r e s : l ' a -
cide en C2g e s t un element important de la d istrib u tio n des acides d erives des
g ly cerid es dans le s s o ls , a lors que le maximum se s itu e a Cjj pour le s p lantes
de su rface. D'une fapon gen erale, ces histogrammes et le s CPI n'indiquent pas
d 'ev o lu tio n . De meme, dans le cas des este rs ä longue chaine, le s histogrammes
des a lc o o ls et des acid es lin e a ir e s derivant de 1 'hydrolyse basique sont pra­
tiquement identiques pour le s s o ls et la vögetation .

3.2 - Drigine des bitumes

Le tableau IV montre qu'apres incubation en presence de p a ille de mais 1I+C,
le taux de r a d io a c tiv ite obtenu dans le s bitumes e x tr a its des deux types de so l
e st plus elev e que c e lu i present au depart dans la p a ille de mais ll*C. Ce taux
e s t plus elev e pour le podzol que pour le so l hydromorphe. Une p a rtie de c e tte
a c t iv it e provient du m ateriel vegetal incorpor§, mais i l semble qu'une fra c tio n
peut provenir de syntheses m icrobiennes.
Les pourcentages de r a d io a c tiv ite obtenus dans le s bitumes apres incubation
de ces s o ls en presence de glucose 11+C montrent qu’en e f f e t , le s microorganismes,
sont su sce p tib les d'61aborer des quan tites notables de produits de nature l i p i -
dique qui s ' incorporent dans la fra c tio n "bitumes". La production de ces compo­
ses e s t plus importante pour l ’horizon Aj de podzol.
по FUSTEC-MATHON et al.

F H ',.2 . H is to g r a m m e s d e r e p a r titi o n d e s k y d r o c a r b u r e s lin e a ir e s .

3.3 - Influence des bitumes sur la m icroflore des so ls

Nous avons mis en evidence un e f f e t d äp ressif des so lu tio n s de bitumes sur
la croissan ce des microorganismes du so l ( 5 ] . Cet e f f e t augmente avec la con­
centration de l'e x t r a it [Figure 3 ).
La t o x ic it e de l'e x t r a it augmente egalement lorsque le pH du m ilieu d ecroit
(Figure 4 ). La m icroflore d’un horizon Aj de podzol parait la plus r e s is ta n te ,
mais on con state qu'ä la valeur du pH du so l considere, l'in d ic e de croissance
e s t de 80 pour la m icroflore du so l brun le s s iv e , de 40 pour c e lle du so l hydro-
morphe e t de 20 seulement pour c e lle du podzol.
 IAEA-SM-211/48

TABLEAU III. LONGUEURS DE CHAINES DOMINANTES

ET VALEURS DU C A R B O N P R E F E R E N T IA L IN D E X
DANS LES HYDROCARBURES

Hydrooarbures

Maximum CPI

Vdgfitation c29 ' C31 " c 33 13,15

Podzol horizon A2 c29 * C31 ' c 33 8,15

Podzol horizon Aj c2 7 “ c 31 c 33 2,94

Sol hydromorphe c29 ' C31 ‘ C33 5,50

TABLEAU IV. DISTRIBUTION DE LA RADIOACTIVITE DANS

LES BITUMES DANS LES SOLS ENRICHIS EN PAILLE DE MAIS
ET EN GLUCOSE APRES TROIS SEMAINES DTNCUBATION
(en %de la radioactivite introduite)

Sol hydro­
Podzol
morphe

P a ille de mals 1!*C

2,6 2,6
non incubee

P a ille de mals 14С

incubde 3,2 2,9

Glucose ll*C 11,8 B, 5

% Bitumes

F I G .3 . I n f l u e n c e d e la c o n c e n t r a t i o n d e s e x t r a i t s d e b i t u m e s s u r l a c r o i s s a n c e d ’u n e m i c r o p o p u l a t i o n de

so i b ru n le s s iv e .
112 FUSTEC MATHON et al.

F I G .4 . V a r ia tio n d e V e f f e t d e p r e s s i f d u n e s o lu t io n d e b itu m e s d 1 % e n fo n c tio n d u p H s u r d e s m ic r o p o p u la tio n s

d e d iff e r e n t e s o r ig in e s .

F I G .5 . I n f l u e n c e d e s d i f f e r e n t e s f r a c t i o n s d e s b i t u m e s s u r l a c r o i s s a n c e d ’u n e m i c r o p o p u l a t i o n d e s o l b r u n l e s s i v e .

F IG .6 . I n f l u e n c e d e l a l o n g u e u r d e s c h a i n e s e t d e s f o n c t i o n s a l c o o l e t a c i d e s u r la c r o i s s a n c e d ’u n e m i c r o -

p o p u l a t io n d e s o l b r u n le s s iv e .
 IAEA-SM-211/48 113

L’e f f e t in h ib iteu r varie en fonction des d iffe r e n te s fa m ilie s de composes

presents dans le s bitumes [Figure 5 ). Certains con stitu a n ts sont peu inhibiteur.s
(ceton es, e s te r s , a lc o o ls ) . La fra ctio n acide et le s hydrocarbures sont le s plus
toxiqu es.
Enfin, s i l'o n compare l'a c tio n d 1hydrocarbures de d iffe r e n te s longueurs,
d 'a lc o o ls et d 'acid es correspondents (Figure 6 ), on con state qu ’ un hydrocarbure
en Cj7 peut exercer un e f f e t stim ulant tandis que l'a lc o o l et surtout 1 'acide
correspondent ont un e f f e t d e p r e ssif. Pour le s composes ä chaine plus longue
[C28 ■ C261, 1 'in h ib itio n e s t nettement plus f o r te , mais la presence de fonc-
tio n s ne semble pas a lors in te rv e n ir.

4 - DISCUSSION ET CONCLUSIONS
Les taux de bitumes mesures ä un moment donne dans un so l representent un b i-
lan entre le s apports par le s debris vegetaux et animaux plus ou m oins|transform es,
l'a d d itio n de produits d’o rig in e microbienne e t la decomposition de ces d ivers cons­
titu a n ts . II e s t generalement admis que le s produits lip id iq u e s se decomposent len -
tement dans le s s o ls , la degradation etant surtout fre in e e par 1 'anaerobiose
dans le s s e ls engorges e t par l'a c id it e du m ilieu .
Dans la region sableuse des Landes du liedoc, on con state que le s podzols ren-
ferment des q uantites de bitumes nettement plus e lev e es que le s s o ls hydromorphes.
Au niveau des deux types de s o l, la composition de la vegetation e s t tr e s v o isin e ,
la nature des produits apportes au so l par le s debris ne peut done pas etre tre s
d iffe r e n te . Les d iffer en ce s peuvent apparaitre s o it au niveau de la transformation
e t de la decomposition de ces produits dans le s o l, s o it au niveau de la production
de composes d 'o rig in e microbienne.
Dans le s horizons s u p e r fic ie ls des podzols, ce rta in es fr a c tio n s s'accumulent
e t sont moins transformees que dans le s horizons correspondants des s o ls hydromor­
phes. C 'est le cas notamment de la fra ctio n acide e t des hydrocarbures a in s i que
le trad uisen t clairem ent le s valeurs du "Carbon P re feren tia l Index". Par contre,
la plupart des fr a c tio n s neutres e x tr a ite s du so l ( a lc o o ls , ceto n es, e s te r s) sem-
blent peu transformees par rapport aux fra c tio n s correspondantes dans le s plantes
mais e l l e s sont plus rapidement biodegradees. La fa ib le t o x ic it e de ces c o n s ti­
tuants a 1 'egard de la m icroflore glob ale du so l peut expliquer c e tte degradation
p r e fe r e n tie lle .
Par a ille u r s , nous avons egalement pu mettre en Svidence la p o s s ib ilit e d'une
production de composes d 'o rig in e microbienne plus importante au niveau des horizons
s u p e r f ic ie ls des podzols. Cette production e st certainement due pour la plus grande
part aux champignons abondants dans ces s o ls , 1 'accumulation d ’acides gras fa v o ri-
sant elle-meme le developpement des champignons (2 ).

En conclusion, i l apparait que dans ces s o ls sableux l'a c id it e du m ilieu joue

un ro le plus important que 1 ’hydromorphie. La texture permet, en e f f e t , une c e r ta i-
ne c ir c u la tio n des nappes e t une aeration sü ffisa n te du m ilieu . Au niveau des pod­
z o ls , on note, d'une part, une transformation et une decomposition moins poussees
des produits d’orig in e vegetale e t , d'autre part, une production plus importante
de composes d 'o rig in e microbienne. Dans ces s o ls , l'a c id it e du m ilieu a cc ro it 1 'e f ­
f e t toxique des composes lip id iq u e s et reduit l ’a c t iv it e biologique g lo b a le.
II semble que le s bitumes puissen t jouer un ro le tr e s important dans la forma­
tio n des humus de type mor, 1 ’a c id ific a tio n progressive du m ilieu a la su ite du le s -
sivage en tra in era it 1 ’accumulation de produits toxiques su sce p tib les de r a le n tir
l ' a c t i v i t e microbienne e t l ’evolution generale de la m atiere organique. II у aurait
notamment p ersistan ce dans le so l d 'acid es organiques sim ples qui fa v o risen t le pro­
cessu s de p o d z o lisa tio n .
1 14 FUSTEC-MATHON et al.

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[2] BRAIDS, O., MILLER, R., in Soil Components, I — Organic components, Springer-Verlag, Berlin (1975) 343.
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des Mines, Serv. Labo., Qu6bec R.P. 281 et 282 (1953).
[4] WANG, T., Int. Rice Commn Newsl. 18 2 (1969) 23.
[5] FUSTEC-MATHON, E„ RIGHI, D., JAMBU, P., Rev. Ecol. Biol. Sol. 12 1 (1975) 393.
[6] JAMBU, P., RIGHI, D„ Sei. Sol, Bull. A.F.E.S. 3(1973) 207.
[7] ARPINO, P., OURISSON, G., Anal. Chem. 43(1971) 1656.
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[9] COOPER, J., BRAY, E„ Geochim. Cosmochim. Acta 27 (1963) 1113.
[10] ARPINO, P., Les lipides de sediments lacustres Eocene, These Sei. Strasbourg (1973) 107 p.
[11] BECK, G., DOMMERGUES, Y„ VAN DEN DRIESSCHE, R., Rev. Ecol. Biol. Sol 12 1 (1975) 393.

DISCUSSION

B. GUILLET: I noted in your first slide that in horizon A2 there were considerable percentages
of carbon coming from bitumen compared with total carbon. Do you think that this is an effect
originating with the roots? In other words, do the plant roots have a larger bitumen content
than the aerial parts?
E. FUSTEC-MATHON: That is a possibility, but we do not think that the figure is very
significant because this horizon is very poor in organic matter, and as a result of the fractionation
it may also be that errors have been introduced. The level is indeed very low.
J. CORTEZ: To what reason do you attribute the fact that the monoglycerides and
diglycerides are inhibitors of the microflora?
E. FUSTEC-MATHON: I have repeatedly observed this in experiments, and have myself
been surprised that, in particular, they are more active inhibitors than the long esters. I cannot
explain it at present.
CHARACTERIZATION
OF HUMIC ACIDS
(S e ssio n 7 b )
 IAEA-SM-211/7

R e v ie w p a p er

R E C E N T F IN D IN G S O N T H E C H A R A C T E R IZ A T IO N
O F H U M IC S U B S T A N C E S E X T R A C T E D F R O M S O IL S
F R O M W ID E L Y D IF F E R IN G C L IM A T IC Z O N E S

M. SCHNITZER
Soil Research Institute,
Agriculture Canada,
Ottawa, Ontario,
Canada

Abstract

RECENT FIN DIN G S ON THE C H ARACTERIZATION OF HUMIC SUBSTANCES EXTRACTED FROM SOILS
FROM WIDELY D IFFER IN G CLIMATIC ZONES.
T o ob tain inform ation o n the effec t o f clim ate o n the analytical characteristics and chem ical structure o f
hum ic substances, h um ic and fulvic acids w ere extracted from soils form ed under A rctic, co o l tem perate, subtropical
and tropical clim ates. A ll hum ic m aterials were extracted, fractionated, purified and analysed b y the same m ethods.
T hese included elem entary and fu nction al group analyses, sp ectrop h otom etry in th e ultra-violet, visible and
i.r. regions, Electron Spin R esonance (E SR ) spectrom etry and chem ical degradation w ith alkaline К М п 04 and
alkaline CuO. The oxid ation products w ere separated b y solvent extraction and chrom atographic m eth od s, and
iden tified b y mass spectrom etry and micro-i.r. spectrop h otom etry. T he analytical, spectrophotom etric and
spectrom etric data, and the in form ation provided b y chem ical degradation, show ed that hum ic substances form ed
under w id ely differing clim atic con d ition s had essentially similar chem ical structures and characteristics. Major
hum ic ‘building b lock s’ were in all instances com p lex phenolic and benzene-carboxylic structures. M ost o f the
aliphatics w ere n-fatty acids w hich appeared to be esterified to phenolic OH groups. R ecent p hysico-chem ical
studies d one in ou r laboratory show that hum ic and fulvic acids behave like linear, flexib le p oly electro ly tes that
are readily aggregated, m ost lik ely w ith th e aid o f H -bonding, at lo w pH but dispersed, because o f increased dissocia­
tio n o f fu nction al groups, at higher pH . Thus, it b ecom es apparent at this tim e that hum ic and fulvic acids are
n o t single m olecules b u t m olecular associations o f p henolic and benzene-carboxylic com p ou nd s ( ‘building blocks’)
o f m icrobiological, p olyp h en olic, lignin and condensed lignin origins. The ‘building b lock s’ appear to be held
togeth er b y weak linkages, m ainly H-bonds.

INTRODUCTION

It is generally accepted by soil scientists that climate exerts profound effects on the genesis as
well as on the physical and chemical characteristics of soils. Considerably less, however, is known
on how this same environmental factor affects the chemical structure and reactivity of organic
matter. Since humic substances, that is, humic acids (HAs), fulvic acids (FAs) and humins, consti­
tute the bulk of the organic matter in all inorganic and in highly humified organic soils, we focused
our efforts on these materials. The question that we wanted to answer was how similar or different
were humic substances extracted from soils formed under widely differing climates such as that
prevailing in the Arctic, the cool temperate, the subtropical and the tropical zones. To provide
the desired information, we extracted, purified, fractionated, analysed and degraded all HAs and
FAs by the same methods. Chemical analyses included elementary analysis (С, H, N, S, O), func­
tional group analysis (C0 2H, phenolic and alcoholic OH, quinonoid and ketonic C = 0, OCH3)
and oxidative degradation with alkaline KMn04 and alkaline CuO. The oxidation products were
separated by solvent extraction, thin-layer and preparative gas chromatography, and identified on

117
1 18 SCHNITZER

TABLE I. CLIMATIC ZONES AND SOILS

FROM WHICH HAs AND FAs WERE EXTRACTED

(a) A r c tic clim a te

Brunic Turbic C ryosol, Ahb horizon, Northern Canada

fb j Cool, te m p e ra te clim ate, acid soils

V olcanic A sh, horizon, Japan

Diluvial, A ! horizon, Japan
Spod osol, 0 2 and Bh horizons, Eastern Canada
Gray Luvisol, A i and B2 horizons, Western Canada

fc) Cool, te m p e ra te clim ate, n eutral soils

Haploboroll, A i horizon, Western Canada
Glossic U dic N atriboroll, A , horizon, Western Canada
U dic N atriboroll, A 2 horizon, Western Canada

(d) S u b tro p ic a l clim a te

Aridic Boroll, A , horizon, Central Argentina

G lossic N atriboroll, A j horizon, Central Argentina
N atriboroll, A t horizon, Central Argentina
A ridic Haploboroll, A 2 and B2 horizons, Central Argentina

(e) T ropical clim ate

Inceptisols, A i and В horizons, D om inica, West Indies

(including Hydrandepts and Dysdrandepts)

a gas chromatographic-mass spectrometric-computer system and by micro-infra-red spectrophoto­

metry. In addition, all humic samples were analysed by ultra-violet, visible and infra-red spectro­
photometry as well as by electron spin resonance spectrometry.
We were hoping that the results of this investigation would throw light on (a) the effect of
climate on the chemical structure and properties of humic materials that occur in soils, and
(b) advance our knowledge of the structural chemistry of these materials, a problem that has
occupied soil scientists for more than 200 years [ 1, 2].

EXPERIMENTAL

Materials

Climatic zones under which the soils examined were formed, and the classification and
geographic origin of soil samples from which HAs and FAs were extracted, are listed in Table I.
In some cases soil samples were collected from both surface and subsurface horizons, but most
soil samples used in this investigation came from surface horizons.

Extraction, separation and purification of humic substances

Alkaline soil samples were first decalcified with dilute HC1 solution, washed and air-dried.
HAs and FAs were extracted from finely ground, air-dry soil samples with 0.5N NaOH under N2
at room temperature, using a solution:soil ratio of 10:1. Following separation of the dark-coloured
supernatants from the residual soils by centrifugation, the alkaline extracts were acidified to pH 2.0
 IAEA-SM-211/7 1 19

and allowed to stand at room temperature for 24 h. The soluble materials (FAs) were separated
from the coagulates (HAs) by centrifugation. The HAs were redissolved in 0.5N NaOH linder N2
and reprecipitated with 2N HC1. Following centrifugation, the HAs (coagulates) were transferred
to dialysis bags, dialysed against distilled water until free of СГ, and then freeze-dried. When
additional purification was needed, this was done by shaking the HAs at room temperature for
36 h with a large excess of 0.5% (vol./vol.) HC1-HF, centrifuging, washing the residues (HAs) with
distilled water until free of СГ, and then freeze-drying. The FAs were passed repeatedly| over
H-resin contained in large glass columns. In some instances further purification such as dissolution
in methanol, high-speed centrifugation and additional resin treatments was necessary to lower
the ash content of the FAs [3].

Analytical methods

C and H were determined by dry-combustion, N by the automated Dumas method,! S by

oxygen-flask combustion, and О was calculated by subtracting %C+ %H+ %N + %S from 100.
The OCH3 content was determined by the Zeisel method. Total acidity and carboxyl groups were
determined by the method described by Schnitzer and Gupta [4], total C =0 by oximation [5]
and quinonoid groups according to Schnitzer and Riffaldi [6 ]. Ketonic C= О groups were con­
sidered as being equal to the difference between total C = 0 and quinonoid C=0. Phenolic OH
groups were assigned to the difference between total acidity and C02H groups. Total OH groups
were measured by acetylation [7]. Alcoholic OH groups were taken as the difference between
total and phenolic OH groups. j
Moisture was determined by heating samples at 105°C for 24 h, and ash by igniting; samples
at 750°C for 4 h. All data are expressed on a moisture-and ash-free basis.
E4/E 6 ratios were determined by dissolving 2—3 mg of HA and 5 mg of FA in 10 ml of
0.05N NaHC03 solution (pH ±9.0), and measuring optical densities at 465 and 665 nm on a
Beckman model В spectrophotometer. The ratio of the optical densities at the two wavelengths
was the E4/E 6 ratio.
Infra-red spectra were recorded on KBr pellets (± 1 mg of HA or FA per 400 mg of KBr) on
a Beckman IR-12 spectrophotometer. Electron Spin Resonance (ESR) spectra were recorded on
1%(wt/vol.) solutions in 0.1N NaOH on a Varian Associates E3 spectrometer, employing 100 KHz
modulation and a nominal operating frequency of 9.5 GHz [8].

Alkaline KMn04 oxidation of methylated HAs and FAs

One gram samples of each humic material were first suspended in 10 ml of methanol and
methylated with an ether solution of diazomethane, generated from Diazald. The methylation
procedure was repeated with fresh diazomethane until the OCH3-content remained constant.
One gram of each methylated humic material was refluxed for 8 h with 250 ml of 4% (wt/vol.)
aqueous KMn04 (pH = 10). Following oxidation, the excess KMn04 was destroyed by the careful
addition of small volumes of methanol. The insoluble Mn02 was removed by filtration and washed
with small aliquots of hot water. The filtrate and washings were acidified to pH 2 with 6N H2S0 4
solution, transferred to a liquid-liquid extractor and extracted for 72 h with 500 ml of ethyl acetate.
The extract (= oxidation product) was first dried over anhydrous Na2S04, then in a rotary
evaporator and weighed.

Alkaline CuO oxidation

To 500 mg of HA or FA, dissolved in 50 ml of 2N NaOH solution in a stainless-steel auto­

clave, surrounded by a heating mantle, and equipped with a magnetic stirrer, 2.5 g of CuO was
added. The system was heated to 170°C with constant stirring over a period of 1 h, and then
120 SCHNITZER

maintained at that temperature for 2 h. Following cooling to room temperature, suspended CuO
was removed by centrifugation and washed with distilled water. The supernatant solution was
acidified first to pH 6 and then to pH 2, and each time extracted for 24 h in a liquid-liquid extraction
with 250 ml of ethyl acetate. The latter extract (= oxidation product) was dried and weighed
as described above.

Separation and identification of oxidation products

Each oxidation product was first methylated to make it sufficiently volatile for subsequent
gas chromatographic and mass spectrometric analyses. As a second step, each methylated fraction
was dissolved in benzene and examined by analytical gas chromatography. Compounds represented
by well-defined peaks were isolated by preparative gas chromatography under similar conditions
except that more concentrated solutions were injected. Materials eluting from the gas chromato­
graph were collected in capillary tubes and analysed by mass spectrometry and micro-infra-red
spectrophotometry. Each fraction was also analysed directly on a gas chromatographic-mass
spectrometric-computer system (Finigan 3100D GC-MS, interfaced with a model 6000 data
system) [9].
In general, preliminary identification of gas chromatographic peaks was first made by
recording ‘mass chromatograms’ for fragments characteristic of specific compounds or groups
of compounds expected to occur in the mixture and searching through stored spectral data for
specific m/e ratios [10]. The identity of the compound in each peak was then confirmed by
( 1) running its mass spectrum; (2) eluting it from the gas chromatograph and recording its micro-
infra-red spectrum; (3) matching its mass and micro-infra-red spectrum with the known com­
pound to which it corresponded; and (4) co-chromatographing (on the gas chromatograph) of
known and unknown.
The general procedure is summarized in Fig. 1.

D e g r a d a t io n

S o lv e n t e x t r a c t io n

M e t h y la t io n

GLC-MS

S e p a r a t io n o v e r
and t . 1 . c . I

G LC-MS-com puter

F IG .l. P rocedure u sed fo r th e chem ical degradation o f H A s a n d F A s, a n d fo r th e id e n tific a tio n o f o x id a tio n p ro d u cts.
 IAEA-SM-211/7 121

RESULTS AND DISCUSSION

Elementary analyses

Data for elementary analyses of HAs and FAs are shown in Tables II and III. When more
than one set of data was available, the results are presented as ranges. In general, ranges for
HAs were narrower than those for FAs, indicating greater homogeneity for the former. The HAs
contained more С, H and N, but less S and О than the FAs. We were unable to detect any distinct
effect of climate on the elementary composition of the HAs and FAs which we analysed.

Functional groups

Data for functional groups in the different HAs and FAs are shown in Tables IV and V. The
total acidity and C0 2H content of the FAs were in all instances considerably higher than those of
HAs. There were variations in the total C = 0 content, especially among HAs. The OCH3 content
was relatively low for all HAs and FAs. E4/E6 ratios of FAs (Table V) were appreciably higher
than those of HAs in accordance with previous findings published in the literature [ 1]. Again,
climate did not appear to significantly affect the distribution of oxygen-containing functional
groups in the HAs and FAs that we examined.

TABLE II. ELEMENTARY ANALYSIS OF HAs EXTRACTED

FROM SOILS FROM WIDELY DIFFERING CLIMATIC ZONES
C lim atic zone

Elem ent
C ool, tem perate
(%)
A rctic Sub Li upicdl Tropical
A cid soils N eutral soils

C 56.2 5 3 .8 - 5 8 .7 5 5 .7 - 5 6 .7 5 3 .6 - 5 5 .0 5 4 .4 - 5 4 .9

H 6 .2 3 .2 —5.8 4 .4 —5.5 4 .4 —5.0 4 .8 —5.6

N 4.3 0 .8 —2 .4 4 .5 —5.0 3 .3 —4.6 4 .1 —5.5

S 0.5 0 .1 - 0 .5 0 .6 —0.9 0 .8 - 1 . 5 0 .6 —0.8

о 3 2 .8 3 5 .4 - 3 8 .3 3 2 .7 - 3 4 .7 3 4 .8 - 3 6 .3 3 4 .1 - 3 5 .2

TABLE III. ELEMENTARY ANALYSIS OF FAs EXTRACTED

FROM SOILS FROM WIDELY DIFFERING CLIMATIC ZONES

C lim atic zon e

Elem ent
C ool, tem perate
(%)
Arctic Subtropical Tropical
A cid soils N eutral soils

c 4 7 .7 4 7 .6 - 4 9 .9 4 0 .7 - 4 2 .5 4 2 .2 - 4 4 .3 4 2 .8 - 5 0 .6

H 5.4 4 .1 - 4 .7 5 .9 —6.3 5 .9 —7 .0 3 .8 —5.3

N 1.1 0 .9 —1.3 2 .3 —2.8 3 .1 —3.2 2 .0 —3.3

s 1.6 0 .1 —0.5 0 .8 - 1 .7 2.5 1 .3 - 3 .6

о 4 4 .2 4 3 .6 - 4 7 .0 4 7 .1 - 4 9 .8 4 3 .1 - 4 6 .2 3 9 .7 - 4 7 .8
122 SCHNITZER

TABLE IV. FUNCTIONAL GROUP ANALYSIS AND E4/E6 RATIOS OF HAs

EXTRACTED FROM SOILS FROM WIDELY DIFFERING CLIMATIC ZONES

Climatic гопе

Functional group
(m eq /g) C ool, tem perate
A rctic Tropical
A cid soils N eutral soils

T otal acidity 5.6 5 .7 —8.9 6 Л - 6 .6 6 .3 —7.7 6 .2 - 7 .5

C 0 2H 3.2 1 .5 - 5 .7 3 .9 - 4 .5 4 .2 —5.2 3 .8 - 4 .5

Phenolic OH 2.4 3 .2 - 5 .7 2 .1 - 2 .5 2 .1 —2.5 2 .2 - 3 .0

A lcoh olic OH 4 .9 2 .7 - 3 .5 2 .4 - 3 .2 2.9 0 .2 - 1 .6

Q uinonoid C = 0 2.3 1 .4 - 2 .6
|o .l - 1 .8 -{ 4 . 5 —5.6 |o .8 - l .S
K eton ic C = 0 0 .3 - 1 .4
1.7

vq
0

0
00
OCH3 0 .4 0 .4 0.3 0 .3 —0.5

1
e 4/ e 6 5.3 3 .8 —5.0 4 .0 —4.3 3 .9 - 5 .1 5 .0 - 5 .8

TABLE V. FUNCTIONAL GROUP ANALYSIS AND E4/E6 RATIOS OF FAs

EXTRACTED FROM SOILS FROM WIDELY DIFFERING CLIMATIC ZONES

Climatic zo n e

Fun ction al group

C ool, tem perate
(m eq /g)
Arctic Subtropical Tropical
A cid soils N eutral soils

T otal acidity 11.0 8 .9 - 1 4 .2 ND 6 .4 - 1 2 .3 8 .2 - 1 0 .3

C 0 2H 8.8 6 .1 - 8 .5 ND 5 .2 —9.6 7 .2 - 1 1 .2

Phenolic OH 2.2 2 .8 —5.7 ND 1 .2 —2.7 0 .3 - 2 .5

A lcoh olic OH 3.8 3 .4 —4 .6 ND 6 .9 —9.5 2 .6 —5.2

Q uinonoid C = 0 2 .0 0.3 —1.5

■J1.7—3.1 ND i 1.2—2.6
K etonic C - 0 2 .0 l l 1.6—2.7
d

0 .9 —1.2
d

OCH3 0 .6 ND 0 .8 —0.9
1
i

VO
OO
jb

e 4/ e 6 11.5 9 .0 ND 7 . 6 - 1 1 .2

Variations in elementary composition and functional group content

Ranges in elementary composition (Tables II and III) and functional group content (Tables IV
and V) for all HAs and FAs are summarized in Table VI. From these data means were calculated
and these are presented in Table VII. The data in this table may be considered as approximations
of the elementary composition and functional group content of the ‘ideal’ or model HA and FA.
A more detailed inspection of the data shows that (a) the ‘ideal’ HA contains approximately 10%
more C but 10% less О than does the ‘ideal’ FA; (b) there is relatively little difference between
 IAEA-SM-211/7 123

TABLE VI. RANGES AND MEANS FOR ELEMENTARY COMPOSITION,

FUNCTIONAL GROUPS AND E4/E6 RATIOS FOR FAs EXTRACTED
FROM SOILS FROM WIDELY DIFFERING CLIMATIC ZONES

Elem ent
HAs FA s
(%)

c 5 3 .6 - 5 8 .7 ( 5 6 .2 ) a 4 0 .7 - 5 0 .6 (4 5 .7 )

H 3 .2 —6 .2 (4 .7 ) 3 .8 —7 .0 (5 .4 )

N 0 .8 —5.5 (3 .2 ) 0 .9 —3.3 (2 .1 )

S 0. 1 - 1 .5 (0 .8 ) 0 .1 - 3 .6 (1 .9 )
0 3 2 .7 - 3 8 .3 (3 5 .5 ) 3 9 .7 - 4 9 .8 (4 4 .8 )

F u n ction al group
HAs FA s
(m eq /g)

T otal acidity 5 .6 —7.7 ( 6 .7 ) a 6 .4 - 1 4 .2 (1 0 .3 )

C 02H 1 .5 - 5 .7 (3 .6 ) 5 .2 - 1 1 .2 (8 .2 )

Phenolic OH 2 .1 —5.7 (3 .9 ) 0 .3 —5.7 (3 .0 )

A lcoh olic OH 0 .2 —4 .9 (2 .6 ) 2 .6 —9.5 (6 .1 )

Q uinonoid C = 0
j o . 1 - 5 . 6 (2 .9 ) jl.2 - 4 .2 (2 .7 )
K etonic C —0

OCH3 0 .3 —0 .8 (0 .6 ) 0 .3 —1.2 (0 .8 )

e 4/ e 6 3 .8 —5.8 (4 .8 ) 7 .6 - 1 1 .5 (9 .6 )

a Means are in brackets.

the two materials in H, N and S contents; (c) the total acidity and C02H content of the FA are
appreciably higher than those of the HA; (d) both materials contain approximately the same
number of phenolic OH, total C = 0 and OCH3 groups per unit weight, but the FA is richer in
alcoholic OH groups than is the HA; (e) as expected, the E4/E6 ratio of the ‘ideal’ FA is much
higher than that of the ‘ideal’ HA. This indicates that the FA has a lower particle or mplecular
weight than does the HA [11], and it also reflects the abundance of C0 2H groups in the FA.

Major oxidative degradation products

Major compounds produced by the oxidation of methylated and unmethylated HÄs and FAs
are usually aliphatic, phenolic and benzene-carboxylic acids [12]. In addition, smaller amounts
of n-alkanes substituted furans and dialkyl phthalates are also isolated. The most abundant aliphatic
degradation products consist of n-fatty acids, especially the n-C16 and n-Cl 8 acids, and di- and
tri-carboxylic acids (Fig.2). Prominent benzene-polycarboxylic acids are the tri-, tetra-!, penta-
and hexa-forms (Fig.3). Major phenolic acids isolated include those with between 1 and 3 OH
groups and between 1 and 5 C0 2H groups per aromatic ring (Fig.4). Chemical structures of major
products formed by the KMn04 oxidation of methylated and unmethylated HAs and FAs extracted
from tropical volcanic soils are shown in Fig.5 [3]. In this investigation 52 degradation products
were identified. In some of these structures OCH3 groups are attached to the rings and must have
124 SCHNITZER

TABLE VII. ANALYSIS OF ‘IDEAL’ HA AND FA

(fro m m eans o f all data)

Elem ent
HA FA
(Jo )

c 56.2 4 5 .7
H 4 .7 5.4
N 3 .2 2.1
s 0.8 1.9
0 3 5 .5 4 4 .8

1 0 0 .4 9 9.7

F u n ction al groups
(m eq /g)

T otal acidity 6 .7 10.3

C 0 2H 3 .6 8.2
Phenolic OH 3 .9 3 .0
A lcoh olic OH 2 .6 6.1
Q uinonoid C = 0
■^2 . 9 |г .7
K etonic C = 0

ocH 3 0 .6 0.8

E4/E 6 4 .8 9 .6

CH3(CH2)i4COaH
CH3(CH2)i6 с о г н

COgH
(CHg)n n = 0 —8

COgH

Rs4 ^0 ^ .Ra
CHg-COgH
CH-COgH
F IG .2 . M ajor a lip h a tic degradation p ro d u cts.
C H g-co2H r4 R3

occurred in this form in the initial HAs and FAs because these compounds were produced by
oxidation of both methylated and unmethylated HAs and FAs [3]. The structures listed in Figs 2—5
may be considered to constitute the major humic ‘building blocks’. These are similar in HAs,
FAs and humins [12].

Products resulting from the КМПО4 oxidation of methylated HAs and FAs

Major types of products resulting from the KMn04 oxidation of 1.0 g of methylated HAs
and FAs are shown in Tables VIII and IX. The Arctic HA (Table VIII) produced considerably
higher yields of aliphatic compounds than did any of the other HAs. Weight ratios of benzene-
 125

F I G .3 . M a jo r b e n z e n e -c a r b o x y lic d e g r a d a tio n p r o d u c ts .

F I G .4 . M a jo r p h e n o lic d e g r a d a tio n p r o d u c ts .

carboxylic to phenolic compounds, which may be considered to reflect the interrelationship

between major humic ‘building blocks’, were, however, of the same general magnitude, except
that the ratio for HAs from subtropical soils was found to be higher than the remaining ratios.
Aliphatic compounds accounted for only a small proportion of the total products resulting from
the degradation of FAs (Table IX). Weight ratios of benzene-carboxylic to phenolic compounds
were similar for all FAs, and were of the same magnitude as those for most HAs. With one exception,
we were unable to detect any significant effect of climate on the weight ratios of major aromatic
structures for the different HAs and FAs. The only distinct effect of climate that we were able
to observe was that the Arctic HA appeared to contain considerably greater amounts of aliphatic
structures than did the other HAs.

Products resulting from the alkaline CuO oxidation of HAs and FAs

Major types of products resulting from the alkaline CuO oxidation of 1.0 g of HA and FA
are shown in Tables X and XI. Again, the Arctic HA produced larger amounts of aliphatic com­
pounds than did any of the other HAs. Aside from the Arctic HA, the most abundant compounds
 F IG . 5. M a jo r c h e m ic a l s tr u c tu r e s in tr o p ic a l v o lc a n ic H A s a n d F A s . C s ta n d s fo r C O 2H g ro u p or g ro u p

fo r m in g C O 2H on К М пО ц o x id a tio n s .

TABLE VIII. MAJOR TYPES OF PRODUCTS (mg) RESULTING FROM THE KMn04
OXIDATION OF 1.0 g OF METHYLATED HA EXTRACTED FROM SOILS FROM
VARIOUS CLIMATIC ZONES

Clim atic zon e

T yp e o f product C ool, tem perate

A rctic Subtropical Tropical
A cid soils N eutral soils

129.2
©

9.8-51.6
7
0

A liphatic 7.0-15.8 8.3-116.7

Phenolic 36.8 70.4-79.0 68.1-96.5 16.2-31.2 32.3-111.7
B en zene-carboxylic 50.8 80.2-122.4 147.5-173.5 9.2-164.1 49.6-183.3

T otal id en tified 224.2 194.4-282.4 248.6-281.3 118.1-198.5 100.2-350.4

Weight ratio
b en zene-carboxylic
1.4
OO
<N
<4

phenolic 1.1-1.6 5.3-6.1 1.2-2.4

1
 IAEA-SM-211/7 127

TABLE IX. MAJOR TYPES OF PRODUCTS (mg) RESULTING FROM THE KMn04
OXIDATION OF 1.0 g OF METHYLATED FA EXTRACTED FROM SOILS FROM
VARIOUS CLIMATIC ZONES

Climatic zon e

T yp e o f product C ool, tem perate

Subtropical Tropical
A cid soils N eutral soils

A liphatic 9.2 6 .8 - 8 .9 0 ^ 1 .6 5 .7 - 2 7 .1
Phenolic 30 .5 4 3 .1 - 4 6 .5 7 .4 - 6 0 .0 1 5 .9 - 2 6 .3
B en zene-carboxylic 46 .9 7 7 .6 - 9 5 .4 2 0 .6 - 8 6 .9 3 7 .9 - 6 8 .4

T o ta l iden tified 8 6 .6 1 2 9 .6 - 1 4 7 .8 2 8 .0 - 1 4 8 .1 6 4 ,6 - 1 0 8 .0

Weight ratio
benzene-carboxylic |
JO

JO
bo
to
1
00

1
phenolic 1.5 114—3.5

TABLE X. MAJOR TYPES OF PRODUCTS (mg) RESULTING FROM THE CuO-NaOH

OXIDATION OF 1.0 g OF HA EXTRACTED FROM SOILS
FROM VARIOUS CLIMATIC ZONES

Climatic zon e

T yp e o f product C ool, tem perate

A rctic Tropical
A cid soils Neutral soils

A liphatic 104.2 2 6 .2 4 6 .2 1 9 .6 - 5 5 .3
Phenolic 63.6 5 1 .0 6 8 .1 6 2 .7 - 9 8 .0
B en zene-carboxylic 24.8 28.4 2 0 .2 2 0 .9 - 3 4 .9

T otal identified 192.6 105.6 135 .0 1 0 3 .2 - 1 8 8 .2

Weight ratio
benzene-carboxylic
phenolic 0 .4 0 .6 0.3 0 .3 —0.5

formed by the alkaline CuO oxidation of HAs and FAs were phenolics. This confirms earlier
findings [13] that alkaline cupric oxide oxidation was an especially efficient method for releasing
phenolic structures. Relatively low yields of benzene-carboxylic acids resulted from the alkaline
CuO oxidation of the HAs and FAs. This is also indicated by the low benzene-carboxylic to phenolic
weight ratios (Tables X and XI). Again, we failed to observe any effect of climate on yields of
phenolic and benzene-carboxylic oxidation products.
]
Maximum yields of principal structures

In order to uncover which method of oxidation would produce the highest yields of major
products, we oxidized a HA, extracted from the A! horizon of a Haploboroll in Western Canada
128 SCHNITZER

TABLE XI. MAJOR TYPES OF PRODUCTS (mg) RESULTING

FROM THE CuO-NaOH OXIDATION OF 1.0 g OF FA
EXTRACTED FROM SOILS FROM VARIOUS CLIMATIC ZONES

Clim atic zone

Type o f product
C ool, tem perate
Tropical
acid soils .

A liphatic 51.6 1 5 .6 - 4 4 .2

Phenolic 4 5 .2 2 2 .7 - 5 5 .4

B enzene-carboxylic 2 4 .0 1 9 .5 - 4 3 .8

Total identified 1 2 0 .8 5 7 .8 - 1 4 3 .4

Weight ratio
benzene-carboxylic
phenolic 0.5 0 .8 —0.9

TABLE XII. MAJOR CHEMICAL STRUCTURES IN

IN ‘IDEAL’ HA AND FA

HA FA
Major products M ethod
(%) (%)

A liphatic 24.0 2 2 .2 C u O -N a O H + K M n 0 4

Phenolic 20.3 30.2 C u O -N a O H + K M n 0 4

B en zene-carboxylic 32 .0 23 .0 CH 3 CO 2 O H ,K M n 0 4

Total ■76.3 75 .4

„ . benzene-carboxylic
R a t io ----------- ;------ n—®—
phenolic 1 .6 0 .8

A rom aticity 69 71

(cool, temperate zone, neutral soil), and a FA, extracted from the Bh horizon of a Spodosol in
Eastern Canada (cool, temperate climate, acid soil), by the following methods or combination of
methods [13]: (a) alkaline CuO oxidation; (b) alkaline CuO and KMn04 oxidation; (c) alkaline
CuO and KMn04 and H20 2 oxidation; (d) alkaline KMn04 oxidation, and (e) peracetic acid
oxidation. All methods were used on methylated as well as on unmethylated HAs and FAs.
Alkaline CuO oxidation of unmethylated HAs and FAs was found to be especially efficient for
releasing phenolic structures, but was relatively inefficient for degrading aromatic structures bonded
by C-C bonds and which were poor in oxygen. The latter types of structures were more readily
degraded by the more drastic peracetic acid oxidation or oxidation of methylated HAs and FAs
with alkaline KMn04. Maximum yields of major oxidation products resulting from the degradation
of the HA and the FA referred to above, and the methods that were most efficient for this purpose,
are listed in Table XII. The data in the table were multiplied by two because we assumed that
losses during the lengthy oxidation, separation and isolation procedures were 50%. Thus, the
data in Table XII show that the ‘ideal’ HA contains about equal proportions of aliphatic and phenolic
structures, but a greater percentage of benzene-carboxylic structures or structures producing
 IAEA-SM-211/7 129

benzene-carboxylic acids on oxidation. By contrast, the ‘ideal’ FA contains more phenolic than
benzene-carboxylic and aliphatic structures. It is interesting that both the HA and FA contain
approximately equal proportions of aliphatic structures. Aromaticity values were approximated
by expressing yields of phenolic and benzene-carboxylic acids as percentages of total yields. The
aromaticity calculated in this manner is about 70% for both materials. Thus, as far as chemical
structure is concerned, the ‘ideal’ HA and FA are quite similar, except that the FA is richer in
phenolic but poorer in benzene-carboxylic structures than the HA. The main difference between
the two materials is that the ‘ideal’ HA contains more C and N, fewer C02H groups per unit weight,
but has a higher particle or molecular weight than the ‘ideal’ FA, and is thus less soluble in aqueous
solutions at pH < 7.0. 1

Spectrophotometric analyses

Ultra-violet and visible spectra of all HAs and FAs examined were featureless, with optical
densities decreasing as the wavelength increased. Major bands in infra-red spectra were at
3400 cm-1 (hydrogen-bonded OH), 2900 cm-1 (aliphatic С—H stretch), 1720 cm" 1 (C=0 of
C0 2H, C =0 stretch of carbonyl), 1620 cm" 1 (aromatic C=C, hydrogen-bonded C =0 of carbonyl,
COO"), 1400 cm" 1(C=0 stretch or OH-deformation of C0 2H) and near 1200 cm" 1 (С—О stretch
or OH-deformation of C0 2H). Infra-red spectra of HAs and FAs that were low in ash showed
troughs in the 2700 to 2400 cm" 1 region, which were due to hydrogen-bonded C02H groups [14].

ESR spectrometry

ESR spectra of all HAs and FAs were single, symmetrical lines devoid of hyperfine splitting.
Free radical concentrations of HA and FA solutions were of the order of I0 17—1018 spins/g,
spectroscopic splitting factors (g-values) ranged from 2.0032 to 2.0050, and line widths from
2.5 to 4.0 G.

The chemical structure of HAs and FAs

The chemical, spectrophotometric and spectrometric data presented here show that humic
substances formed under widely differing climatic conditions have similar chemical structures.
Recent work [15] has indicated that at least 50% of the aliphatic structures consist of n-fatty
acids esterified to phenolic OH groups (Fig.6). The remaining aliphatics are made up of more
‘loosely’ held fatty acids and n-alkanes that appear to be physically adsorbed on the humic
materials and are not structural components. The principal ‘building blocks’ of all HAs and FAs
that we have so far examined are, however, complex phenolic and benzene-carboxylic acids. The
major questions that need to be answered at this time is how do the ‘building blocks’join together
and what type(s) of structural arrangement is produced?
Some clues as to what structural arrangements may be involved here are provided by recent
work done in our laboratory [ 16] on HAs and FAs with the aid of a Scanning Electron Microscope.

F I G .6 . S tr u c tu r e o f p h e n o l- f a tty a c id e s te r s in H A s a n d F A s .
130 SCHNITZER

ОН

F IG . 7. P a r tia l s tr u c tu r e f o r F A .

We found that at low pH (2-3) FA occurred mainly as elongated fibres or bundles of fibres, forming
a relatively open structure. With increase in pH (4—7), the fibres tended to mesh into a finely
woven network to yield a sponge-like structure. At pH 8, the FA formed sheets which tended to
thicken at pH 9. At pH 10, fine, homogeneous grains were visible. The effect of pH on the HA
structure was similar to that observed on FA, except that because of low solubility in water at
pH < 6, the pH range at which the major transitions could be observed had to be narrowed to
between 6 and 10. In a subsequent study [ 11 ] we observed similar distinct effects of pH on the
viscosity, particle shapes and dimensions and on the polyelectrolytic behaviour of HAs and FAs.
At low pH, HA and FA behaved like uncharged polymers, at higher pH they both exhibited
characteristics typical of linear, flexible polyelectrolytes. This suggests that humic substances
are not made up of condensed rings but contain structures with bonds about which free rotation
can occur. Similar results have been obtained from X-ray studies [17] which show loose, open
structures for humic materials. Other considerations that enter into this discussion are long-term
observations that indicate that HAs and FAs are readily aggregated and dispersed not only by
changes in pH but also by changes in salt concentrations or valence of cations. A meaningful
concept of the chemical structure of HAs and FAs must take all available evidence into account.
It becomes more and more apparent that humic substances are not single molecules but rather
associations of molecules of microbiological, polyphenolic, lignin and condensed lignin origin.
These are the benzene-carboxylic and phenolic compounds that we have isolated, and which appear
to be held together by relatively weak linkages such as hydrogen-bonding, Van der Waals forces
and 7r-bonding. In higher molecular weight humic fractions, that is, HAs and FAs, associations
between ‘building blocks’ appear to be more stable than in FAs, and may in addition to the weaker
linkages mentioned above also involve more energetic bonding via C -0 and C-C bonds. Our data
suggest that aggregation of HA and FA particles at low pH results from intermolecular interactions,
mostly likely via hydrogen-bonds, whereas dispersion at higher pH arises from intermolecular
repulsion due to increased ionization of oxygen-containing functional groups.
A chemical structure that is in harmony with most of the requirements listed above has been
proposed by the author [2]. This molecular arrangement (Fig.7) may account for a significant
portion of the FA structure. Each of the compounds that make up the structure has been isolated
from FA without chemical degradation [2]. Bonding between ‘building blocks’ is exclusively
by hydrogen-bonding, which makes the structure flexible, permits the ‘building blocks’ to aggregate
and disperse reversibly, depending on environmental conditions, and also allows the FA to react
 IAEA -SM -211/7 131

with other inorganic and organic soil constituents either via oxygen-containing functional groups
on the large external and internal surfaces, or by trapping them in internal voids, provided that
the molecular sizes are of the appropriate dimensions and that charges are complementary.

ACKNOWLEDGEMENTS

I am grateful to G. Ogner, S.U. Khan, M.I. Ortiz de Serra, R. Riffaldi, K. Matsuda, J.A. Neyroud,
S.M. Griffith, N. Senesi, Y. Chen, S.I.M. Skinner, D. Skinner and E. Vendette for valuable colla­
boration and assistance with the work reported in this paper.

REFERENCES

[1] KONONOVA, M.M., Soil Organic Matter, Pergamon Press, New York (1966) 13, 101.
[2] SCHNITZER, M., KHAN, S.U., Humic Substances in the Environment, Marcel Dekker, New York (1972) 1, 196.
[3] GRIFFITH, S.M., SCHNITZER, M„ Can. J. Soil Sei. 55 (1975) 251.
[4] SCHNITZER, M., GUPTA, S.U., Soil Sei. Soc. Am., Proc. 29 (1965) 274.
[5] FRITZ, J.S., YAMAMURA, S.S., BRADFORD, E.C., Anal. Chem. 31 (1959) 260.
[6] SCHNITZER, M„ RIFFALDI, R., Soil Sei. Soc. Am., Proc. 36 (1972) 301.
[7] OGG, C.L., PORTER, W.L., WILLITS, C.O., Ind. Eng. Chem. (Anal. Ed.) 17 (1945) 394.
[8] RIFFALDI, R„ SCHNITZER, M., Geoderma 8 (1972) 1.
[9] NEYROUD, J.A., SCHNITZER, M„ Can. J. Chem. 52 (1974) 4123.
[10] SKINNER, S.I.M., SCHNITZER, M„ Anal. Chim. Acta 75 (1975) 207.
[11] CHEN Y., SCHNITZER, M., Soil Sei. Soc. Am., Proc. (submitted for publication).
[12] SCHNITZER, M., “The chemistry o f humic substances”, Environmental Biogeochemistry (NRIAGU, J.O., Ed.),
Ann Arbor Science Publishers Inc., Ann Arbor, Michigan (1976) 89.
[13] SCHNITZER, M., ORTIZ DE SERRA, M.I., Can. J. Chem. 51 (1973) 1554.
[14] SCHNITZER, M., GRIFFITH, S.M., Can. J. Soil Sei. 55 (1975) 491.
[15] NEYROUD, J.A., SCHNITZER, M., Soil Sei. Soc. Am., Proc. 38 (1974) 907.
[16] CHEN, Y., SCHNITZER, M., Soil Sei. Soc. Am., Proc. (in press).
[17] KODAMA, H., SCHNITZER, M., Fuel 46 (1967) 87.

DISCUSSION

W. FLAIG (S cien tific S ecretary): We must all thank Mr. Schnitzer for his outstanding talk.
He succeeded in giving us a remarkably clear summary of a great many data. We acknowledge
that his work represents an enormous step forward in humus chemistry.
According to the data on carboxyl groups in HA (3.6) and FA (8.2), one might perhaps
conclude that FAs are formed by oxidative cleavage of HA. Another explanation might be that
the part of the initial material with aromatic structure is decarboxylated during the polycondensation
processes. What do you think?
M. SCHNITZER: It is not clear yet whether HA is formed from FA or whether the reverse
reaction occurs. I am inclined to favour your first suggestion, although I have no experimental
evidence for either mechanism. My answer is based more or less on a feeling I have about this
matter.
B.R. NAGAR: The work you have reported is most interesting and impressive. Have you
done or are you planning to do work on the ‘nitrogen fraction’ of humus extracted from different
types of soil?
132 SCHNITZER

M. SCHNITZER: Yes, we are now starting work on the ‘unknown’ nitrogen in soils and humic
materials.
T. WEICHELT: In your model structure of humic substance, you have only shown hydrogen
bonds between the various components (units) of the system. What do you think about the
chemical importance of the covalent bonds and Van der Waals bonds in the nature of the substance
in question.
M. SCHNITZER: Although I showed only H-bonding, I believe that Van der Waals inter­
actions, 7Г-7Г bonding and covalent bonding also occur. But I wanted to emphasize that H-bonding
was very important.
E. FUSTEC-MATHON: When the extraction of the humic components from a soil after
extraction of the bitumens with ethanol-benzene is compared with the direct extraction of the
humic components from the same soil, one finds, in the second case, that almost all the bitumens
appear in the HA fraction. Do you not think that the preliminary extraction of products soluble
in ethanol-benzene would modify some of the relationships present, particularly after oxidative
degradation of the HAs?
M. SCHNITZER: In your soils, bitumens appear to be associated with the HA fraction.
This association appears to be relatively loose because you can extract the bitumens with organic
solvents. In order to study the ‘true’ structural HA components, it would be a good thing to
remove the bitumens before oxidative degradation.
 IA E A -SM -211/39

STRUCTURE ET GENESE DES

ACIDES FULVIQUES DES SOLS
DES LANDES DU MEDOC (FRANCE)

D. RIGHI, P. JAMBU, T. DUPUIS

Laboratoire de pedologie,
Universite de Poitiers,
Poitiers,
France

Abstract-Risum£

STRUCTURE AND GENESIS OF FULVIC ACIDS IN THE MEDOC LANDES SOILS (FRANCE).
The structure o f fulvic acids formed in sandy soils with permanent ground water, ranging from podzols to
hydromorphic soils, was studied. The acids fall into three classes: Type I corresponds to А ц surface horizons;
it is characterized by a high polysaccharide content. The nitrogen is present chiefly in the alpha-amino form;
the total acidity is lower than in the following types and the phenol functions predominate. Type II applies to
the hard pans (B2h) in the best-drained podzols; it is differentiated by the absence of polysaccharides and a lower
C/N ratio, since the nitrogen is distributed mainly in two forms: alpha-amino and heterocyclic nitrogen; the total
acidity is strong and the COOH functions predominate. Type III, which comes somewhere between the other
two, relates to the most moist podzol pans.

STRUCTURE ET GENESE DES ACIDES FULVIQUES DES SOLS DES LANDES DU MEDOC (FRANCE).
Nous avons cludie la structure des acides fulviques formes dans des sols sableux ä nappe permanente allant
des podzols aux sols hydromorphes. Ces acides se classent selon trois types: Le type I correspond aux horizons
superficiels A n ; il est caracterise par une forte teneur en polysaccharides; l’azote у est principalement sous forme
Q-ainmce, l’acidite totale est plus faible que dans les types suivants et les fonctions phenols dominent. Le type II
correspond aux horizons d’accumulation indures (B2h) des podzols les mieux draines; il se differencie par
Tabsence de polysaccharides et un rapport C/N plus faible, l’azote se repartissant en deux formes principales:
o-aminee et heterocyclique; Tacidite totale est forte et les fonctions COOH dominent. Le type III, intermediaire
entre les deux precedents, correspond aux horizons d’accumulation des podzols les plus humides.

I NTRO DU CT I O N

Nous avons сЬегоИё ä preciser 1 'influence d ’ un engorgem ent plus ou moins

p r o n o n c e s u r l a n a t u r e d e s a c i d e s f u l v i q u e s (AF) p r o d u i t s a u c o u r s d e s p r o c e s s u s
1
d h u m i f i c a t i o n d a n s l e s s o l s a c i d e s d e s L a n d e s du Medoc. Ces s o l s s o n t r e p a r t i s
selon des sequences d ' hydromorphie c r o is s a n te dont le s po les extrem es sont des
p o d z o l s b i e n d i f f e r e n c i e s ( h o r i z o n B2 h i n d u r e ) e t d e s s o l s h y d r o m o r p h e s p e u h u -
m i f e r e s . Les te rm e s i n t e r m e d i a i r e s s o n t d e s p o d z o l s h u m i q u e s ä h o r i z o n B2 h
m e u b le . Ces s o l s s e d e v e l o p p e n t s u r l e meme s a b l e g r o s s i e r q u a r t z e u x e t p o r ­
tent une lande humide ä sem i-hum ide , uniform em ent plant^e en pins m aritim es.

□ans ces sequences, 1 ' evolution de la m atiere organique est beaucoup plus
d ire c te m e n t c o n d itio n n S e p a r l e rögim e h y d r i q u e q u e p a r l a v e g e t a t i o n И ) . Des
ötudes a n te rie u re s ont m ontre que la tra n sfo rm a tio n des d e b ris vegetaux s u it
deux voies distinctes aux extrem ites des sequences : dans le s podzols, la frag­
m entation de la lignine serait lim itee et celle-ci se transform erait directe­
ment en humine ( 2) ; dans les sols hydromorphes, ou l'a c tiv ite biologique est
p lu s f o r t e , la l i g n i n e se f r a c t i o n n e r a i t en u n ites plus sim ples qui sont a l'o -
r i g i n e , en p a r t i c u l i e r , d ' a c i d e s h u m i q u e s peu e v o lu e s (3).

133
 134
TABLEAU I. CARACTERISTIQUES ANALYTIQUES DES HORIZONS ETUDIES

Podzols Podzols
Sol Sol
Types de sols
ä Bh hydrom. a Bh hydrom.
a Bh indure ä Bh indure
meuble m euble

H orizons
3 LAG 1 3 LAG 5 3 LAG 6 4 LAG 3 1 LAG 1 1 BER 1 1 BER 5 1 BER 6 4 BER 2 6 BER 1
(Ац ) (B h) 22 (B3) 2
(B h) (А ц 1 CAn ) (B 22
h) (B3) 2
[B h] (А ц)

pH e a u 4,2 4,8 5,0 5,4 4,5 3,5 4,5 5,1 4,6 4,9

RIGHI et al.
S/T ( %) 12,3 4,5 9,9 7,5 18,2 17,2 1,5 1,1 2,1 23,8

A rgiles [< 2 y) (%) 1,1 1,4 0,8 1,6 2,8 1,9 0,9 0,8 3,1 6,5

C to tal ( %. ) • 31,7 15,1 15,1 14,1 16,7 66,0 13,0 5,6 21,9 32,2

C/N 40 30 30 20 17 34 26 - 18 16

C AH/ C t o t a l (%) 25,4 29,2 22,9 33,6 27,1 30,6 37,9 9,1 49,5 19,8

C AF/C total C%) 2,1 59,7 64,6 39,5 14,3 1,7 60,5 69,7 25,3 13,3

C humine/C total (%) 21,3 - - - 6,9 12,8 - - - 3,0

 IA E A -SM -211/39 135

M AT E R I E L ET METHOBES D ’ ETUDE

Nous avons compare les AF d e cinq types d 'h o rizo n s rS partis sur deux se­
quences :

- horizons Aj i d e s p o d z o l s l e s m i e u x d r a i n e s [ 1 BER 1 , 3 LAG 1 ) ;

- horizons Aj j d es s o l s hydrom orphes peu h u m ife re s BER 1 , 1 LAG [6 1) ;
- horizons B2 2 h i n d u r e s d e s p o d z o l s l e s m o i n s humides (1B E R 5, 3LAG5) ;
- horizons B 3
d e s p o d z o l s p r e c e d e n t s [ 1 BER 6 , 3 LAG ) ; 6 ;
- horizons B2 h des podzols les plus humides [4 BER 2 ; 4 LAG 3 ) . |

Les caractSristiques analytiques des horizons, donnees au tableau I, m et-

t e n t en e v i d e n c e u n c e r t a i n n o m b r e d e d i f f e r e n c e s . De p l u s , l e s h o r i z o n s A n d e s
podzols se d i s t i n g u e n t des h o r iz o n s А ц d es s o l s hydrom orphes peu h u m ife re s p a r
le u r regim e hydrique e t le u r a c t i v i t e b io lo g iq u e. Pour le s prem iers, le point
de f l e t r i s s e m e n t perm anent e s t a . t t e i n t au c o u rs de l ’e t e , a lo rs que le s deu-
xiem es r e s t e n t p lu s hum ides. L ' a c t i v i t e m i n e r a l i s a t r i c e du c a r b o n e ( m e s u r e e p a r
re s p ir o m e tr ie je s t de 1,5 a 2 f o i s p lu s f o r t e pour le s s o ls hydromorphes (4).

L 'etude m icrom orphologique permet £galement de diffS rencier les divers

2
h o r i z o n s B h. Ceux d e s p o d z o l s l e s m i e u x d ra in e s p r e s e n te n t une m i c r o s tr u c t u re
en re v e te m e n ts fo rm e s p a r ' illuviation 1 e t le depot de la m a tie re organique
soluble ou pseudo-soluble tandis que les horizons B2 h d e s podzols plus humi­
des ont des m icroagregats dans le sq u e ls une p a r t ie de la m atiere organique
p r o v ie n d r a it de la fra g m e n ta tio n e t de 1
' hum ification s u r p la c e de ra c in e s
(5) (61.

Nous a v o n s d i s p e r s e l e s AF d e c e s d i v e r s h o r i z o n s s e l o n l a m e t h o d e d e
Flubert e t G o n z a l e z ( 7 ) , q u i u t i l i s e u n e r e s i n e e c h a n g e u s e d ' i o n s DOWEX 5 0 W -
X 8 f o r m e Fl+ e n m i l i e u aqueux. La t e n e u r en cendres des produits obtenus est
de 3 a 5 p. c e n t.

L 'analyse а ete effectuee en u tilisa n t les techniques dejä em ployees pour

les a c i d e s h u m i q u e s d e c e s memes s o l s ( 3 ) . N e a n m o i n s , q u e l q u e s m o d i f i c a t i o n s
ont e t e a p p o r t e e s . L ' a z o t e a am in e a e t e d o s e p a r l a m S thode de Moore e t S t e i n
( ) 8 q u i u t i l i s e l a r e a c t i o n ä l a n i n h y d r i n e . Une p a r t i e de l 'a z o t e s o lu b le
apres hydrolyse par HC1 6N n 'est i d e n t i f i a b l e n i comme a z o t e d is tilla b le (en
p r e s e n c e d e m a g n ö s i e o u meme d e s o u d e ) , n i c o m me a z o t e a a m i n e . N o u s c o n s i d e -
r o n s , comme G o n z a l e z e t a l. ( 9 ) , q u 'il s ’a g i t d 'u n e forme h e te ro c y c liq u e .
L 'h y d ro g e n e a e t e d ose en d e t e r m i n a n t l ' e a u o b te n u e a p r e s o x y d a tio n d e s AF
ä /1 0 0 0 ° C e n p r i s e n c e d e c a t a l y s e u r .

L e s c o u r b e s d ’ ATD s o u s a z o t e o n t e t e e n r e g i s t r § e s selon une te c h n iq u e

d S j a d e c r i t e ( 1 0 ) . Le d o s a g e d e s p o l y s a c c h a r i d e s а e f f e c tu e p a r la m etho­
d e d e B r i n k et a l. ( 1 1 ) .

RES U L T A T S ANALYTI QUES ET C A R A C T E R I S A T I O N DES AF

Les r£sultats sont regroupgs dans les tableaux II et III et sur;les figu­
res 1 et 2.

- Spectres infrarouges (Figure 1)

Ils perm ettent de classer les AF s e l o n tro is types distin cts :

T y p e I - AF d e s h o r i z o n s A i 1
des podzols e t des so ls hydromorphes. I l s sont
c a r a c t § r i s e s p a r u n e t r e s f o r t e a b s o r p t i o n e n t r e 1150 e t 1000 cm- 1 , d u e a u x
v i b r a t i o n s de v a l e n c e C-0 d e s p o l y s a c c h a r i d e s e t d e s b a n d e s d ' i n t e n s i t i mo-
yenne a 2850, 2930 et 2960 cm- 1 , relatives aux vibrations de valence C-H d e s
g r o u p e m e h t s CH 2 3
e t CFI . On r e l e v e S g a l e m e n t d e s b a n d e s faibles a 1540 e t 1650
сгтг* a t t r i b u e e s a la p resen ce de li a is o n s p e p tid e s .

Т у р е I I - AF d e s h o r i z o n s B 22h e t B 3 d e s p o d z o l s l e s m o i n s h u m i d e s . Ils dif-

fä re n t des precedents par 1 ' absence des bandes c a ra c tö ris tiq u e s des polysac­
charides et des liaisons peptides. C ependant, la bande d 'ab so rp tio n des OH
 u>
04

TABLEAU II. EVALUATION DU DEGRE D’AROMATICITE - ANALYSE ELEMENTAIRE ET DOSAGE DES POLYSACCHARIDES

Podzols Podzols
Sol Sol
Types de sols
ä Bh hydrom. ä Bh hydrom.
a Bh indure a Bh indure
m euble m euble

H orizons
3 LAG 1 3 LAG 5 3 LAG 6 4 LAG 3 1 LAG 1 1 BER 1 1 BER 5 1 BER 6 4 BER 2 6 BER 1
(Ai i ) (ВзгЮ (B3) 2
CB h ) (Ац ) (А ц) (B 22h ) (B3 ) 2
(B h) (Ai i )

D.O. 2920 cm -1 1,79 1,48 1,62 1,47 2,04 1,66 1,61 1,45 1,61 1 ,B0
D.O. 1510 cm “1
Räsidu apres chauffage sous

RIGHI et al.
40,2 42,5 41,4 43,0 40,2 40,2 41,5 41,9 40,3 40,7
N 2 ä B00°C

Carbone total ( %) 45,0 45,2 45,1 44,6 46,4 46,3 45,7 44,3 44,1 42,9

Hydrogene (%) 5,0 3,6 3,8 3,9 4,6 4,9 3,6 3,5 3,9 4,8

Azote to tal (%) 0,90 0,97 0,87 0,98 1,14 1,07 1,08 0,75 0,98 1,03

Rapport C/N 50,0 46,6 51,9 45,4 40,6 43,3 42,2 59,0 45,0 41,7

P olysaccharides ten mg
33 749 2 055 1 985 5 969 21 625 30 151 2 694 1 843 7 846 29 6B 0
p . c e n t g AF)

C polysaccharides ,
C AF
31,4 1,9 1,8 5,6 19,4 26,8 2,4 2,0 7,4 29,1
 IA E A -SM -211/39 137

v e r s 3 4 0 0 cm “1 s'e la rg it vers les hautes fröquences avec de nombreux pointös

a 3220, 3180 e t 3 0 8 0 cm“ 1 , ce qui in d iq u e la p resen ce de g r o u p e s a m i d e s ou
am ines l i e s . Contrairem ent au type p reced en t, le s bandes de v ib r a tio n de v a­
l e n c e C-H v e r s 2 8 5 0 - 2 9 5 0 cm '1
sont toujours tre s f a i b l e s . L ' a b s o r p t i o n d e s OH
fortem ent И ё э d e s a c i d e s e n t r e 2 7 0 0 e t 2 4 0 0 cm “1 est tre s intense.
Type I I I - AF d e s h o r i z o n s B ? h d e s p o d z o l s t r e s h u m i d e s . C ' e s t u n t y p e i n t e r ­
m ed iate e n t r e l e s d e u x p r e c e d e n t s . L e u r s p e c t r e e s t c e l u i du t y p e I I a v e c , en
plus, les bandes faibles caracteristiques des polysaccharides.

- Courbes ATO s o u s atm osphere inerte (Figure 2)

T o u t e s l e s c o u r b e s d e b u t e n t p a r un p i c e n d o t h e r m i q u e d e d e p a r t d e 1 ' ea u
h y g ro sc o p iq u e , s u i v i d 'u n e f f e t e x o th erm iq u e e t a l § e t com plexe d o n t l e m axi-
mu n s e s i t u e e n t r e 3 7 0 e t 5 4 0 ° C . S u r c e m a s s i f e x o t h e r m i q u e s e g r e f f e n t p l u -
sieurs pics endothermiques dont le prem ier, maximum ä 150°C, est reduit ä un
Spaulem ent. A plus h a u t e t e m p e r a t u r e , on o b s e r v e d i f f e r e n t s p i c s e n d o t h e r m i­
ques qui sont lie s ä la d e s tr u c tio n des d iv e r s groupem ents oxygenös s ta b l e s .
Ces pics perm ettent de distinguer tro is types de courbes correspondent aux
tro is types d'AF differencies par infrarouge.

Type I - Leurs courbes sont caracterisees par la prösence d'un öpaulem ent a
235-250°C, suivi d'un pic endotherm ique brutal et assez fo rt, maximumä 490°C.
L 'i n t e n s i t e de ce d e r n ie r d i f f e r e avec l 'o r i g i n e des AF : il est beaucoup plus
i n t e n s e p o u r l e s AF d e s o l s h y d r o m o r p h e s .

Type II - Leurs courbes prösentent un accident endotherm ique assez faible cul­
m inant ä 450°C, s u iv i d'un p ic tres faible, maximum ä 490°C. Ce dernier n ’ap-
parait pas dans tous les cas.

Type I I I - Leurs co u rb es sont i n t e r m e d i a t e s e n tr e le s deux ty p e s p r£ c£ d en ts.

De p l u s , e l l e s p r e s e n t e n t t o u j o u r s un p i c e n d o t h e r m i q u e s u p p l e m e n t a i r e c u l m i n a n t
ä 515°C.

- Poids m oleculaire apparent

Les c o u rb e s o b te n u e s a p r e s p a s s a g e s u r g e l S e p h a d e x G 25 e t G 50 s o n t
tres s e m b l a b l e s d ' u n AF ä l ' a u t r e . S eu le une p e t i t e f r a c t i o n e s t retenue sur
le gel G 25. Par contre, la quasi to ta litö des AF e s t retenue sur le gel G 50,
c e q u i ö t a b l i t un p o i d s m o l e c u l a i r e a p p a r e n t c o m p r i s e n tre 5.000 et 10.000. Ces
r e s u l t a t s sont proches de ceux obtenus p a r H ubert e t G onzalez (7) p o u r d e s AF
de p o d z o l s du Q uebec.

- Degrä d ' arom aticite (Tableau II)

I I p e u t e t r e e s t i m e d e p l u s i e u r s m a n i e r e s . Le r a p p o r t D . 0 . 2 9 2 0 c m " 1/
D. Ü. 1 5 1 0 cm -1
m e s u r e s u r l e s s p e c t r e s i n f r a r o u g e s (C-H a l i p h a t i q u e s / C = C a r o -
m a t i q u e s ) d o n n e u n d e g r e d ' a r o m a t i c i t e p l u s ё 1 е у ё p o u r l e s AF d e s h o r i z o n s
s p o d i q u e s . Le t a u x du " r e s i d u f i x e " ( e s s e n t i e l l e m e n t a r o m a t i q u e ) o b t e n u a p r S s
c h a u f f a g e s o u s a z o t e ä 6 0 0 ° C c o n d u i t a u meme r ö s u l t a t , m a i s l ' e c a r t e n t r e l e s
AF r e s t e faible. Les difförences observees doivent etre liees ä 1 ' im portance
de la fraction polysaccharidique prösente dans les AF d e s horizons А ц.

- Taux de polysaccharides (Tableau II)

Les dosages refleten t les differences observöes sur les spectres infra-
r o u g e s . La t e n e u r en p o l y s a c c h a r i d e s v a r i e dans d 'a sse z grandes pro p o rtio n s
e t d i f f ö r e n c i e n e t t e m e n t l e s AF s e l o n l e u r p r o v e n a n c e . Le r a p p o r t c a r b o n e d e s
p o l y s a c c h a r i d e s / c a r b o n e t o t a l d e s AF d o n n e :
- 20 a 30 p . cent pour les AF d e s horizons А ц (Type I)
5 a 7 p. cent pour les AF d e s 2
h o riz o n s B h meubles (Type III)
2 p. cent environ dans les horizons 2
B h indures et B 3 (Type II)
 u>
00

TABLEAU III. REPARTITION DES FORMES DE L’AZOTE ET DOSAGE DES FONCTIONS ACIDES

Podzols Sol Podzols Sol

Types de sols hydrom. hydrom.
ä Bh ä Bh
ä Bh indure ä Bh indure
m euble m euble
C
D 3 LAG 5 3 LAG 6 4 LAG 3 1 LAG 1 1 BER 5 1 BER В 4 BER 2 6 BER 1
H
4
□
(B 22h ) CB3 ) 2
(B h) (Ац ) (B 22h ) (B3 ) (B h)2 (Au )
I

LD
NJ
О
N to tal (%) 0,97 0,87 0,98 1,14 1,08 0,98 1,03

N non hydrolysable (1) 21,0 10,5 20,2 21,9 20,3 11,0 15,2 12,7

RIGHI et al.
N hydrolysable tl) 79,0 89,5 79,8 78,1 79,7 88,9 84,8 87,3

N a amine (1) 31,9 28,1 43,2 54,5 24,8 30,1 36,4 57,5

N d istilla b le Cl) 17,8 14,0 22,9 25,6 12,6 18,2 19,3 31,8

N heterocyclique tl) 29,3 47,4 13,7 - 42,3 40,6 29,1 -

A cidite totale COOH + OH ( 2) 909 881 959 B 00 955 893 962 827

COOH ( 2) 674 705 616 488 6B7 708 644 483

OH p h e n o l i q u e s ( 2) 235 176 343 312 268 185 318 344

tl) R esultats exprimds en p. cent de 1 'azote total

(2) R e s u lta ts exprimes en meq p . cent g d 'acid e s f u l v i q u e s _s a n S cendres
s e c h e s ä 80°C
 I AE A-SM -211 /3 9 139
tr a n s m is sio n

P o u r l e s h o r i z o n s Ац , ces c h i f f r e s s o n t s u p ö r ie u r s a oeux o b te n u s p ar
G u c K s r t C 1 2 ) p o u r d e s AP d e p o d z o l s . Au n i v e a u d e s h o r i z o n s E ^ h , l e s c h i f f r e s
sont assez sem blables.

- A nalyse elSm entaire (Tableau II)

L a t e n e u r e n h y d r o g e n e e s t n e t t e m e n t p l u s S l e v S e p o u r l e s AF d e s h o r i ­
zons А ц q u i c o n t i e n n e n t u n e f r a c t i o n p o l y s a c c h a r i d i q u e i m p o r t a n t e . Le t a u x
d e c a r b o n e v a r i e t r e s p e u d ' u n AF a 1 ' a u t r e a l o r s q u e l ’ on o b t i e n t d e s d i f -
f ö r e n c e s a s s e z s e n s i b l e s p o u r l ' a z o t e . Le r a p p o r t C/N e s t t o u j o u r s t r e s ё 1 е -
уё (40 ä 6 0 ) . I I e s t maximum d a n s l e s h o r i z o n s B 3 d e s p o d z o l s e t minimum p o u r
l e s AF d e s h o r i z o n s А ц d e s s o l s h y d r o m o r p h e s .

- Formes de l'azo te (Tableau III)

Apr s 6 hydrolyse, environ 80 p. cent de 1 ’a z o te se trouve dans la fraction

hydrolysable. La proportion d ’a z o t e a aminö, d i s t i l l a b l e et hötörocyclique de
cette fraction varie avec le type d'AF considörö :
- pour les AF d u type I, l ’azote а аггйпё d o m i n e largem ent alors que l'azo te
hötörocyclique n 'est pas representö j
- p o u r l e s AF d u t y p e I I , l ' a z o t e а а п и п ё e t l ’ a z o t e h ö tö ro c y c liq u e prödomi-
n e n t d a n s l e s h o r i z o n s В г г Ь i n d u r ö s ; p o u r l e s AF d e s 3
h o riz o n s B , la forme
hötörocyclique en solution represente presque la m oitiö de l'a z o te to tal ;
140 RIGHI e t al.

F I G .2 . C o u r b e s d ’a n a l y s e t h e r m i q u e d i f f e r e n t i e l l e s o u s a t m o s p h e r e i n e r t e .

- pour les AF d u type III, les formes a amine e t d istilla b le augm entent aux
depens de 1 ' azote heterocyclique.

C e s r e s u l t a t s d i f f e r e n t d e c e u x o b t e n u s p a r G o n z a l e z e t H u b e r t [91 q u i
o n t o b s e r v i s u r d e s AF d e p o d z o l s d u Q u e b e c u n e r e p a r t i t i o n i d e n t i q u e d e s f o r ­
mes d e l ’a z o t e q u e l q u e s o i t l e p r o f i l ou l ' h o r i z o n e t u d i e .

- Fonctions oxygenees (Tableau III)

La r e p a r t i t i o n des fonctions С00Н e t OH p h e n o l i q u e s est differente dans

c h a q u e t y p e d ' A F . L e s AF d e s h o r i z o n s A 31
des s o ls hydromorphes (type I) ont
1 ' a c i d i t e c a rb o x y liq u e la p lu s f a i b l e , mais so n t le s p lu s r i c h e s en f o n c t io n s
p h e n o l s . L e s AF d e s h o r i z o n s E ^ h i n d u r e s e t B 3
(type II) ont l 'a c i d i t § carb o ­
x y l i q u e l a p l u s f o r t e , m a i s p e u d e f o n c t i o n s p h e n o l . L e s AF d e s h o r i z o n s B h 2
m e u b l e s ( t y p e I I I ) o n t ä l a f o i s u n e f o r t e a c i d i t ö p h e n o l i q u e comme c e u x d u
type I et une f o r t e a c i d i t e c a r b o x y l i q u e comme c e u x du type II. C 'est done a
ce niveau que 1 ’a c i d i t e t o t a l e e s t l a p l u s f o r t e .

CONCLUSI ON

L ' e t u d e d e s AF p r o v e n a n t d e d i v e r s h o r i z o n s d e p o d z o l s e t d e s o l s h y d r o ­
m orphes peu h u m i f e r e s ne s em b le p a s m e t t r e en S v id e n c e de d i f f e r e n c e s f o n d a -
m e n t a l e s d a n s l a s t r u c t u r e d e s AF p r o p r e m e n t d i t s . L e s d i f f e r e n c e s o b s e r v e e s
apparaissent essentiellem ent liees ä la ргёэепсе en proportions variables se-
lon l ' o r i g i n e d e s AF d e s u b s t a n c e s n o n specifiquem ent humiques, te lle s que
les p o l y s a c c h a r i d e s ou l e s p r o t e i n e s .

La rS partition des groupes fonctionnels azotfis et oxygenes differencie

nettem ent les AF. L 'azo te "höterocyclique" domine dans les AF d e s horizons
spodiques les mieux draines alors que l'a z o te а ат!пё devient plus im portant
 IA E A -SM -211/39 141

dans les profils plus humides et q u 'il est maximum d a n s les sols hydromor-
phes. De т ё т е , dans les podzols, l'a c id ite est principalem ent due adx grou-
pem ents c a rb o x y liq u e s a l o r s que dans le s s o l s hydromorphes l 'a c i d i t e pheno-
l i q u e e s t m a j o r i t a i r e . C e s c a r a c t e r e s c o n f e r e n t a u x AF d e s h o r i z o n s !s p o d i -
ques i n d u r e s u n c a r a c t e r e p l u s " m a t u r e " q u ' a u x AF d e s s o ls hydromorphes.
Ceci e s t en a c c o rd avec l ' a g e a p p a re n t e le v e (= 3.0 0 0 ans B .P.) donne par
la d a ta tio n des horizons spodiques in d u r e s (1 3 ). D 'a u t r e p a r t , c e t t e "matu-
r i t e " a p p a r a i t p l u s g r a n d e du f a i t de 1 ' e l im i n a ti o n p r e f e r e n t i e l l e p a r la
nappe des petites molecules hydrosolubles.

La teneur plus elevee en groupem ents phenoliques des AF d e s sols hydro­

morphes peu hum iferes tra d u irait un caractere d'hum us jeune, mais aussi un
a u t r e mode d ’ h u m i f i c a t i o n : d a n s c e s s o l s , l a lignine serait p lu s fragm en-
t e e (3) e t s e s monomeres p a r t i c i p e r a i e n t p l u s largem ent ä la Synthese des
p r o d u its humiques.

REFERENCES

[1 ] JA M B U , P ., R IG H I, D ., Sei. Sol 3 ( 1 9 7 3 ) 207.

[2 ] D U PU IS, T ., JA M B U , P ., R IG H I, D ., T rans. In t. S ym p. (H u m u s e t P lan ta VI> P rague (1 9 7 5 ) 4 4 1 .
[3 ] R IG H I, D ., JA M B U , P ., M A T H O N , E ., D U PU IS, T ., C.R. I erColl. in t. (B io d e g rad atio n e t H u m ificatio n !,
N an cy , 1974, P ierro n e d . (1 9 7 5 ) 3 5 8 .
[4 ] K H O X A Y O .C ., T hese U niv. P o itiers (1 9 7 5 ) 68 p.
[5 ] R IG H I, D ., D E C O N IN C K , F ., P roc. 4 th In t. W orking M eeting o n Soil M icro m o rp h ., K in g sto n , ;1973, Soil
M icroscopy (R U T H E R F O R D , G .K ., E d .), K ingston, C anada (1 9 7 4 ) 567.
[6 ] R IG H I, D ., Sei. Sol 4 (1 9 7 5 ) 315.
[7 ] H U B E R T , G ., G O N Z A L E Z , A ., Can. J . Soil Sei. 51 (1 9 7 1 ) 157.
[8 ] M O O R E , S „ S T E IN , W .,J . B iol. C hem . 211 ( 1 9 5 4 ) 9 0 7 .
[9 ] G O N Z A L E Z , A ., H U B E R T , G ., C an. J . Soil Sei. 52 (1 9 7 2 ) 1.
[1 0 ] D U P U IS, T ., JA M B U , P ., D U P U IS , J ., A n n . A gron. 21 1 (1 9 7 0 ) 75.
[1 1 ] B R IN K , R „ D U B A C H , P ., LY N C H , D ., Soil Sei. 89 (1 9 6 0 ) 157.
[1 2 ] G U C K E R T , A ., T hese D o c t. E ta t U niv. N an cy (1 9 7 3 ) 124 p .
[1 3 ] R IG H I, D ., G U IL L E T , B ., (D a ta tio n s p a r le ca rb o n e -1 4 n a tu re l de la m a tie re o rg an iq u e d ’h o riz o n s sp o d iq u es de
p o d z o ls d es L a n d es d u M edoc (F ra n c e )!, p re se n ts c o m p te s re n d u s , IA E A -SM -211 /7 0 .

DISCUSSION

P. L O SS A IN T : It is w ell k n o w n th a t th e fulvic acids are ra th e r u n sta b le sub stan ces w hose

p ro p e rtie s an d c o n c e n tra tio n in soils change fro m season to season. D oes y o u r p a p e r re la te to a
single sam pling? D o y o u in te n d to stu d y th e p ro p e rtie s o f y o u r fulvic acids as a fu n c tio n o f
g eo clim atic v ariatio n s? j
P. JA M B U : A ll th e sam plings w ere d o n e a t th e sam e tim e a t th e en d o f spring. T h e seasonal
v a ria tio n s in th e p ro p e rtie s o f fulvic acids have n o t y e t b e en stu d ied . It is a n in te restin g su b ject,
an d w e in te n d to u n d e rta k e th is w ork.
 IAEA -SM -211 /7 2

AGGREGATION