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J Appl Phycol (2010) 22:7177

DOI 10.1007/s10811-009-9429-6

The allelopathy and allelopathic mechanism of phenolic acids


on toxic Microcystis aeruginosa
Ting-Ting Zhang & Chun-Yan Zheng & Wei Hu &
Wen-Wu Xu & Hao-Fen Wang

Received: 2 November 2008 / Revised and accepted: 27 February 2009 / Published online: 20 March 2009
# Springer Science + Business Media B.V. 2009

Abstract Cyanobacterial contamination of water has been


a serious problem in recent years. Thus, the effective
control of undesired cyanobacteria has become an urgent
issue. We studied therefore the effects of -coumaric acid
and vanillic acid on toxic Microcystis aeruginosa and the
allelopathic mechanisms. The results showed that the growth
of toxic M. aeruginosa was significantly inhibited by coumaric acid and vanillic acid, with an EC50 of 0.260.07
and 0.340.05 mmol L1, respectively. Our data also
demonstrated that both -coumaric acid and vanillic acid
triggered the generation of superoxide anion radicals (O2).
The O2 might induce a lipid peroxidation which may
change cell membrane penetrability, thereby leading to the
eventual death of M. aeruginosa. Our current studies
further provide evidence that some phenolic acids such as
-coumaric acid and vanillic acid may be a potential
effective solution for aquatic management.
Keywords Phenolic acids . Toxic Microcystis aeruginosa .
Allelopathy . Allelopathic mechanism

Introduction
Frequent outbreaks of cyanobacterial blooms in eutrophic
water bodies have been a great threat to water resources in
recent years. To efficiently manage the aquatic environT.-T. Zhang (*) : C.-Y. Zheng : W. Hu : W.-W. Xu : H.-F. Wang
The Key Laboratory of Biotic Environment and Ecological Safety
in Anhui Province, College of Life Science,
Anhui Normal University,
Wuhu 241000, China
e-mail: cyhztt@mail.ahnu.edu.cn

ment, effective measures should be taken to control


undesired cyanobacterial growth in an aquatic ecosystem.
Over the past 20 years, there has been significant interest
for the growth inhibition of cyanobacteria by allelochemicals released by aquatic macrophytes (Gross et al. 2003;
Zhang et al. 2007a) and it has been demonstrated that many
aquatic macrophytes secrete allelopathic substances against
cyanobacteria (Van et al. 2002). Some of these bioactive
substances have been extracted and purified successfully.
Among all allelochemicals, phenolic acids have been
studied most widely. As early as in 1984, Rice (1984)
pointed out that phenolic acids were water-soluble and with
a property of plant growth inhibition. Gross et al. (1996)
showed that Myriophyllum spicatum releases the allelopathic compound hydrolyzable tannin (eugeniin), and its
derivatives, ellagic and gallic acids. Nakai et al. (2001)
found the pyrogallic acid, (+)-catechin, from M. spicatum.
All these phenolic acids have been shown to significantly
inhibit the growth of Microcystis aeruginosa. Zhang et al.
(2007b) demonstrated that the ferulic acid and hydroxybenzoic acid inhibit the growth of Anabaena flosaquae and Chlorella pyrenoidosa. Taken together, these
findings point to the possibility of cyanobacteria control by
using appropriate allelochemicals.
Vanillic acid (4-hydroxy-3-methoxy benzoic acid) and coumaric acid ((E)-3-(4-hydroxyphenyl)-2-propenoic acid)
have been found in a variety of plants and have
demonstrated a potent allelopathic inhibition on other plant
growth (Blum 1996; Inderjit et al. 2002; Djurdjevic et al.
2004). In order to further study the allelopathic inhibition of
phenolic acids and to look for cyanobacteria control agents,
we studied the effect of vanillic acid and -coumaric acid
on toxic M. aeruginosa and explored their possible
mechanisms.

72

Preliminary experiments
-Coumaric acid and vanillic acid were dissolved in BG-11
medium. The -coumaric acid or vanillic acid solution was
inoculated with 10 mL M. aeruginosa in exponential growth
in sterilized 50 mL flasks. The final concentration of either
-coumaric acid or vanillic acid in the M. aeruginosa/
phenolic acid mixture was adjusted to 0, 0.2, 0.4, 0.6, 0.8,
and 1.0 mmol L1 and the final M. aeruginosa density used
was approximately 105106 cells mL1. The M. aeruginosa/
phenolic acid mixture was cultivated for 8 days under the
above conditions. The concentrations that showed the
optimal inhibitory effect were determined by calculating
how many M. aeruginosa cells survived after the 8-day
culture (i.e., the solution in which the minimal number of
cells survived is the one with the most potent effect).
M. aeruginosa growth inhibition experiments
Based on the preliminary experiments, M. aeruginosa
growth inhibition experiments were conducted. Briefly, a
200-mL culture solution of M. aeruginosa in exponential
growth phase was filled into a 1,000-mL sterilized flask,
followed by the addition of -coumaric acid or vanillic acid
(pre-dissolved in BG-11 medium and filtered through
0.22 m autoclaved membrane filters). The final volume
of the solution was 800 mL and the final concentration of
-coumaric acid or vanillic acid in the mixture solution was
adjusted to 0, 0.1, 0.3, 0.5, and 0.7 mmol L1 using BG-11
medium. The final M. aeruginosa density was 2.19105
cells mL1. Each concentration of the two phenol acids had
15 replicates: five were used for M. aeruginosa cell
number, chlorophyll a content, electrical conductivity
(EC), soluble proteins, and nucleic acid assays; five for
superoxide anion radical (O2) determination; and the other
five for cellular lipid peroxidation (LPO) assays. All the
flasks were cultivated for 8 days under the conditions
described above, and all the assays were tested once every
other day.

Im t 100  mc  mt =mc

m ln Nn  ln N0 =tn  t0

where c represents the mean control specific growth rate,


t the mean specific growth rate for the test concentration t,
N0 the number of cells per milliliter at time t0 (beginning of
the test), and Nn the measured number of cells per milliliter
at time tn (Xian et al. 2006).
The effective concentration for 50% reduction in M.
aeruginosa growth (EC50) was calculated by probit
analyses (Statistical software SPSS13.0 Inc., USA) using
the relative inhibition of the specific growth rate and the
nominal concentration of phenolic acid (Rassoulzadegan
and Akyurtlakli 2002; Xian et al. 2006).
The content of chlorophyll a was evaluated using high
performance liquid chromatography (HPLC; Millie et al.

12
Cell density (105 cellmL-1)

Toxic Microcystis aeruginosa FACHB-942 was provided


by the Institute of Hydrobiology, Chinese Academy of
Sciences; -coumaric acid and vanillic acid were purchased
from Sigma (USA); other chemicals were the products of
Beijing Chemical Plant (Beijing, China).
Prior to the initiation of the experiments, M. aeruginosa
was grown in 250 mL sterilized flasks with 100 mL BG-11
medium and placed in an incubator, an irradiance of
60 mol photons m2 s1 at 251C and lightdark regime
14:10 h for 7 days. All flasks were shaken four times
everyday (Zhang et al. 2008).

The number of M. aeruginosa was counted using a


hemocytometer. The relative inhibition (It) of the specific
growth rate at certain concentration of phenolic acid was
calculated based on the formula:

A. -Coumaric acid

10
8
6
4
2
0

10

4
6
Time (days)

10

14

B. Vanillic acid
Cell density ( 105 cellmL-1)

Materials and methods

J Appl Phycol (2010) 22:7177

12
10
8
6
4
2
0

2
0

0.1

0.3

0.5

0.7

Fig. 1 a, b Effect of phenol acid (millimoles per liter) on the growth


of M. aeruginosa (bar = SD, n=5)

J Appl Phycol (2010) 22:7177

1.2
1

*
*

0.8

0.6

0.4

0.2
0

Chlorophyll a contents (mgg-1 FW)

A. -Coumaric acid

1.4

B. Vanillic acid
1.2
1

0.8

0.6

0.4
0.2
0

4
Time (days)

2
0

0.1

0.3

6
0.5

group was used to evaluate the level of nucleotides and


nucleic acids released.
Total soluble protein was determined using the Bradford
method using an assay kit (Nanjing Bioengineering
Institute, Nanjing, China).
O2 was monitored using a spectrophotometric method
(Xiao et al. 1999). The reaction of hydroxylamine and O2
produces NO2, which was developed by adding sulfanilic
acid and -naphthylamine. The absorption at 530 nm of the
sample quantitatively correlates with the O2 amount. In
brief, 0.5 g cells were homogenized with ice-cold phosphate buffered saline (PBS) (6 mL, 65 mM, pH 7.8),
filtered with filter paper, and centrifuged at 3,400 rpm for
10 min at 4C. A 1 mL supernatant was added to 0.9 mL
PBS (65 mM, pH 7.8) and 0.1 mL hydroxylamine
hydrochloride (10 mM), followed by incubation in a 25C
water bath for 20 min. After incubation, 0.5 mL of the
mixture was added to 0.5 mL sulfanilic acid (17 mM) and
0.5 mL -naphthylamine (17 mM), incubated for 20 min at
25C, and extracted with an equal volume of butanol. The
samples were settled for 10 min at room temperature and
the butanol phase was taken for measurement at 530 nm.
LPO was assayed according to the method of Cavas
(2005). M. aeruginosa cells (0.5 g) were homogenized in
2 mL ice-cold 1.15% KCl. The homogenate was placed in a

3
2.5

0.7

Fig. 2 a, b Effects of phenol acid (millimoles per liter) on the


chlorophyll a contents of M. aeruginosa (bar = SD, n=5). *p<0.05,
compared with the control

EC.ratio

Chlorophyll a contents (mgg-1 FW)

1.4

73

A. -Coumaric acid

*
*

2
1.5

* **

**
*

0.1

0.3

*
**

**

1
0.5
0

0.5

0.7

2.5
2

B. Vanillic acid

*
*

EC.ratio

2002; Liu et al. 2003). The M. aeruginosa sample solution


(100200 mL) was centrifuged at 3,500g for 20 min, 0.1 g
M. aeruginosa was ground and extracted with 90% acetone,
and the supernatant removed into a 10-mL volumetric flask
and diluted with 90% acetone to the volume, then the
solution was injected into the HPLC (Agilent 1100 Series,
Agilent Technologies, USA) through a 0.2-m micropore
filter. Based on the standard regression curve established
using a chlorophyll a standard series, the contents of
chlorophyll a were determined.
EC was analyzed using a portable conductivity meter
(Cole-Parmer Instrument Company, USA). Twenty milliliters of each sample was taken from the culture flask once
every other day and was immediately filtered with a 0.22-m
Millipore. The supernatant was used for analysis.
Nucleic acids and nucleotides were detected using a
UVVis spectrophotometer (Sun et al. 2004). The supernatant was prepared as described above. The optical density
(OD) of the supernatant was measured at 260 nm. The
OD260 ratio between the experimental group and the control

* *

1.5

* *

**

**

1
0.5
0
0

0.1
0.3
0.5
Concentration (mmolL-1)

2d

4d

6d

0.7

8d

Fig. 3 a, b Effect of phenol acid on the release of M. aeruginosa


intracellular electrolyte. Data are expressed as the ratio between the
EC of the experimental group and the control group (bar = SD, n=5).
*p<0.05, compared with the control

Protein content ratio

J Appl Phycol (2010) 22:7177

2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

Protein content ratio

74

1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

A. -Coumaric acid
*

*
**

0.1

0.3

B. Vanillic acid
*

*
*

* **

0.5

* *
*

0.7

0.1
0.3
0.5
Concentration (mmolL-1)
2d

4d

*
* *

6d

*
* *
*

0.7

8d

Fig. 4 a, b Effect of phenol acid on the release of M. aeruginosa cellular


proteins. Data are expressed as the ratio between the protein content of
the experimental group and the control group (bar = SD, n=5). *p<
0.05, compared with the control

EC50 of -coumaric acid for M. aeruginosa was 0.26


0.07 mmol L1 on day 8. At the lowest concentration
(0.1 mmol L1), -coumaric acid inhibited the growth of M.
aeruginosa from day 6, whereas the maximal inhibition
was found on day 8 at a concentration of 0.7 mmol L1,
with a 100% inhibitory ratio.
Vanillic acid was also found to inhibit the growth of M.
aeruginosa (p<0.05) in a concentration-dependent manner
except for the group at the lowest concentration (0.1 mmol L1;
Fig. 1b). The EC50 was 0.340.05 mmol L1 and the
maximal inhibition (87.890.5%) was found at 0.7 mmol L1
on day 8. However, in contrast to the control group, vanillic
acid at 0.1 mmol L1 showed a slight growth-promoting
effect on M. aeruginosa (p<0.05).
The inhibitory effect of -coumaric acid and vanillic acid
at the concentrations of 0.5 and 0.7 mmol L1 was also
shown by the decrease of chlorophyll a contents from the
fourth day (Fig. 2a), compared with the controls (p<0.05),
while, at 0.1 and 0.3 mmol L1, the effect of the two
phenolic acids on chlorophyll a contents did not have a
significant effect.
Figure 3a shows an effect of the treatments with coumaric acid on the EC ratio between the treated groups
and the control groups, with the effect mainly related to the
concentration of the phenol acid and the acting time. The
2.5

A. -Coumaric acid
*

All experiments were quintuplicate. Data are expressed as


mean SD and analyzed by SPSS 13.0. One-way ANOVA
followed by LSD test was used to test for the differences
between individual means. The 0.05 level or less was
selected as the point of minimum statistical significance in
every comparison.

Results
As shown in Fig. 1a, -coumaric acid inhibited the growth
of M. aeruginosa at all concentrations tested (p<0.05). The

*
1.5

* *

* *

**

0.5

0.7

1
0.5
0
0

OD260 ratio

Statistical analysis

OD260 ratio

glass tube to which was added a solution containing 0.2 mL


8.1% SDS, 1.5 mL 20% acetic acid solution adjusted to
pH 3.5 with NaOH, 1.5 mL 0.8% aqueous solution of 2thiobarbituric acid, and 0.7 mL distilled water. The mixture
was heated at 95C for 60 min. After cooling with tap water,
1 mL distilled water and 5 mL of n-butanol/pyridine (15:1, v/v)
were added and shaken vigorously. After centrifugation at
4,000 rpm for 10 min, the n-butanol layer was taken for
absorbance measurement at 532 nm (Yagi 1994).

2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

0.1

0.3

B. Vanillic acid

*
*

* *

* *

0.1
0.3
0.5
Concentration (mmolL-1)
2d
4d 6d 8d

0.7

Fig. 5 a, b Effect of phenol acid on the release of M. aeruginosa


intracellular nucleic acids. Data are expressed as the ratio between the
OD260 of the experimental group and the control group (bar = SD, n=5).
*p<0.05, compared with the control

J Appl Phycol (2010) 22:7177

75

OD530 ratio

3.5

A. -Coumaric acid
*

3
2.5
2

* *

1.5
1
0.5
0
0

0.1

0.3

0.5

0.7

3.5

OD530 ratio

B. Vanillic acid

*
*

2.5

*
*

1.5

1
0.5
0
0

0.1

0.3

0.5

0.7

Concentration (mmolL-1)
2d

4d

6d

8d

Fig. 6 a, b Effect of phenol acid on M. aeruginosa O2 generation.


Data are expressed as the ratio between the OD530 value of the
experimental group and the control group (bar = SD, n=5). *p<0.05,
compared with the control

0.05), the OD260 values, except for the values of the second
day.
As can be seen in Fig. 6a, the OD530 ratio between the coumaric acid group and the control group increased
significantly starting on day 2, with peak ratio of 3.41
0.22 on day 6 at the concentration of 0.7 mmol L1; due to
the almost complete death of the M. aeruginosa, the
superoxide anion radical could not be tested on day 8.
Vanillic acid also induced an increase in OD530 ratio, except
for the lowest concentration group (0.1 mmol L1), with a
peak ratio of 2.670.28 on day 8 at 0.7 mmol L1 (Fig. 6b).
All groups treated with phenolic acid, except for the lowest
vanillic acid concentration group (0.1 mmol L1), were
significantly different from the control groups (p<0.05).
Figure 7 shows that the malondialdehyde (MDA)
contents of the control group (test for lipid peroxidation)
remained relatively steady, but the phenolic acid treatment
groups exhibited a significant increase in LPO level at
concentrations from 0.1 to 0.7 mmol L1. For -coumaric
acid, the maximal MDA value was 11.6 mol g1 fresh
weight (FW) on day 6 at the concentration of 0.7 mmol L1,
which was 5.03 times that for the control groups; for
vanillic acid, the maximal MDA value was 10.5 mol g1FW
on day 8 at the concentration of 0.7 mmol L1, which was
4.82 times that for the control groups. Almost all groups
treated with phenolic acid, except for the groups treated with

14
MDA (umolg-1 FW)

12

A. -Coumaric acid

**
*

10
8
**

*
*

*
*
*

*
*

4
2
0
0

0.1

0.3

0.5

0.7

14
MDA (umol g-1 FW)

maximal effect of -coumaric acid was found at 0.7 mmol L1


with an EC ratio of 2.270.16 on day 8. Vanillic acid also
elevated the EC values in a concentration-dependent manner,
with an EC ratio of 1.960.25 at 0.7 mmol L1 on day 8
(Fig. 3b). The results of all the treated groups, either with coumaric acid or with vanillic acid, were significantly
different from the control groups (p<0.05).
Upon addition of -coumaric acid, we also noticed a
concentration-dependent increase in the contents of
soluble protein and OD260 (nucleic acid) starting on
day 2 (Figs. 4a and 5a). At lower concentrations (0.1
0.3 mmol L1), -coumaric acid induced a gradual
elevation in soluble protein contents and OD260 values,
whereas at higher concentrations (0.50.7 mmol L1), it
caused a quick increase, with a maximal ratio of 1.630.21
for protein content and 1.780.17 for OD260 between the
treated groups and the control groups on day 8. Similarly,
vanillic acid also caused a gradual increase in soluble
protein contents and OD260 values in a concentrationdependent manner, with a maximal ratio of 1.530.09 for
protein contents and 1.660.18 for OD260 between the
treated groups and the control groups on day 8 (Figs. 4b
and 5b). The soluble protein contents of the treated groups
were significantly different from the control groups (p<

12

B. Vanillic acid

*
*

10
8

**
*

*
*

4
2
0
0

0.1
0.3
0.5
Concentration, mmolL-1
0

2d

4d

6d

0.7

8d

Fig. 7 a, b Effect of phenol acid on the MDA contents of M.


aeruginosa (bar = SD, n=5). *p<0.05, compared with the control

76

vanillic acid on day 2, were significantly different from the


control groups (p<0.05).

Discussion
Phenolic acids in different ecosystems have been widely
studied and invariably identified as the allelopathic or
phytotoxic substances (Rice 1984; Inderjit et al. 2002; Park
et al. 2006). Our studies further show that -coumaric acid
and vanillic acid both inhibited Microcystis aeruginosa
growth, and that -coumaric acid was more potent than
vanillic acid. Moreover, vanillic acid also exhibited a
biphasic effect on the growth of M. aeruginosa, that is, a
growth-promoting effect at low concentration and an
inhibiting effect at high concentration. This phenomenon
is consistent with previous reports (He and Ye 1999; Nakai
et al. 1996). However, we found that the reduction of M.
aeruginosa cell number was not in keeping with the
decrease of chlorophyll a content, and this implies that
the main mechanism of the two phenol acids on M.
aeruginosa growth inhibition might not be by inhibiting
chlorophyll a synthesis but by some other mechanisms.
The cell membrane is the target for many antimicrobial
agents (Denyer and Stewart 1998; Sun et al. 2004) and the
release of intracellular components is a good indicator of
membrane integrity (Chen and Cooper 2002). Small ions
such as potassium and phosphate tend to leach out first,
which contribute to EC, then large molecules such as DNA,
RNA, and other materials leak out (Chen and Cooper 2002;
Sun et al. 2004). Our experiment results showed an increase
of EC, OD260, and protein content, which indicated the
leakage and release of electrolytes, nucleic acids, and proteins
from the cyanobacteria upon the addition of either coumaric acid or vanillic acid, and suggested that the cellular
membrane was irreversibly damaged by these compounds
resulting in the eventual disintegration of M. aeruginosa.
These results agree well with our previous findings that hydroxybenzoic acid inhibited the growth of M. aeruginosa
by destroying the cell wall structure (Zhang et al. 2008).
Einhellig (1995) proposed membrane-associated disturbances as the common mode of action of phenolic acids. He
suggested that after their entry through the membrane,
phenolic acids may cause depolarization of the cell
membrane and change ion influx and retention. Recent
studies suggest that some allelochemicals, which act as an
environmental stress, can increase the production of O2 in
cells (Vardi et al. 2002; Zhang et al. 2008). O2 is the
precursor of active free radicals that have the potential for
reacting with biological macromolecules and thereby
inducing cell damage. O2 also plays an important role in
the formation of other reactive oxygen compounds including hydrogen peroxide, hydroxyl radical, and singlet

J Appl Phycol (2010) 22:7177

oxygen, which induce oxidative damage in lipids, proteins,


and DNA. Our former report (Zhang et al. 2007b) indicated
that antioxidant enzyme (superoxide dismutase) activities
and specific activities of A. flos-aquae were enhanced at the
beginning of -hydroxybenzoic acid and ferulic acid
oxidative stress conditions. However, this increase did not
match the production of reactive oxygen species, thus
resulting in increased lipid peroxidation in A. flos-aquae. In
the present study, we found that -coumaric acid or vanillic
acid resulted in an increase in O2 and MDA contents in
M. aeruginosa cells, which suggests that these two phenol
acids did trigger O2 radical generation.
Further analysis of the relationship between O2 and
all the other results above showed a significant correlation
between O2 and LPO (r=0.937, p<0.05). The O2 level
correlates well with EC (r=0.895, p<0.05). In addition,
the O2 level is correlated soluble protein (r=0.884, p<
0.05). Finally, O2 and nucleic acids in M. aeruginosa
culture solution are also closely correlated (r=0.893, p<
0.05). Altogether, these results suggest that the O2 radical
generation under the conditions of phenol acid oxidative
stress might be the most important event contributing to the
molecular mechanisms underlying the action of the two
phenolic acids on cyanobacteria. To verify the reliability of
the experimental results, all the experiments were carried out
twice, and good repeatability was obtained.
Based on the above, we conclude that -coumaric acid and
vanillic acid can inhibit the growth of M. aeruginosa in a
dose-dependant manner, with -coumaric acid being more
potent with EC50s for -coumaric acid and vanillic acid of
0.260.07 and 0.340.05 mmol L1 on day 8, respectively.
The allelopathic mechanism was involved in the O2
generation in M. aeruginosa, which may induce lipid
peroxidation, thereby causing cell wall damage and release
of intracellular components. Our study suggests that these
two phenol acids might be used in the development of
potential effective biological algicides.
Acknowledgments This study was supported by the National
Natural Science Foundation of China (No.30870429) and the
Provincial Key Laboratory of Biotic Environment and Ecological
Safety in Anhui.

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