Soil Biology & Biochemistry 43 (2011) 628e637

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Soil Biology & Biochemistry

journal homepage: www.elsevier.com/locate/soilbio

Tannic acid and Norway spruce condensed tannins can precipitate

various organic nitrogen compounds
Bartosz Adamczyk*, Sylwia Adamczyk, Aino Smolander, Veikko Kitunen
Finnish Forest Research Institute, Vantaa Research Unit, P.O. Box 18, FI-01301 Vantaa, Finland

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 23 June 2010
Received in revised form
23 November 2010
Accepted 29 November 2010
Available online 14 December 2010

There is increasing evidence that tannins affect degradation of soil organic matter and nitrogen cycling.
It is assumed that the inuence of tannins on biochemical processes is partly related to their ability to
precipitate proteins. However, there is almost no information about precipitation of organic nitrogencontaining compounds other than proteins. A few studies indicate that tannins can precipitate arginine,
choline or chitosan. In this study we used commercial tannic acid and condensed tannins extracted from
Norway spruce (Picea abies (L.) Karst) needles to investigate precipitation of a wide range of organic
nitrogen compounds including amino acids (all 20 proteinaceous), peptides (insulin, oxidized glutathion,
reduced glutathion, AlaAla, GlyGlu, GlyPhe, GlyGlyGly), proteins (bovine serum albumin, Rubisco i.e.
D-ribulose 1,5-diphosphate carboxylase), nitrogen bases, polyamines and aminosugars (N-acetyl-Dglucosamine, chitin and chitosan). Our results showed that tannins can precipitate a subset of these
compounds e of the amino acids only arginine, of the peptides studied only insulin, all the proteins,
polyamines, nitrogen bases, chitin and chitosan, but not N-acetyl-D-glucosamine. Concentrations of
organic nitrogen compound and tannins affected amount of these compounds in precipitates. Moreover,
pH value affected precipitation. The amount of precipitated organic nitrogen compound and the amount
of precipitated tannins showed positive correlation across different pH. Precipitation of organic
N-containing compounds other than protein by tannins can potentially affect reactions in all biochemical
mixtures including tannins and these organic nitrogen compounds, and affect soil N cycling.
2010 Elsevier Ltd. All rights reserved.

Keywords:
Organic nitrogen
Precipitation
Proteinetannin complex
Tannins

1. Introduction
Tannins are an abundant group of plant secondary metabolites,
which are usually divided into two basic classes, condensed tannins
(CT) and hydrolysable tannins. Condensed tannins have procyanidins and prodelphinidins as monomers. Hydrolysable tannins are
grouped into gallotannins and ellagitannins, which are composed
of gallic acid or hexahydroxydiphenic acid esters, respectively,
linked to a sugar moiety (Kraus et al., 2003a). The abundance of
tannins in plants is reected in their concentrations in soil organic
horizon (in boreal forest humus layer up to 0.04 mg MGE (methyl
gallate equivalents) g
1 SOM of hydrolysable tannins, Adamczyk
et al., 2008; 2009b; 5 mg g
1 SOM of CT, Kanerva et al., 2008).
Among many potential functions, tannins may affect degradation of
soil organic matter and nitrogen cycling (Fierer et al., 2001; Kraus
et al., 2003a; Kanerva et al., 2006). The inuence of tannins on

Abbreviations: CT, condensed tannins; OrgN, organic nitrogen; TA, tannic acid.
* Corresponding author. Tel.: 358 10 211 2595; fax: 358 10 211 2206.
E-mail address: bartek_adamczyk_79@o2.pl (B. Adamczyk).
0038-0717/$ e see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.soilbio.2010.11.034

biochemical processes is suggested to be partly related to their

ability to precipitate proteins. Creation of such proteinetannin
complexes is dependent on the biochemical and physical characteristics of proteins and tannins, such as chemical structure, protein
charge and protein isoelectric point, but solution pH value is also an
important factor (Hagerman and Butler, 1981; Fierer et al., 2001;
Kraus et al., 2003a).
Only a few studies have examined whether tannins precipitate
OrgN compounds other than proteins. Kalina and Pease (1977)
showed formation of a complex of tannic acid (TA) with choline,
an amine and the precursor of the neurotransmitter acetylcholine
(Ilcol et al., 2005). Adsorption of TA to chitosan beads and chitosanemontmorillonite has been demonstrated (Chang and Juang,
2004; An and Dultz, 2007). Mole and Waterman (1987) showed
precipitation of the amino acid arginine by TA and numerous
extracts from plants rich in CT, and also precipitation of histidine
but only by CT-rich extracts of bracken (Pteridium aquilinum).
Hagerman and Butler (1981) showed in competitive study that
afnities of alanine, glycine, proline for CT are at least a thousandfold lower than those of proteins and that peptides with fewer than
six residues interacted very weakly with tannins. Moreover, these

B. Adamczyk et al. / Soil Biology & Biochemistry 43 (2011) 628e637

authors presented results suggesting that also some other

compounds, like polyamino acids, polyvinylopyrrolidone and
formamide can inhibit precipitation of proteins by CT.
The possibility of tannins to form complexes with arginine and
chitosan, not only with peptides/proteins, raised the question about
precipitation of other OrgN compounds in a tannin-rich environment, like boreal forest soil. In addition to protein and peptides soil
may contain other OrgN compounds such as amino acids (reported
values of amino acids fall in the range of 0.04e24 mg N g
1 soil;
Lipson and Nsholm, 2001), polyamines (concentrations below
2 nmol g
1 soil; Young and Chen, 1997), nucleic acids and nitrogen
bases (concentrations of purines and pyrimidines ranged from 21 to
138 mg g
1 of dry soil; Schulten and Schnitzer, 1998), aminosugars
(5e6% of soil OrgN; Lipson and Nsholm, 2001) and chitin
(concentrations from 1 to 30 mg g
1 soil; Pacovsky and
Bethlenfalvay, 1982). These compounds enter the soil as plant,
microbial and animal residues.
The aim of this study was to determine whether tannins
(commercial TA and CT extracted from Norway spruce needles)
have the ability to precipitate different OrgN compounds. As
nitrogen-containing compounds we used twenty amino acids,
seven peptides, two proteins, three polyamines, ve nitrogen bases
and aminosugars (N-acetyl-D-glucosamine, chitin and chitosan). As
pH affects precipitation of proteins, we also measured precipitation
of OrgN compounds across various pH (from pH 3 to 9).
2. Materials and methods
2.1. Organic nitrogen compounds, tannins and buffers used
A list of the OrgN compounds and their chemical structures are
presented in Table 1, and in Supplementary data. All these reagents
and tannic acid (TA) were purchased from Sigma. Commercial TA is
a mixture of compounds, mainly polygalloylglucose, with average
Mw of 1701.2 Da, and molecular formula C76H52O46. Condensed
tannins were extracted from Norway spruce (Picea abies (L.) Karst)
needles, which were taken representatively from three replicate
spruce plots of a tree species experiment in Kivalo, northern
Finland (described in detail by Smolander and Kitunen, 2002) in
March 2007. Undamaged green needles were dried. Extraction and
fractionation was done as described by Fierer et al. (2001), modied
by Kanerva et al. (2006). This method provides four fractions of
tannins. Fractions number 1, 2, 3 contain CT but also monomers of
CT and even compounds different from tannins. However, fraction
number 4 has the highest purity in comparison with others and
does not have monomers of CT (see Kanerva et al., 2006). Therefore
we decided to use only fraction number 4, and we characterized it
using method as described in Karonen et al. (2004) with liquid
chromatography (with reversed-phase and normal-phase columns)
coupled with mass spectrometry (Thermo-Electron, Finnigan LXQ).
Our study revealed that CTs of spruce from fraction number 4 were
built up mainly from procyanidin units (no prodelphinidin units
were found), mainly from tetramers, pentamers and hexamers as
well as higher polymers (Mw 1154e1826 Da, and also higher).
Condensed tannins were dissolved in water (1 mg ml
1), mixed
and, to remove undissolved particles (11.8% by mass of whole
material), centrifuged (5000g, 15 min) before use.
Most OrgN compounds were dissolved in pure water. However,
compounds with poor solubility in water, namely aspartic acid,
asparagine, glutamic acid, tryptophane, insulin, proteins and chitosan were dissolved in buffers (described below). Insulin was
dissolved in buffer (pH 3 and 3.5), but for pH 4 and 4.5 insulin was
rst dissolved in the acidic ingredient of the buffer and alkaline
compound of buffer was then added drop by drop (as described in
Lens, 1945). Such a narrow range of pH was used because of the

629

inability of insulin to dissolve at higher pH. Chitosan was dissolved

in buffers (pH from 3 to 5.5) using the method of Shepherd et al.
(1997), in higher pH chitosan cannot be dissolved (Shepherd
et al., 1997). Briey, chitosan was placed in a test-tube with
buffer; the suspension was mixed and allowed to hydrate for
30 min at room temperature, then placed in a boiling water bath for
20 min, mixed and cooled to room temperature.
To examine the effect of pH on precipitation, three different
buffers were used: formic acideammonium hydroxide (3e3.5 pH),
acetic acidetriethanoloamine (4e6 pH) and ammonium hydroxideeformic acid (7e9 pH).
In our study, the amount of Org N and tannin precipitated out of
solution was studied after mixing tannins with OrgN compounds.
Thus precipitation was dened as loss of the compound from the
supernatant following centrifugation of this mixture (tannins with
OrgN compounds), relative to a control which received only tannin
or OrgN. All precipitation studies were conducted in 3 replicates.
2.2. Determination of amount of precipitated TA or CT
by organic N-containing compounds
The concentrations of tannins were measured on the basis of
their reaction with FeCl3 (Hagerman and Butler, 1978), forming
a colored ironephenolate complex, which is determined spectrophotometrically. Tests with all of the OrgN compounds used in this
study in the absence of tannins did not give colored reaction with
FeCl3, demonstrating that this method was specic to the tannins.
OrgN compound (0.2 ml in water, initial concentrations as
shown in the gures) and TA (0.5 ml, initial concentration
10 mg ml
1, in water) were added to buffer (0.5 ml, 0.2 M, pH 3e9).
For CT studies, 0.1 ml of OrgN compound, and CT (0.25 ml,
1 mg ml
1 initial concentration, in water) were added to buffer
(0.25 ml, 0.2 M, pH 3e9). To OrgN compounds dissolved already in
buffer (see Section 2.1), only tannins were added, keeping the same
nal concentrations of all reagents as in the above description. After
30 min of mixing in a planar shaker at room temperature, samples
were centrifuged (5000g, 15 min). In studies with TA, from the
supernatant 0.02 ml of liquid was taken and mixed with triethanoloamine (1 ml, 5% v/v, in water), and later with FeCl3 (0.05 ml,
0.01 M dissolved in 0.01 N HCl); the same procedure was used for
CT studies, but 0.2 ml of supernatant was mixed with 0.5 ml triethanoloamine and 0.05 ml FeCl3. After 20 min, absorbance at
510 nm was read. In the case of the controls, instead of OrgNcontaining solutions, ultrapure water was used. The results are
given as % loss of initial amount of TA or CT. Standard curves were
prepared separately for TA and CT, ranging from 0.1 mg ml
1 to
5 mg ml
1 and from 0.05 mg ml
1 to 1 mg ml
1, respectively.
In the case of chitin, which is almost insoluble (Hu et al., 2007),
and nitrogen bases (insoluble in low and neutral pH), powdered,
solid form was used. Nitrogen bases were used in amount from
10 mg to 100 mg; chitin was used for CT studies in the range
5e50 mg, and in TA studies in the range of 5e200 mg. To that
material, TA (1 ml, 5 mg ml
1, in water) or CT (0.5 ml, 0.5 mg ml
1,
in water) was added. In order to measure the impact of pH on
precipitation, 25 mg and 10 mg (chitin) or 50 mg (nitrogen bases)
was mixed with buffer (0.5 ml, 0.2 M, pH 3e9) and 0.5 ml of TA
(10 mg ml
1, in water); in the case of CT studies, only 0.25 ml of
buffer and 0.25 ml CT (1 mg ml
1, in water) were used. The decrease
in tannin concentrations was measured as above. In the case of
controls, no OrgN compound was added.
2.3. Determination of the amount of insulin precipitated by tannins
To measure insulin concentration, LC-MS and the method
described by Ho et al. (2008) were used, with modications. To

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B. Adamczyk et al. / Soil Biology & Biochemistry 43 (2011) 628e637

Table 1
Organic nitrogen compounds used.
Compound

Mw, Da

pI

Molecular formula

N content, %

Additional information (e.g. functional groups)

Amino acids
Alanine
Arginine
Asparagine
Aspartic acid
Cysteine
Glutamic acid
Glutamine
Glycine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenyl-alanine
Proline
Serine
Threonine

89
174
132
133
121
147
146
75
155
131
131
146
149
165
115
105
119

6.0
11.1
5.4
2.7
5.0
3.2
5.6
5.9
7.4
5.9
5.9
9.5
5.7
5.4
6.3
5.6
5.6

C3H7NO2
C6H14N4O2
C4H8N2O3
C4H7NO4
C3H7NO2S
C5H9NO4
C5H10N2O3
C2H5NO2
C6H9N3O2
C6H13NO2
C6H13NO2
C6H14N2O2
C5H11NO2S
C9H11NO2
C5H9NO2
C3H7NO3
C4H9NO3

15.73
32.18
21.21
10.52
11.57
9.52
19.18
18.67
27.09
10.68
10.68
19.17
9.39
8.48
12.17
13.33
11.76

Tryptophan
Tyrosine

204
181

5.8
5.6

C11H12N2O2
C9H11NO3

13.72
7.73

Valine

117

5.9

C5H11NO2

11.96

1 amine group, 1 methyl group, 1 carboxyl group

4 amine groups, 1 carboxyl group
1 amide groups, 1 amine group, 1 carboxyl group
1 amine group, 2 carboxyl groups
1 amine group, 1 carboxyl groups, 1 thiol group
1 amine group, 2 carboxyl groups
1 amide group, 1 amine group, 1 carboxyl group
1 amine group, 1 carboxyl group
imidazol group, 1 amine group, 1 carboxyl group
1 amine group, 1 carboxyl group, 1 methyl group
1 amine group, 1 carboxyl group, 1 methyl group
2 amine group, 1 carboxyl group
1 amine group, 1 carboxyl group, 1 thiol group
1 amine group, 1 carboxyl group, 1 phenyl group
1 carboxylic group, 1 pyrrolidine group
1 amine group, 1 carboxyl group, 1 hydroxyl group
1 amine group, 1 carboxyl group, 1 hydroxyl group,
1 methyl group
1 amine group, 1 carboxyl group, 1 indole group
1 amine group, 1 carboxyl group, 1 phenyl group,
1 hydroxyl group
1 amine group, 1 carboxyl group, 2 methyl groups

Peptides
Alanyl-alanine

160

6.1

C6H12N2O3

17.50

Glycyl-glycyl-glycine
Glycyl-glutamic acid
Glycyl-phenyalanine

189
204
222

6.1
3.8
6.1

C6H11N3O4
C7H12N2O5
C11H14N2O3

22.22
13.72
12.61

Insulin
Oxidized glutathione

5733
613

5.6
3.8

C254H377N65O75S6
C20H32N6O12S2

15.87
13.70

Reduced glutathione

307

3.8

C10H17N3O6S

13.68

Proteins
Bovine serum albumin (BSA)
Rubisco, from spinach

66430
557000

4.7
4.2

607 amino acids

8 large and 8 small subunits

15.50
15.46

26 Arg residue per molecule

256 Arg residue per molecule

Polyamines
Putrescine
Spermidine
Spermine

88
145
202

C4H12N2
C7H19N3
C10H26N4

31.81
28.96
27.72

2 amine group
3 amine group
4 amine group

Nitrogen bases (used as solid)

Adenine
Cytosine
Guanine
Uracil
Thymine

135
111
151
112
126

C5H5N5
C4H5N3O
C5H5N5O
C4H4N2O2
C5H6N2O

51.85
37.86
46.35
25.00
22.22

1
1
1
2
1

Aminosugars
Chitin, (used as solid)
Chitosan, 75% deacetylated
N-acetyl-D-glucosamine

(203)n 400000
(161)n
221

(C8H13NO5)n
(C6H11NO4)n
C8H15NO6

(6.89)n
(8.69)n
6.33

2 amide group, 4 hydroxylic group, 2 methyl group

1 amine group, 2 hydroxyl group
1 amide group, 4 hydroxyl groups, 1 methyl group

avoid high molarity of buffer from the supernatant, samples were

diluted with water 10 times before measurements. Briey, 0.01 ml
aliquots were injected into a reversed-phase column (Phenomenex
Proteo, 100  2.0 mm, 4 mm C12, 90 A), and eluted with a linear
gradient of A e (0.1% v/v, formic acid in water:acetonitrile 98:2) and
B (0.1% v/v, formic acid in water:acetonitrile 2:98); ow rate was
0.15 ml min
1. Elution started with 100% A, in 10 min e 10% A,
14 min e 100% A, 15 min e 100% A. The mass spectrometer was
operated in positive-ion mode, spray needle voltage 5 kV, capillary
temp 300  C. For detection, ion chromatogram was used. The
external standard of commercial insulin (prepared as studied
sample, but without tannins) was used.

0
1
0
0
0
1
1
0
4
0
2

Arg residue, 1 amide group, 1 amine group,

carboxyl group, 2 methyl group
Arg residue, 2 amide group, 1 amine group, 1 carboxyl group
Arg residue, 1 amide group, 1 amine group, 2 carboxyl group
Arg residue, 1 amide group, 1 amine group,
carboxyl group, 1 phenyl group
Arg residue
Arg residue, 2 amine group, 4 amide group,
carboxylic group
Arg residue, 1 amine group, 1 thiol group,
amide group, 2 carboxylic group

amine group, 4 N in heterocyclic rings

amine group, 1 ketone group, 2 N in heterocyclic ring
amine group, 1 ketone group, 4 N in heterocyclic ring
ketone group, 2 N in heterocyclic ring
methyl group, 2 ketone group, 2 N in heterocyclic ring

2.4. Determination of amount of protein precipitated by tannins

To avoid problems with measurements of protein concentration
in mixtures containing tannins (described in Kilkowski and Gross,
1999), protein concentration was measured according to Bradford
(1976) but with modications by Owusu-Apenten (2002); 1 ml of
supernatant was mixed with trichloroacetic acid (0.25 ml, 72%,
w/v). In the case of CT, only 0.2 ml of supernatant was taken and
added to 0.05 ml of 72% trichloroacetic acid. After 1 h of incubation
at 2  C and centrifugation (12,000g, 15 min) the supernatant was
removed and the precipitate dissolved in NaOH (1 M, 0.1 ml). Then
3 ml of Bradford reagent was added and the absorbance was

B. Adamczyk et al. / Soil Biology & Biochemistry 43 (2011) 628e637

measured at 595 nm. Standard curves were prepared separately for

BSA and Rubisco.
2.5. Determination of the amount of polyamines precipitated
by tannins
Concentrations of putrescine, spermidine, spermine (initial
concentration 50 mM) in the supernatant were measured as
described by Sanchez-Lopez et al. (2009) with LC-MSeMS
(Thermo-Electron, Finnigan LXQ), with some modications. To
avoid high molarity of buffer from the supernatant, samples were
diluted with water 10 times. Samples were then centrifuged
(10000g, 5 min), and 0.9 ml of supernatant was mixed with heptauorobutyric acid (0.1 ml of 250 mM in water). After centrifugation (10000g, 5 min), 0.7 ml of supernatant was put into an LC
bottle; 0.01 ml aliquots were injected into a reversed-phase column
(Phenomenex Luna, 100  2.0 mm, 5 mm C18, 100 A). Polyamines
were eluted with a gradient of methanol containing propionic acid
(1%, v/v) and water with heptauorobutyric (1 mM) acid with
propionic acid (1%, v/v). The gradient started with 10:90 and linearly reached 90:10 in 9 min; for another 1.5 min it was kept at 90:10
and then returned to initial conditions in 3 min; it was then kept at
10:90 for 4.5 min for equilibration (summary 18 min). The ow rate
was 0.15 ml min
1. The mass spectrometer was operated in positive-ion mode, spray needle voltage 5 kV, capillary temperature
was 300  C. For detection, an ion chromatogram was used. External
standards of commercial polyamines (prepared like the studied
sample, but without tannins) were used.

631

concentration of arginine (200 mM) caused signicantly greater

precipitation of TA compared with lower concentrations of arginine
(100 and 50 mM). For CT studies, detectable precipitation was
found at pH 5.5e6 (Fig. 1B). Maximally, only 15% of tannins was
precipitated with arginine. It is possible that higher arginine andor
tannin concentrations would cause higher precipitation but,
to keep pH on a stable level, we could not increase initial
concentrations.
3.2. Peptideetannin precipitation
Of all the peptides studied, only insulin showed an ability to
form precipitates with TA and CT (Fig. 2AeC). At the pH range
studied, there was always precipitation, but the largest amounts of
TA or CT in the insulinetannins precipitates were obtained at pH 4
and 4.5. However, for lower insulin concentrations (20 and 50 mM)
precipitation of TA was on the same low level nevertheless pH
value. The amount of tannins (CT and TA) and the amount of insulin
precipitated across pH was signicantly correlated (r 0.89,
P 0.00, n 12). Higher pH was not studied, because of the
inability of insulin to dissolve at higher pH in the conditions used
here.
3.3. BSAetannin precipitation

To determine the differences between means of values, one-way

ANOVA was used, followed by Tukeys test. If needed, the results
were transformed to fulll the assumptions of the analysis of
variance. If there were fewer than 3 groups of results, t-test was
used. P < 0.05 was considered as statistically signicant. To study
correlation, the Pearson test was used. For all statistical analyses we
used Statistica (Statsoft, Inc.).

Precipitation of BSA by tannins was dependent on pH as well as

concentrations of BSA and tannins (Fig. 3AeB). Amount of precipitated BSA and TA or CT was usually signicantly highest at a pH
4.5e5.5. Amount of tannins in precipitate was correlated across pH
with amount of BSA in precipitate (for CTeBSA r 0.97, P 0.00;
for TAeBSA r 0.98, P 0.00, n 30).
Higher concentrations of TA (5 mg ml
1, 10 mg ml
1) were also
used; patterns of precipitation of TA and BSA were still similar,
showing low level of precipitation at pH 3 but high precipitation at
higher pH, and again low at pH 9 (data not shown). Lower
concentrations of BSA (50 mM, 20 mM, 10 mM) were also used, but
the pattern of precipitation with 10 mg ml
1 TA was the same,
merely showing a lower level of precipitated TA (data not shown).

3. Results

3.4. Rubiscoetannin precipitation

3.1. Amino acidetannin precipitation

At a pH range of 3e5.5 the amounts of precipitated TA and

Rubisco showed a tendency to decrease with an increase of pH
(Fig. 4A); differences between Rubisco amounts in precipitates
were mainly not statistically signicant, however, amount of TA in
precipitate was the highest at pH 3. Amounts of Rubisco and CT in
precipitates depended only slightly on pH, usually not showing

2.6. Statistical analysis

Of all 20 amino acids, precipitation with tannins was observed

only for arginine. Precipitation was dependent on pH as well as
arginine concentration. Precipitation occurred only at pH 4.5e7,
with maximum at 5e5.5 pH (Fig. 1A). At pH 5.5, higher

Fig. 1. Arginineetannin precipitation at different pH, expressed as percent of initial tannin amounts. A) Percentage of tannic acid (10 mg ml
1 initial concentration) precipitated by
arginine in different concentrations, B) Percentage of condensed tannins (1 mg ml
1 initial concentration) precipitated by 100 mM arginine. Values are means of 3
replicates  standard deviation.

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B. Adamczyk et al. / Soil Biology & Biochemistry 43 (2011) 628e637

Fig. 2. Insulinetannin precipitation at different pH, expressed as percent of initial compound amounts. A) Percentage of tannic acid (10 mg ml
1 initial concentration) precipitated
by insulin in different concentrations. B) Percentage of condensed tannins (1 mg ml
1 initial concentration) precipitated by 0.1 mM insulin. C) Percentage of insulin (0.1 mM initial
concentration) precipitated by 10 mg ml
1 TA or 1 mg ml
1 CT (initial concentrations). Values are means of 3 replicates  standard deviation.

statistically signicant differences between values (Fig. 4B). There

was a signicant positive correlation between concentration of
precipitated Rubisco and precipitated TA across pH (r 0.62,
P 0.01, n 30).
3.5. Polyamineetannin precipitation

detected for thymine in comparison with other nitrogen bases,

which all showed higher results (Fig. 6B). Higher amount of N base
added caused higher precipitation with both TA and CT. Differences
in pH conditions had no clear inuence on the amount of precipitated TA, except the low values for cytosine and adenine at the
highest pH of 9 (Fig. 6C).

All the polyamines studied formed precipitates with both

tannins in a pH dependent manner (Fig. 5AeD); pH below about 4.5
inhibited precipitation (Fig. 5AeB). The amounts of precipitated
polyamines and the amount of precipitated TA or CT showed
positive correlation across pH (e.g. spermidineeTA r 0.85,
P < 0.01; putrescineeCT r 0.97, P < 0.00, n 3).
Concentrations of polyamines other than 50 mM were also used
(100 mM, 20 mM, 10 mM); but the pattern of precipitation was
similar, merely showing lower results for lower concentrations of
polyamines and higher results for 100 mM concentrations (data not
shown). The only exception was precipitation of 100 mM spermine
by TA, here amounts of precipitated TA were high (75e80%),
regardless of pH value (data not shown).

N-acetyl-D-glucosamine did not form a precipitate with either

TA or CT. Its polymer, chitin, precipitated with TA and CT, even if
only 5 mg of chitin had been used (Fig. 7AeB). In conditions
without adjustment of pH, higher amounts of chitin added
increased precipitation of TA and CT. In general, pH did not affect
precipitation signicantly (Fig. 7B).
Chitosan (deacetylated chitin) also formed a precipitate with TA
in a pH- and concentration-dependent manner, showing the largest
amount of precipitated TA at the highest pH studied (5e5.5) (Fig. 8).

3.6. Nitrogen baseetannin precipitation

4.1. Precipitation of organic N compounds with tannins

In conditions without adjustment of pH, for thymine and uracil

the amount of precipitated TA was always close to detection limit,
higher for cytosine and the highest for 100 mg of adenine and
guanine (Fig. 6A). The lowest amount of precipitated CT was

Of all organic N compounds studied, precipitation with tannins

(TA and CT) was observed for arginine as the only amino acid
studied, insulin as the only peptide studied, both proteins (BSA and
Rubisco), all polyamines, nitrogen bases, chitin and chitosan. In the

3.7. Aminosugaretannin precipitation

4. Discussion

Fig. 3. Bovine serum albuminetannin precipitation at different pH, expressed as percent of initial compound amounts. A) Percentage of precipitated tannic acid and BSA. B)
Percentage of precipitated condensed tannins and BSA. Bovine serum albumin was used in initial concentration of 25 mM, tannins in 1 mg ml
1 initial concentration. Values are
means of 3 replicates  standard deviation.

B. Adamczyk et al. / Soil Biology & Biochemistry 43 (2011) 628e637

633

Fig. 4. Rubiscoetannin precipitation at different pH, expressed as percent of initial amount of compounds. A) Percentage of precipitated tannic acid and Rubisco. B) Percentage of
precipitated condensed tannins and Rubisco. Rubisco was used in initial concentration of 1 mM, tannins in 1 mg ml
1 initial concentration. Values are means of 3
replicates  standard deviation. Rubisco e D-ribulose 1,5-diphosphate carboxylase.

case of these interactions, pH and concentration of compounds

were important factors inuencing the amount of precipitated
compounds. The amount of precipitated OrgN compound and the
amount of precipitated tannins showed similar patterns and there
was usually a high correlation between them across pH. Qualitatively, reactivity of OrgN compounds to CT and TA was similar, with
one exception; CT precipitated with all the nitrogen bases studied,
whereas TA precipitated mainly with guanine, adenine and cytosine. Not all compounds could be dissolved; nitrogen bases and
chitin were used in solid form. For these compounds only the loss of
tannin from solution was measured.
It would be tempting to compare results of different OrgN
compounds. However, it cannot be done directly, because we used

different initial concentrations of both tannins and OrgN

compounds; some compounds were used in dissolved form, some of
them in solid form, which can to some extent inuence the results,
but not substantially. We summarized our study by calculating,
how much orgN could the same amount of TA and CT precipitate.
When different pH conditions had an effect, we chose the highest
precipitation value. In case of studies with undissolved OrgN, we
took into calculations results for 10 mg of OrgN compound and
calculated as described above. This comparison (Fig. 9) showed that
1 mg of CT always precipitated more OrgN compounds than 1 mg of
TA. This is in agreement with studies of Mutabaruka et al. (2007)
who found that ratio in precipitates of BSA and quebracho tannin
(CT) or TA was 4:1 and 3:2, respectively. Also Kraus et al. (2003b)

Fig. 5. Polyamineetannin precipitation at different pH, expressed as percent of initial amount of compounds. A) Percentage of tannic acid precipitated by 50 mM (initial
concentration) putrescine, spermine or spermidine. B) Percentage of condensed tannins precipitated by 50 mM putrescine, spermine or spermidine. Percentage of polyamines
precipitated by C) Tannic acid. D) Condensed tannins. Tannins were used in 10 mg ml
1 initial concentration (TA) or 1 mg ml
1 concentration (CT). Values are means of 3
replicates  standard deviation.

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B. Adamczyk et al. / Soil Biology & Biochemistry 43 (2011) 628e637

Fig. 6. Nitrogen baseetannin precipitation, expressed as percent of initial amount of tannins. A) Percentage of tannic acid (10 mg ml
1 initial concentration) precipitated by
different amounts of nitrogen bases without pH adjustment. B) Percentage of condensed tannins (1 mg ml
1 initial concentration) precipitated by different amounts of nitrogen
bases without pH adjustment. C) Percentage of tannic acid (10 mg ml
1 initial concentration) precipitated by 50 mg of nitrogen bases at different pH. Values are means of 3
replicates  standard deviation.

found the same ratio between BSA and TA. Fig. 9B shows also that
high amounts of N base eN or polyamine eN are needed to form
a complex with tannins. Probably these compounds seldom occur in
boreal forest soil in such concentrations in relation to tannin
concentration. May be formation of these complexes, although
possible, is not very relevant in boreal forest soils.
In our study, among all 20 amino acids only arginine formed
precipitate with tannins. Precipitation of arginine by CT-rich
extracts of many plant species was already demonstrated by Mole
and Waterman (1987), who also showed precipitation of histidine
but only by extracts from bracken (P. aquilinum). Moreover,
Hagerman and Butler (1981) showed that afnities of alanine,
glycine and proline for CT are at least a thousand-fold lower
than those of proteins and large polymers. We did not observe

precipitation of these amino acids. Our studies cannot be directly

compared with Hagerman and Butler (1981) because they used
a mixture of tannins, BSA and other compounds which were
inhibiting BSA precipitation. Precipitation of arginine in our study
may have arisen from the fact that this amino acid is able to form
multiple hydrogen bonds (Borders et al., 1994). Hydrogen bonds are
important in the formation of complexes with tannins, as was
shown for proteinetannin complexes (Kraus et al., 2003a). In our
study, precipitation was observed at pH 4e7, in which amino acids
have protonated amine group (NH
3 ) (at higher pH there is no
protonation) and tannins have negative charge (at lower pH
tannins possess charge close to zero) (An and Dultz, 2007), which
makes them reactive towards themselves and explain precipitation
observed in our study.

Fig. 7. Chitinetannin precipitation, expressed as percent of initial amount of tannins. A) Percentage of tannic acid (10 mg ml
1 initial concentration) or condensed tannins
(1 mg ml
1 initial concentration) precipitated by different amounts of chitin without pH adjustment. B) Percentage of tannic acid and condensed tannins precipitated by 10 mg or
25 mg of chitin at different pH. Values are means of 3 replicates  standard deviation.

B. Adamczyk et al. / Soil Biology & Biochemistry 43 (2011) 628e637

Fig. 8. Chitosanetannic acid precipitation at different pH, expressed as percent of

initial amount of tannic acid. Percentage of tannic acid (10 mg ml
1 initial concentration) precipitated by different concentrations of chitosan. Values are means of 3
replicates  standard deviation.

From all the peptides studied, only insulin precipitated with

tannins. One explanation could be the lack of arginine in the rest of
the peptides; however, out of a total of 51 amino acid residues
insulin contains only one arginine residue. Insulin, which has
higher molecular mass (5.7 kDa) than other peptides studied
(<0.7 kDa), has more functional groups (not only arginine-derived)
which can potentially interact with tannins. Our results are in
agreement with Hagerman and Butler (1981), who showed in
competitive studies that peptides with fewer than six residues
interacted very weakly with CT. Yan and Bennick (1995) showed
that tannins precipitated peptides, histatins, having arginine in the
amino acid sequence and a molecular mass above 3 kDa.
The proteins used in our study, Rubisco and BSA (molecular
mass of 557 and 66 kDa, respectively), have many functional
groups, also arginine-derived, with potential to interact with
tannins. Therefore it was not surprising that these proteins
precipitated with TA and CT. Such precipitation of proteins has been
described previously for BSA (e.g. Hagerman and Butler, 1978) and
Rubisco (e.g. McAllister et al., 2005). Precipitation of proteins is
included in the denition of tannins (Kraus et al., 2003a). The
highest precipitation of BSA was obtained at pH close to the
isoelectric point (pH 4.7), which is in accordance with the results of
other studies (e.g. Hagerman and Butler, 1981). The reason for
increased precipitation of proteins by tannins at pH close to their
isoelectric point is decreased electrostatic repulsion of protein
molecules (Hagerman and Butler, 1978).

635

Polyamines formed precipitates with TA and CT mainly at pH

higher than 4.5, which can be explained by positive charge of
polyamines (polyamines possess positive charge under a wide range
of pH; Ma et al., 2010), and negative charge of tannins at pH higher
than 4.5 (An and Dultz, 2007). Positively charged polyamines can
precipitate negatively charged compounds, as it was proven for
protein precipitation by polyamines at high pH (Ma et al., 2010).
Precipitation of polyamines by tannins seems to be inuenced also
by polyamine structure; the amounts of precipitated tannins and
polyamines were highest for spermine (Mw 202), intermediate for
spermidine (Mw 146) and lowest for putrescine (Mw 88).
Nitrogen bases also differed in their reactivity to tannins, which
can be explained by structural differences. Adenine, guanine and
cytosine all possess an amine group outside heterocyclic ring and
reacted strongly with both TA and CT. On the contrary, uracil
and thymine have N only in a heterocyclic ring and they have lower
molecular mass, which could cause lower reactivity to tannins.
N-acetyl-D-glucosamine, having one amide group, did not show
precipitation by tannins, whereas its polymer chitin and chitosan
(deacetylated chitin) showed substantial precipitation with TA and
CT. It was not surprising that chitosan is able to precipitate tannins;
it has been shown that chitosan can x many molecules such as
pesticides, proteins, dyes and heavy metals (Rhazi et al., 2002). The
reactivity of chitosan towards tannins is probably connected with
presence of amine groups (Ritthidej et al., 2002). Higher precipitation of chitosan at highest pH studied could be caused by
decreased solubility of chitosan at higher pH (Gyliene et al., 2003).
In addition to different reactivity of different OrgN compounds
towards tannins, tannins have also been shown to vary in their
structure and reactivity (Kraus et al., 2003a). Various tannins may
potentially provide different results to those reported here.
4.2. Ecological signicance of the interaction between
tannins and OrgN compounds
Precipitation of several kinds of organic N compounds,
described in this paper, may inuence soil-decomposition
processes. According to the theory presented by Northup et al.
(1995), soil proteinetannin complexes can decrease N leaching
and may act as a source of N, which is recovered by the mycorrhizal
symbionts of certain plant species. As our study revealed that
proteins are not the only OrgN compounds that can be precipitated
by tannins, perhaps this theory should be extended also to include
other OrgN compounds. Tannins decrease protein-N losses by
joining them into relatively recalcitrant complexes (Kraus et al.,

Fig. 9. Comparison of ability of condensed tannins and tannic acid to precipitate different OrgN compounds, presented as mg of OrgN precipitated with 1 mg of tannins (TA or CT).
A) Results for OrgN compounds which were used as dissolved. When pH had an effect, we chose the highest precipitation value. B) Results for OrgN compounds which were used in
solid state; calculations were made here on the basis of results for 10 mg of OrgN compound. Values are means of 3 replicates.

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B. Adamczyk et al. / Soil Biology & Biochemistry 43 (2011) 628e637

2003a). Tannin-protein complexes are very difcult to degrade

enzymatically (Adamczyk et al., 2009b), but release of N from such
recalcitrant complexes has been shown (Mutabaruka et al., 2007;
Joanisse et al., 2009). However, the degradability of tannineOrgN
complexes other than tannineprotein complexes is unknown.
All OrgN compounds studied in our paper may be a direct or
indirect source of nitrogen for plants. Tetraselmis striata cells
showed an ability to use some nitrogen bases (Ricketts, 1990).
Chitin can be used by mycorrhizal fungi as an N source (Leake and
Read, 1990). Bagni et al. (1978) showed that polyamines can act as
an N source for explants taken from dormant tubers of Helianthus
tuberosus. Moreover, certain plants have been shown to take up
amino acids (Nsholm et al., 2009), short peptides (PaungfooLonhienne et al., 2009), and even have access to a protein by the
use of root-secreted proteases (Godlewski and Adamczyk, 2007;
Adamczyk et al., 2009a).
As mentioned above, precipitation by tannins is inuenced by
pH. Typical agricultural soil has relatively high pH (sometimes even
more than 8, as found by e.g. Floch et al., 2009), but boreal forest
soils usually have low pH (less than 4, Tamminen and Derome,
2005). Moreover, the amount and structure of tannins in various
soils also differ (Kraus et al., 2003a). Thus the formation and role of
complexes between organic N-containing compounds and tannins
are probably strongly inuenced by the type of soil.
The high ability of Rubisco to precipitation by tannins at low pH
could have a great impact on N cycling in boreal forest soils, as
Rubisco is a very abundant protein in plant leaves and needles (Parry
et al., 2003). It is not known if such complexes of Rubisco with
tannins are stable in acidic soil, but if they are stable, they may act as
a source of slowly sequestered nitrogen (according to the abovementioned theory of Northup et al., 1995). On the other hand, at low
pH the precipitation of polyamines in our study was not impressive.
However, perhaps part of the polyamines introduced into the soil
may remain there as precipitates. Moreover, as polyamines can act
as phytohormones (Young and Chen, 1997), the phenomenon of
precipitation by tannins can also inuence polyamine phytohormonal activity, and perhaps increase their life-time in the soil, in
this way causing prolongation of their hormonal inuence.
The possibility of precipitation of chitosan with TA may have an
inuence on numerous applications of this compound. For
example, to remove heavy metals, Guo et al. (2009) added chitosan
to the soil. Possible precipitation of chitosan by tannins in the soil
can affect removal of heavy metals by decreasing formation of
heavy metal complexes with chitosan or by stabilization of chitosan
in the soil without inuencing the reactivity towards heavy metals.
Moreover, tannins can also act as metal chelators (Kraal et al.,
2006), which makes the problem of metal removal from the soil
by chitosan more complicated.
In conclusion, not only proteins/peptides can form precipitates
with tannins, but arginine, polyamines, nitrogen bases, chitin and
chitosan also showed this ability. Precipitation with tannins seems
to be inuenced by molecular mass and number/type of functional
groups of OrgN compounds. Concentrations of OrgN compounds
and tannins, and pH of solution are important factors affecting the
interactions of tannins with OrgN compounds. Precipitation of
OrgN by tannins may potentially affect N cycling. As precipitation of
an OrgN compound is not restricted to proteins, studies concerning
biochemical reactions of mixtures that include these N compounds
and tannins should take into account possible formation of
precipitates between these substances.
Acknowledgements
We are grateful to Dr Joann von Weissenberg for checking the
English language of this paper and to Matti Vihakas for superb

fractionation of CT. In addition, we thank Anneli Rautiainen for

helping with laboratory work. This study was funded by the
Academy of Finland.

Appendix. Supplementary data

Supplementary data related to this article can be found at doi:
10.1016/j.soilbio.2010.11.034.

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