Volume 11 – No.

9b – 2002
REPRINT pp. 636 – 641

DETERMINATION OF CONDITIONAL STABILITY CONSTANTS

METAL-Trichoderma viride COMPLEXES
OF METAL- COMPLEXES
BY THE POTENTIOMETRIC TITRATION METHOD
G. Sanna – G. Alberti – P. Castaldi – P. Melis

Angerstr. 12
85354 Freising - Germany
Phone: ++49 – (0) 8161-48420
Fax: ++49 - (0) 8161-484248
e-mail: parlar@psp-parlar.de
www.psp-parlar.de
© by PSP Volume 11 – No 9b. 2002 Fresenius Environmental Bulletin
DETERMINATION OF CONDITIONAL STABILITY CONSTANTS
OF METAL-Trichoderma viride COMPLEXES
BY THE POTENTIOMETRIC TITRATION METHOD
Giovanna SANNA1, Guido ALBERTI2, Paola CASTALDI1 and Pietro MELIS1
1
Università di Sassari, Di.S.A.A.B.A., Chimica Agraria, viale Italia 39, I - 07100 Sassari, Italy
2
Università di Cagliari, Dipartimento di Scienze Chimiche, Monserrato, I - 09042Cagliari, Italy
SUMMARY
In this paper the binding ability of functional groups
present in the cell walls of an ubiquitous soil fungus,
Trichoderma viride Pers.: Fr., towards some divalent
heavy metals, Cd, Co, Cu, Pb and Zn, was studied. Proc-
essing the data from potentiometric titrations of T. viride
by the McCallum, Midgley and Gran's combined meth-
ods, the surface of the cell wall was characterised in terms
of functional group total acidity, distinguishing among
strong, weak and very weak acidity. The dissociation
constant values of weak and very weak acid, pKa1 and
pKa2, respectively, were graphically determined using the
Henderson-Hasselbach equation. Titration data of T. viride
in the presence of metal were processed with the Bjer-
rum's method as adapted by Gregor for determining the
conditional stability constants of metal complexes with
synthetic polyelectrolytes in terms of log K1 and log K2.
Here has been used for 1:1 and 1:2 metal-T. viride com-
plexes, respectively.
KEYWORDS:
Heavy metals, fungi, Trichoderma viride, potentiometric titration.
INTRODUCTION
Soil is a biologically complex environment, where the
great mass of micro-organisms present cannot be ignored.
The importance of micro-organisms depends on their
ability to transform animal and vegetable remains into
simpler compounds that enter again the ecosystem life
cycle. Furthermore, they are mainly responsible for the
mobilisation, bio-transformation and bio-accumulation
processes of micronutrient and/or pollutant metals present
in traces in the soil.
The micro-organism ability, in particular bacteria and
fungi, to interact with metals by means of adsorption
phenomena is well-known [1 - 5]. The cell wall is the place
636
where the interactions between metal and fungus occur,
forming bonds with different energy. As the chemical
composition of the cell wall may vary considerably de-
pending on the fungus species, remarkable differences in
the metal absorption capacity and mechanism may exist.
Heavy metals were studied, in particular, because of
their accumulation in soils, uptake by plants and contami-
nation of groundwater. Certain metal compounds show
negative effects in high doses, but are essential for some
organisms in small doses. The heavy metal adsorption
behaviour varies among soils types and depends on the
physical and chemical soil properties [6]. In the last years
interesting researches have been conducted in order to
understand the role played by the soil living biomass on
the dynamics of those metals that are both micronutrient
and toxic for the plants [7, 8].
The purpose of this study was to examine how an
ubiquitous soil fungus, Trichoderma viride Pers.: Fr.,
interacts with some heavy metals. Another aim was to
improve the knowledge on the absorption capacity of the
soil living biomass by methods enabling to evaluate the
contribution of the different mechanisms operating on the
metal-fungus interaction.
MATERIALS AND METHODS
Separation and breeding of T. viride have been carried
out according to the method reported by Sanna [5]. Chitin
was purified according to the method reported by Muz-
zarelli [9].
Potentiometric Titration
The polyelectrolyte nature of the absorbent studied
and the hypothesis that each site occupied by a proton
may be available for the absorption of a metal atom, allow
the application of a potentiometric titration method, gener-
© by PSP Volume 11 – No 9b. 2002 Fresenius Environmental Bulletin

ally used in the study of organometallic complexes in solu-
tion and, more recently, in the study of the dead microbial
biomass [10, 11, 12]. Potentiometric titration of T. viride
was carried out in the following way: 0.3 g of freeze-dried
fungus were dispersed in 40 mL water containing 1 mL of
5N NaNO3, a known quantity of 0.05N HNO3 was added
until a pH value of approx. 3 was reached. The suspension
was titrated with 0.1N NaOH from pH 3 to pH 10.5 after
correction of the volume to 50 mL with water. For com-
parison, a blank titration was performed using a similar
system without fungus. Titration in the presence of metal
was carried out bringing the fungus/water suspension to
pH 5.0 by 0.05N HNO3 and adding the metal at this point.
After an hour for equilibration, the suspension was
brought to pH 3.0 using a known quantity of 0.05N HNO3
and titrated with 0.1N NaOH from pH 3.0 to pH 5.5,
starting from an initial volume of 50 mL. The titration was
stopped at pH 5.5 to avoid the possible formation of metal
hydroxides. This procedure was repeated at 1.07 meq/L
concentration for each metal examined (Pb, Co, Cu, Cd, Zn).
A Radiometer Copenhagen VIT 90 automatic titration
system connected to an ABU 93 automatic burette was
used. The titrating solutions were of high purity grade
Normex at 25 °C in nitrogen atmosphere. All determina-
tions were carried out using fungal material soaked for
three hours in water at room temperature before use. The
T. viride so obtained resulted fully vital. The acidity
value for the fungal substrate is the average of six deter-
minations and the standard deviation is lower than 5%.
RESULTS AND DISCUSSION
Titration Curves
Examining the T. viride titration curve (Fig. 1) in the
pH range between 3 and 10.5, in absence of metals, a
progressive dissociation of the protonated sites present on
the fungus walls can be observed.

TRICHODERMA VIRIDE
12,00
10,00
8,00
pH

6,00
4,00
2,00
0,00
0,00 1,00 2,00 3,00 4,00 5,00
mL NaOH 0.1 N

FIGURE 1 - Trichoderma viride titration curve.

7 ,0 0 T r ic h o d e r m a
Z n - T ric h o d .
C d - T r ic h o d .
6 ,0 0 C u - T ric h o d .
P b - T r ic h o d .
5 ,0 0 C o - T r ic h o d .
pH

4 ,0 0

3 ,0 0

2 ,0 0
0 ,0 0 0 ,5 0 1 ,0 0 1 ,5 0 2 ,0 0 2 ,5 0 3 ,0 0
m L N a O H 0 .1 N

FIGURE 2 - Metal-T. viride titration curves.

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© by PSP Volume 11 – No 9b. 2002 Fresenius Environmental Bulletin

The curve does not show pronounced flexes that al-
low to locate the equivalence points indicating the neu-
tralisation of different acidic groups, since the acidity of
the latter vary with pH. Similar behaviour was observed
with other natural substances such as humic and fulvic
acids [13]. T. viride titration curves in the presence of
metal (Fig. 2), obtained using the same concentration of
the tested metals, are shifted toward the right side with
respect to the ones obtained with the fungus without
metal. This shift shows that the presence of metal favours
the acidic site dissociation, lowering the solution pH and,
consequently, increasing the quantity of titrating solution
necessary to bring the system to the same pH value. In
fact, during the absorption process, the metal ions com-
pete with the protons for the binding sites present on the
fungi walls. The curve slope is correlated to the intensity
of protons released in the presence of a specific metal.
The differences observed among the curves give, therefore,
an indication about the ability of the various metals to
compete with the protons. The proton shift order is Cu >
Co > Zn>Pb > Cd.
conditions the interference due to the protons freed from
the hydration water is negligible. Total acidity and the
contribution of the various acidic functions present in the
adsorbing substrate were evaluated by processing the
titration data by the McCallum and Midgley's method
[14], combined with Gran's [15], as described by Bizri et
al. [10] and Brunelot et al. [11]. The data were processed
according to relations 1 and 2:
(V0+V).[H+] = f (V); (1)
(V0+V).[OH-] = f (V) (2)
V0 = initial volume; V = titrant volume added;
[H+] = free proton concentration;
[OH-] = free hydroxyl concentration.
Equation (1) is relative to the titration data before the
equivalence point, while equation (2) is relevant after the
equivalence point.

At pH values lower than the hydroxide formation
point for the metals used in this work, the pH decrease
corresponds only to the proton release from the reactive
groups present in the surface of the fungus cell walls. At
higher pH values also the protons, freed from the hydration
water of the metal, contribute. For the stability constant
evaluation of the metal-T. viride complexes the curve sec-
tion below the hydroxide precipitation pH was considered,
assuming, accordingly to Stevenson [13], that in such
The intercepts of the two lines intersecting the ab-
scissa (V) in Fig. 3, calculated considering the point of the
two parts of the parabola and the value of the intercept of
the blank test (∆), allow to evaluate the different kind of
acidity.
The total acidity for T. viride is 88.90 meq/100 g, a
high value when compared with the literature data for
adsorbent substrata naturally present in the soil (Table 1).

0,060 0,006
(V o +V I )(H +)

0,050 (V o +V H N O 3 )(H +) 0,005

(V 0 +V I )(O H -)

0,040 0,004
(Vo+V1)(OH )
-
(Vo+V) (H )
+

0,030 0,003

0,020 0,002

0,010 0,001

0,000 0
0,000 1,000 2,000 3,000 4,000 5,000
m L N aO H 0.1 N

FIGURE 3 - Gran’s plot

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© by PSP Volume 11 – No 9b. 2002 Fresenius Environmental Bulletin

TABLE 1 - Total acidity and types of acidity of T. viride in comparison with other absorbent substrata.

Total Strong Weak Very weak

Sample acidity acidity % acidity % acidity %
meq/100g meq/100g meq/100g meq/100g
Trichoderma viride 88.90 16.67 19.08 28.33 31.87 43.60 49.04
Aspergillus niger* 57.00 15.00 26.30 19.00 33.30 23.00 40.30
Rhizopus arhizus* 227.00 48.00 21.10 38.00 16.70 138.00 60.80
Humic acid ° 323.00 128.00
Chitin 29.70 3.20 10.96 12.80 43.84 13.17 45.21
(*) Dead mycelium (4) (°) Humic acid extracted from a podsol (13)

The concentration of the different acidic functional
groups has been calculated according to Bizri [10]. This
leads to evaluate the strong acidity (pH < 4) attributed to
mineral and organic acids (phosphoric and carboxylic),
the weak acidity (4 > pH < 6), attributed to weaker car-
boxylic groups, and the very weak acidity (6 > pH < 9)
attributed to phenolic or amidic groups. These groups,
present in the cell wall, are responsible for the fungi bind-
ing ability toward metals.
The differences in acidity, found between T. viride
and the other reference natural substrata, are due, besides
to the more quantity of acidic functions, to the per cent
distribution of the functional groups: the very weak acid-
ity is 49% ca. of the total acidity, whereas weak and
strong acidity are 32 and 19 %, respectively. A similar
distribution is present in Aspergillus niger and Rhizopus
arhizus [12], but the humic acid shows a high weak acid-
ity and the chitin shows, almost exclusively, weak and
very weak acidity [16]. The high percentage of very weak
acidic sites (49%) in T. viride could be attributed to the
amidic groups, hence to the high quantity of chitin and
chitosans present in the fungus cell wall.
Using the Henderson-Hasselbach's equation the ap-
parent dissociation constants of the weak acidity sites,
pKa1, and very weak acidity sites, pKa2, of the substratum
were determined (Table 2).
TABLE 2 - pKa values for the functional groups of
T. viride in comparison with other absorbent substrata.
the dissociation of the functional group is a pH function.
To a higher dissociation of the acid sites corresponds a
higher adsorption of the metals, because the latter com-
pete with the proton present on the ligand sites.
Formation Curves
Titration data for determination of partial and total sta-
bility constants of metal-T. viride complexes were proc-
essed using the Bjerrum's method modified by Gregor and
colleagues, as reported by Stevenson [17]. This method
assumes that the complexation occurs in two stages, form-
ing the two species (MA+) and (MA2), and does not alter
the site dissociation reaction. MA+ and MA2 represent the
metal bound to one and two acid sites, respectively.
The Bjerrum's formation degree “r” represents the mean
number of complexing sites for metal ions at different pH
values and can be determined by the following relation:
C a − [HA] − [A − ] [MA + ] + [MA 2 ]
r= − 2+
CM [M ] + [MA + ] + [MA 2 ]
where Ca = total concentration of weak acid sites; CM =
total concentration of metallic ions; [M2+], [MA+], [MA2] =
concentration of free metal ions, of metal bound to one
acidic site and to two acidic sites, respectively. Moreover,
the concentrations of the non-dissociated weak acid HA
and the dissociated weak acid A- can be obtained from the
relations a) and b), respectively:

Sample pKa1 pKa2 a) [HA] = (1 − α M )C a

Trichoderma viride* 4.9 7.92
− [HA]
Aspergillus niger* 7.0 >10 b) [A ] = K a1
Rhizopus arhizus* 6.5 9.20 [H + ]
Humic acid ° 4.8 9.05
Chitin 5.1 8.02 where αM = ligand dissociation grade; Ka1 = apparent
dissociation constants of the weak acidity sites.
(*) Dead mycelium (4)
(°) Humic acid extracted from a podsol (13)
The "r" values determined for each metal are intro-
duced into the following relation:
The dissociation constants are called "apparent", be- n
æ [HA] ö
cause they refer to well-defined reaction conditions: ionic å (r − n)Bn çç [H + ] ÷÷ = 0
force NaNO3 0.1 N and temperature 25 °C; furthermore, è ø

639
© by PSP Volume 11 – No 9b. 2002 Fresenius Environmental Bulletin

for evaluating the formation constant for the metal-T. viride b2

complex under the conditions of n = 1 and n = 2, which
represent the formation conditions for 1:1 and 1:2 com-
plexes, respectively. To simplify the calculation, the rela-
tion has been rearranged, obtaining a straight line equa-
tion, whose slope and intercept parameters represent,
respectively, the total conditional constant Bn and partial
conditional constant b1. As an example, the graphic reso-
lution for the Cu-T. viride complex is reported (Fig. 4)
using the relations:
b1
c) B 2 = b1b 2 d) K1 =
K a1
e) K2 =
K a1
It is possible to convert the stability constants b1 and
b2 into the classic complexation constants (Tables 3, 4).
The values so obtained are slightly higher than the ones
reported by other authors (Stevenson [13], Bizri et al. [10])
for humic acid. Moreover, the stability order for metal-T.
viride complexes follows neither the Irving-Williams’
series reported by Stevenson [13] for metal-humate com-
plexes.

4
r/(r-1) (HA)/(H )
+

2
y = 0 ,0 1 7 7 x + 0 ,6 2 3 5
1 2
R = 0 ,9 9 9 9

0
-1 0 0 0 100 200 300
-1

-2

+
( 2 - r ) ( H A /H ) /( r - 1 )

FIGURE 4 - Graph used for determining the complexation conditional constant of Cu-T. viride complex.

TABLE 3 - Conditional complexation constants for metal-T. viride complexes (metal concentration 1.071 meq/L).

Metal b1 b2 B2 log K1 log K2

Zn 0.808 0.024 0.019 4.787 3.253
Cd 6.921 0.322 2.229 5.72 4.387
Cu 0.689 0.025 0.017 4.718 3.279
Co 3.675 0.092 0.338 5.445 3.843
Pb 0.388 0.181 0.070 4.469 4.137

TABLE 4 - Conditional complexation constants Cu-chitine (Cu concentration 1.071 meq/L).

Metal b1 b2 B2 log K1 log K2

Cu 598.08 137.39 82171 7.83 7.19

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CONCLUSIONS
No simple equilibrium can adequately describe the
complexation process of metals with complicated absorbent
systems like the one we have studied. Among the different
methods, till now used for the study of complex natural
systems, the potentiometric method allows a good evalua-
tion of the ligand dissociated form, a feature that deeply
affects the results validity. This approach does not require
the determination of free metal in solution, not an easy
parameter to evaluate during the potentiometric titration.
Anyhow, it is clear that the experimental results ob-
tained cannot be interpreted with the same severity as
when applying the same method to simple systems. Be-
cause of the heterogeneous nature of the fungi cell wall,
not every site can be considered equal. In reality, two sites
of the same nature cannot be identical, because of the
different chemical surroundings in which they occur.
The results obtained underline the role that micro-
organisms and their cellular constituents can play in the
soil system and their potential influence on the mineral
nutrition of plants.
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Received for publication: December 28, 2001
Accepted for publication: March 15, 2002
CORRESPONDING AUTHOR
Pietro Melis
Università di Sassari
Di.S.A.A.B.A.
Chimica Agraria
viale Italia 39
07100 Sassari - ITALY
e-mail: pmelis@uniss.it
FEB/ Vol 11/ No 9b/ 2002 – pages 636 - 641