Clay Minerals (1997) 32, 341-349
Study of the humic-like compounds formed
from L-tyrosine on homoionic clays
M. B O S E T T O ,
P. A R F A I O L I ,
O. L. P A N T A N I
AND G. G. R I S T O R I *
Dip. Scienza del Suolo e Nutrizione della Pianta - Universit& di Firenze, and *Centro di Studio per i Colloidi del Suolo,
CNR - Firenze- Piazza& Cascine, 28 - 50144 Firenze, ltaly
(Received 22 July 1996; revised 16 December 1996)
ABSTRACT: The ability to produce humic-like polymeric compounds, with L-tyrosine as the
starting material, was evaluated using different mineral systems, e.g. on Ca-, A1- and Cu(II)-saturated
montmorillonite, nontronite and kaolinite, and on quartz. Clay minerals proved to be effective in the
formation of these compounds, but not quartz, except in the presence of Cu(II). The newly formed
compounds were fractionated by alkaline extraction. With clay systems, the amounts of substances
produced appear to be related more to the interlayer cation than to the clay type. Copper cations
appear to be more effective when not associated with the clay structure.
Humic substances, the major organic constituents of
soils and sediments, are known to affect the
physical, chemical and biological properties and
the fertility of soils. Several biotic and abiotic
pathways have been proposed for their synthesis in
soils: the classic Waksman (1932) theory suggests
that humic substances originate from modified
lignin, while, at present, many authors indicate a
mechanism involving quinones. An important step
in this process is the oxidative polymerization of
phenols, polyphenols or similar compounds to
quinones or semi-quinones. These polymers
undergo polycondensation with amino compounds,
which probably also involve ring cleavage
(Stevenson, 1982). Deamination and decarboxylation reactions can also occur (Wang, 1991).
Furthermore, the formation of dark brown
substances of high molecular weight by reactions
involving quinones is a well known step in the
formation of melanins, as described by Raper
(1927). The reaction pathway starts from the
oxidative polymerization of tyrosine, according to
the scheme shown in Fig. 1.
Filip et al. (1976), Linhares & Martin (1979),
Saiz-Jimenez & Shafizadeh (1985), Schnitzer &
Khan (1986) and Senesi et al. (1987) reported that
melanins are strictly similar to humic substances
found in soil and sediments, in terrestrial and
marine environments.
As is widely known in the literature, many
polymerization processes can be catalysed, to
different extents, by the presence of some mineral
substrates, as reported by Shindo & Huang
(1985a,b). Amongst these substrates, clay minerals,
virtually ubiquitous in soils and sediments, show a
peculiar catalytic activity, probably depending on
their structure and properties and on the environmental conditions (Solomon, 1968; Thompson &
Moll, 1973; Wang et aL, 1984; Sawhney et al.,
1984; Wang et al., 1985; Theng, 1971; Wang &
Huang, 1989; Huang 1990).
In previous works, Bosetto et al. (1994, 1995)
pointed out the positive influence of several clays
on the abiotic formation of dark brown melanoidic
compounds from L-tryptophan plus D-glucose or Ltryptophan alone.
The aim of this work was to study the influence
of some homoionic clays on the formation of
humic-type compounds, with L-tyrosine as a
starting material. This amino acid, containing both
a hydroxyphenyl and an s-amino group, could
follow the polymerization pathway involving the
condensation of quinone-type compounds with
amino groups, and/or that suggested by Raper,
O 1997 The Mineralogical Society
342
M. Bosetto et al.
.•CH2-CH-COOH
I
NH2
CH2"CH'GOOHI
NHz
1/202
L-Tyrosine
HO
H O ~
I~
1/202
HO
GOOH
H
LeucoDOPAcbrome
H
H
DOPAchmme
Melanochrome
(red)
N
5,6 - d i o x y i n d o l e
I
NH2
DOPA - quinone
DOPA
( D i h y d r o x y - ortho - phenylalanine)
5, 6 - D i o x y indolyl - carboxylic acid
CH2"CH-COOH
~_
COOH
Melanine
(black)
H
lndolyl - 5,6 - q u i n o n e
F]c. 1. Diagram of melanine formation from tyrosine (Lotti, 1985).
through the formation of indole-type compounds.
Montmorillonite, nontronite and kaolinite, among
the most important clay minerals in soils and
sediments, were tested as catalysts for these
reactions. The clays were made homoionic with
transition and non-transition metals as Ca, AI and
Cu(II), which have been found to be effective qn
transforming simple c o m p o u n d s as phenols
(Isaacson & Sawhney, 1983). In order to investigate
the possible influence of clay and cations
separately, experiments were also carried out in
non-clay systems (quartz), in the presence of the
same cations.
A possible development of this research could be
its study in natural conditions, where these abiotic
processes could represent a possible pathway for the
formation of humic substances.
These abiotic reactions may take place in
vertisols, very dark soils characterized both by
high clay contents and by a large number of pores
which are inaccessible to microbes (<0.1 p.m). In
these environments, simple compounds like amino
acids can escape the microbial attack and undergo
polycondensation and polymerization reactions.
MATERIALS
AND
METHODS
Materials
Montmorillonite (SWy-I, Crook County,
Wyoming, USA), and nontronite (Hohen Hagen)
were supplied by the Source Clay Repository of the
Clay Minerals Society; the kaolinite came from
Zettlitz in the Czech Republic.
A suitable amount of the <2 mm fraction of clay
was dispersed in distilled water overnight, using a
magnetic stirrer. The <2 ~tm fraction of the clays
was collected by sedimentation, then made
homoionic by adding a 1 M solution of Ca, A1
and Cu(II) ions, as chlorides. The samples were
then washed with distilled water to remove excess
electrolytes until the test for C1- was negative.
The homoionic clays were then air-dried and
stored.
Pure w h i t e q u a r t z , s u p p l i e d by M e r c k
(Darmstadt, Germany) was passed through sieves
to obtain the <0.2 mm fraction, digested with HCI,
washed rapidly with 0.01 N HF to remove any oxide
coating, and oven-dried.
Formation q[ humic-like compounds on clays
Suwannee River humic and fulvic acid standards
were supplied by the International H u m i c
Substances Society (IHSS).
Experimental set-up
A 500 mg quantity of L-tyrosine was added to
5 g of the different homoionic clays and to quartz,
in the presence of different amounts of the above
mentioned cations (added as chlorides) corresponding to the cation exchange capacity (CEC)
of smectites or kaolinite. Both L-tyrosine and cation
chlorides were added as powders, mixed thoroughly
and water was immediately added to start the
wetting-drying cycles. These cycles were performed
at 70~ for 30 days, to achieve the reaction
products in a reasonable time. To the samples
were added 2 ml of water three times a day
throughout the period of the experiment.
The clay-organic systems are indicated as C a - K X, A1-K-X, C u - K - X , C a - M - X , A1-M-X, C u - M X, C a - N - X , A1-N-X, C u - N - X , where M, N and K
indicate montmorillonite, nontronite and kaolinite
respectively. The quartz-organic systems are
reported as Ca-Qk-X, Ca-Q~-X, A1-Qk-X, A1Qs-X, CU-Qk-X, and Cu-Q~-X, where Q is for
quartz and the indexes k and s stand for the amounts
of salt added, corresponding to the CEC of kaolinite
and smectite respectively. In both cases, X
represents the newly formed unknown substances.
After 30 days, at the end of the experiment, the
following analyses were carried out.
Elemental analysis
The carbon and nitrogen residual contents in
mineral-organic systems were determined on the
powders by dynamic dry flash combustion (1800~
under 02 flow), using a Carlo Erba Model NA 1500
Series 2 nitrogen/carbon/sulphur Autoanalyzer.
Extraction of humic substances
One gram of e a c h mineral-organic system was
extracted under nitrogen stream with 100 ml of
0.1 M NaOH + 0.1 M Na4P207 at room temperature,
overnight. After centrifugation (5000 g for 20 min.)
and filtration through 0.45 gm Millipore filters, the
extract (20 ml) was transferred to a centrifuge tube
and acidified until pH 2 with -0.5 ml of 50%
H2SO4 solution. After a further centrifugation at
5000 g for 20 rain, the precipitate (humic acids,
343
HA), was collected, redissolved in 0.5 M NaOH and
stored. The supernatant was fed on a small column
packed with - 6 cm 3 of solid polyvinylpirrolidone
(PVP Aldrich, Germany), previously equilibrated in
0.01 N H2SO4, using the Nagoya method (Watanabe
& Kuwatsuka, 1992). The brown-coloured fraction
retained on the resin (fulvic acids, FA) was eluted
with 0.5 M NaOH and stored. The non-humified
fraction (NH) was discarded. The carbon content
was determined both on the extract as well as on
humic and fulvic fractions according to dichromate
acid oxidation procedures.
The terms 'humic acid' and 'fulvic acid' as well
as 'humin' are operationally defined, being related
to the extraction procedure. The substances in our
systems may be different from those present in
soils (at least because the starting material is a
molecule alone, the environment is abiotic, and the
reaction time is shorter than in the soil environment), so strictly speaking it would be better to
refer to them as humic-like, fulvic-like etc., but for
discussion, the terms humic acid, fulvic acid, etc.
will be used.
Infrared analysis
The FT-IR spectra of the HA and FA extracted
were scanned after evaporation on AgC1 disks by a
Perkin-Elmer model 1710 spectrophotometer, interfaced to a microcomputer and a digital plotter.
X-ray diffraction analysis
Diffraction patterns were obtained in the 20
interval from 3 ~ to 15~ using Co-K~, radiation, with
a Philips 1710 diffractometer, at room temperature.
The analyses were performed on pure smectites and
on smectite-organic systems, on samples both air
dried and heated for 18 h at 190~ The samples
were kept in a dry atmosphere and protected against
rehydration with a Mylar plastic film. Moreover,
the smectite-organic systems were analysed both
unwashed and after water washing.
RESULTS
AND
DISCUSSION
At the end of the experiment, the browning
intensity varied remarkably, depending on the
particular system. During the 30 day reaction, all
the Cu systems promptly developed a deep brown
colour, while with other cations only the clay
systems turned brown. On the other hand, quartz
344
M. Bosetto et al.
100%
AI
Ca
AI
Ca
Cu
_---Iiiiiiiilliiil,
Q
i
Q
k
Q
s
Q
k
C volatilized
AI
100%
M
N
K
M
N
K
Q
s
Q
k
M
N
K
N
K
9 C residue
Ca
AI
Ca
Cu
90%
80%
70%
60%
Q
s
Q
k
I~
10.0
9.5
9.0
8.5
8.0
7.0 r
Q
s
Q
k
N volatilized
M
[]
N
K
M
N
K
Q
s
Q
k
M
N residue
9.8
[ O,N=atio I
8.3
9=
8.1 7.9
7.9
-7
7.7
~
]
7.9
7.0
6.5
6.0
F~c. 2. Losses by volatilization and residues of carbon and nitrogen on clayey and quartz systems, together with
their respective C/N ratio. The L-tyrosine C/N ratio (7.7) is represented by the arrow.
appeared colourless (except with Cu) even after 30
days.
In general, the colour was deeper for smectites
than for kaolinites: this could be ascribed to the
different number of active centres in the minerals
and their relative distribution between crystal edges
and planar surfaces, as suggested by Theng (1971).
In Fig. 2 the carbon and nitrogen losses and
residues are reported together with C/N ratio, after
30 day reaction. The carbon losses can be ascribed
to o x i d a t i v e d e c a r b o x y l a t i o n of t y r o s i n e as
suggested by Andreux (1982), while nitrogen can
volatilize as ammonia, due to the deamination of
the amino acid, as noted by Naidja & Siffert (1989).
It is not possible to unequivocally associate C
and N losses with one of the considered variables,
e.g. to a quartz system or to the type of clay or
cation. The only probable exception is A1 and Ca
clayey systems, which show lower C losses than the
analogous quartz systems. On the contrary, an
effect of the clay can be observed in C/N ratio
values. In fact, all the clayey systems ( C u - N
excluded) show values on or above the horizontal
arrow representing the original C/N tyrosine ratio
(7.7) while the quartz systems (except A1-Qs) lie
below the line.
345
Formation q f humic-like compounds on clays
Ca
AI
AI
Ca
Cu
100%
80%
60%
40%
20%
0% 9
o
s
,,
Q
k
Q
s
I m Unextractable
fraction
Q
k
[]
M
Humic-like
N
K
~
,,
M
Fulvic-like
N
K
Q
s
Q
k
[ ] Non hurnified
M
[]
N
K
Volatilized
FIG. 3. Carbon distribution on clayey and quartz systems.
From these data it can be inferred that, in clayey
systems, deamination and decarboxylation processes
are balanced, or in some cases the first is enhanced.
The C distribution in the systems is shown in
Fig. 3. The synthesis of humic substances (HA, FA
and humin fraction) appears to be promoted by all
clays. Actually these compounds are found in larger
amounts on clays than on quartz, except or. Cu
systems. The transition cation seems to be much
more effective in humification processes when not
associated with clays.
For A1 and Ca clay systems, kaolinites show
lower catalytic power, compared with smectites,
probably due to the presence of oxidizing sites only
at the crystal edges (Shindo & Huang, 1985b;
Zubkova et al., 1989).
On Cu systems, the production of HA, FA and
humin could be due to the formation of interlayer
complexes in which the aromatic ring of tyrosine can
act as a donor of ~ electrons to Cu(II) ions (Mortland
& Pinnavaia, 1971; Pinnavaia & Mortland, 1971).
The successive polymerization proceeds through the
formation of radical cations, for aromatic molecules
polymerized in the interlayer of transition-metal ionexchanged smectites, as suggested by Fenn et al.
(1973), Mortland & Halloran (1976), Soma et al.
(1984, 1985) and Soma & Soma (1988). This
behaviour on clay surfaces depends on the nature
of the interlayer cations and the degree of hydration
of the minerals (Moreale et al., 1985).
The large amounts of humic substances found on
Cu-Q systems could be ascribed to the enhanced
action of the free cation.
The relative amounts of humic substances
obtained with each clay can be related to the
cation used, in the order Ca > Cu > A1. Stable
complexes could form with tyrosine, as has been
demonstrated for organic acids (Kwong & Huang,
1979) probably preventing further polymerization
reactions.
A coupled clay-cation effect can be suggested in
the Ca-N system where the production of humic
substances was one of the largest in the whole
experiment. This could be due to Fe(III) on the
edges of the clay structure and to other Lewis acid
sites, as suggested by Wang & Huang (1989).
In Fig. 4 the FT-IR spectra of the FA fractions
extracted from some mineral-organic complexes are
shown. These spectra appear rather similar to each
other and to the spectrum of the Suwannee River
FA Standard (SwRFAS). All of the samples show a
band at 1720 cm -~ ascribed to COOH groups and a
shoulder at 1600 cm -1, ascribed to a lesser amount
of C=O and C O 0 - groups. The absorption in the
range 1400-1440 cm -1 seems to confirm the
presence of carboxylic groups.
The fulvic acids extracted from two Cu systems
(montmorillonite and quartz associated with CuC12,
i.e. Cu-M and Cu-Q~) show different spectral
features in the 1400 cm -1 region (Fig. 4), and this
is probably due to complexation reaction with the
transition metal.
Figure 5 shows the spectra of the HA fraction
from some mineral-organic complexes compared
with the spectrum of Suwannee River HA Standard
(SwRHAS). As found for the FA, the spectra are
346
M. Bosetto et al.
Ca N
[-
Cu Qs
I
4000
i
i
i
i
i
!
i
I
3000
i
i
1
I
I
1000
2000
WAVENUMBER
I
!
I
450
( c m -1)
FIG. 4. FT-IR spectra of extracted fulvic-like compounds.
rather similar to each other and to the standard. As
expected, the low absorption at -1720 cm -1, together
with the strong absorption at 1650 cm -1' indicates
the presence of few -COOH groups in this fraction.
The X-ray data are reported in Table 1 and show,
even after water washings, the persistence of
organic substances in the interlayer of C a - M - X ,
A 1 - M - X and A I - N - X . This behaviour may be
due to the formation on these clays of low
molecular weight polymers, which can easily
penetrate the internal surfaces.
CONCLUSIONS
(1) All clays, starting from L-tyrosine, can develop
dark brown substances extractable by classic humic
347
Formation of humic-like compounds on clays
Sw RltAS
t'q
Ca M
r~
Z
<
t'~
',o
N N
Ca N
[-
Cu Qs
r-.-tt~
N
Cu N
wmt
-
4000
I
I
1
I
3000
1000
2000
450
WAVENUMBER (cm -1)
Ft~. 5. FT-IR spectra of extracted humic-like compounds.
and fulvic acids extraction procedures. (2) The
quartz systems (quartz + cations) are not effective
in this synthesis, except when Cu(II) is present. (3)
The interlayer cations appear to influence the
amount of newly produced substances (HA, FA,
humin) in the order Ca > Cu > A1, but it is difficult
to evaluate the effect of clay type on this amount.
(4) Copper (II) appears more effective when free
(and not associated with the clay structure).
ACKNOWLEDGMENTS
This research was partially supported by the funds of
Ministero dell'Universit~ e della Ricerca Scientifica e
M. Bosetto et al.
348
TABLE 1. Values of d(001) (in nm) for homoionic smectites and their organic complexes at the end of
experiment.
Pure clay
25~
190~
At the end of the experiments
Unwashed
Water washed
25~
190~
25~
190~
1.40
1.47
1.41
1.41
1.25
1.23
1.19
0.99
1.00
0.99
0.99
0.99
1.51
1.51
1.52
1.51
1.50
1.39
Sample
AIM
A1 N
Ca M
Ca N
Cu M
Cu N
Tecnologica - Italy. The authors wish to thank Mr
Alessandro Dodero for his helpful co-operation in
performing the elemental analysis, and Mr Fabrizio
Filindassi for his technical assistance.
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