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A B S T R A C T
Harmful algal blooms are one of the greatest challenges when
preserving water sources, especially when involving cyanobacteria such
as Microcystis aeruginosa. Finding remediation possibilities is needed,
and one of them has been the use of macrophytes such as the species
Myriophyllum, which have presented allelopathic mechanisms of
phytoplankton control. Thus, this work aimed to evaluate the inhibition
of M. aeruginosa cell growth in a co-exposure with Myriophyllum
aquaticum and the influence on microcystin-LR concentration.
The experiments were carried out using a culture of M. aeruginosa
(1x106 cells mL-1) in a co-exposure with M. aquaticum for seven days.
The inhibitory effects were investigated by counting the cells; the
effects on photosynthetic pigments were measured and microcystin-
LR was quantified in the culture medium on the last experimental
day. To evaluate the possible effects of competition for nutrients and
space, the concentration of total orthophosphate was quantified
and treatment with plastic plants was used. The experiments with
Myriophyllum aquaticum achieved the total inhibition of M. aeruginosa
growth and a significant reduction of the photosynthetic pigments
(> 98%). Additionally, we observed a reduction of microcystin-LR
concentration (79%) in the tests with macrophytes when compared
to the control. Competition for space and nutrients was not observed,
demonstrating that the effects on M. aeruginosa were caused by
aquatic macrophyte presence. These results may indicate that M.
aquaticum causes inhibitory effects on cyanobacteria growth by
allelopathic effects and removes microcystin-LR.
Keywords: allelopathy; cyanobacteria; cyanotoxins; submerged aquatic
macrophytes; nature-based solutions.
R E S U M O
Florações de cianoabactérias são consideradas um dos maiores desafios
na preservação de fontes hídricas, especialmente quando estão
presentes espécies como Microcystis aeruginosa. A descoberta de
alternativas de remediação faz-se necessária, e uma delas é o uso de
macrófitas aquáticas, como as espécies do gênero Myriophyllum, que
apresentam atividades alelopáticas para o controle fitoplanctônico.
Diante disso, este trabalho teve como objetivos avaliar a inibição do
crescimento de células de M. aeruginosa em uma coexposição com
Myriophyllum aquaticum e avaliar a remoção de microcistina-LR.
Os experimentos foram conduzidos com cultivos de M. aeruginosa (1x106
células mL-1) em coexposição com M. aquaticum por sete dias. Os efeitos
inibitórios foram investigados por contagem celular. Os efeitos nos
pigmentos fotossintéticos foram mensurados, além da quantificação de
microcistina-LR no último dia experimental. Para avaliar possíveis efeitos
de competição por nutrientes e espaço, realizou-se a quantificação da
concentração de ortofosfato total e utilizou-se um tratamento com
planta de plástico. Os experimentos com M. aquaticum apresentaram
inibição total do crescimento de M. aeruginosa e redução significativa
na concentração dos pigmentos fotossintéticos (> 98%). Além disso,
foi constatada redução na concentração de microcistina-LR (79%) nos
testes com macrófitas quando comparadas ao grupo controle. Não foi
observada competição por espaço e nutrientes, o que demonstra
que os efeitos sobre M. aeruginosa foram causados pela presença da
macrófita aquática. Dessa forma, estes resultados podem demonstrar
que M. aquaticum gerou a inibição no crescimento de cianobactérias
por efeitos alelopáticos, além de remover a microcistina-LR das águas.
Palavras-chave: alelopatia; cianobactérias; cianotoxinas; macrófitas
aquáticas submersas; soluções baseadas na natureza.
Use of Myriophyllum aquaticum to inhibit Microcystis aeruginosa
growth and remove microcystin-LR
Uso de Myriophyllum aquaticum para inibir o crescimento de Microcystis aeruginosa e remover microcistina-LR
Rafael Shinji Akiyama Kitamura1 , Ana Roberta Soares da Silva2 , Thomaz Aurelio Pagioro3 , Lúcia Regina Rocha Martins3
1Universidade Federal do Paraná – Curitiba (PR), Brazil.
2Instituto Água e Terra – Curitiba (PR), Brazil.
3Universidade Tecnológica Federal do Paraná – Curitiba (PR), Brazil.
Correspondence address: Rafael Shinji Akiyama Kitamura – Laboratório de Fisiologia de Plantas sob Estresse, Departamento de Botânica,
Setor de Ciências Biológicas, Universidade Federal do Paraná – Avenida Coronel Francisco H. dos Santos, 100 – Centro Politécnico Jardim das
Américas – Caixa postal: 19031 – CEP: 81531-980 – Curitiba (PR), Brazil.
E-mail: rafaelkitamura@hotmail.com
Conflicts of interest: the authors declare no conflicts of interest.
Funding: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) – Finance Code 001.
Received on: 01/10/2022. Accepted on: 07/26/2022.
https://doi.org/10.5327/Z2176-94781309
Revista Brasileira de Ciências Ambientais
Brazilian Journal of Environmental Sciences
Revista Brasileira de Ciências Ambientais
Brazilian Journal of Environmental Sciences
ISSN 2176-9478
Volume 56, Number 1, March 2021
This is an open access article distributed under the terms of the Creative Commons license.
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Use of Myriophyllum aquaticum to inhibit Microcystis aeruginosa growth and remove microcystin-LR
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Introduction
Anthropic activities such as agriculture and industry have been
responsible for negative impacts on aquatic ecosystems (Ramya et al.,
2020; Kakade et al., 2021). Increased atmospheric carbon dioxide, the
incorrect use and disposal of fertilizers, and the dumping of domestic
sewage have significantly contributed to the eutrophication of water
bodies (Ramya et al., 2020; Kakade et al., 2021). As a result, increased
harmful algal blooms, mainly of cyanobacteria, have been noted, be-
coming an emergent environmental concern (Touzet et al., 2016; Ka-
kade et al., 2021).
Harmful algal blooms cause negative impacts on environments,
and they negatively affect human health and activities such as aqua-
culture (Zohdi and Abbaspour, 2019; Tanvir et al., 2021). Cyanobacte-
ria bloom causes problems in aquatic ecosystems, including alteration
of the trophic chain structure and local functionality due to the deox-
ygenation of the water and disturbance in odor and color (Lu et al.,
2017). Also, the release of cyanotoxins during the bloom may decrease
ecosystem biodiversity because they can cause toxicity to non-target
organisms (Ramya et al., 2020; Munoz et al., 2021). Similarly, cyano-
toxins are also toxic to humans, and their occurrence in water may be-
come a public health problem (Ramya et al., 2020; Munoz et al., 2021).
Microcystis aeruginosa is one of the most common cyanobacteria that
establish blooms in reservoirs and other aquatic environments, degrad-
ing them mainly by producing microcystins (Li et al., 2016). These chem-
icals are hepatotoxic cyanotoxins that impact human and animal health
(Ramya et al., 2020), and they are associated with cancer, hepatic dys-
function, and acute episodes of intoxication (Kudela et al., 2015).
The ecological, aesthetic, and economic problems generated by cy-
anobacteria are commonly treated by applying conventional algaecides,
oxidative chemical compounds such as potassium permanganate, hydro-
gen peroxide, and anti-algae flocculants (Wang et al., 2017b; Torres et al.,
2020; Munoz et al., 2021). However, many of these compounds present
environmental toxicity and the potential to generate secondary pollut-
ants (Meng et al., 2015; Tazart et al., 2021). Therefore, the development
of economic and environmentally friendly technologies to control cya-
nobacteria blooms is needed, and the use of allelopathic strategies could
be a possibility (Chen and Guo, 2014; Tazart et al., 2021; Zhu et al., 2021).
Allelopathy consists of the positive or negative effect that an or-
ganism exerts on other organisms by releasing compounds (named
allelochemicals) into the environment (Li et al., 2021; Zhu et al., 2021)
Studies have shown that these substances may be a feasible alternative
to combat cyanobacteria blooms because they are biodegradable and,
depending on the concentration used, they do not present toxicity to
the environment (Meng et al., 2015; Li et al., 2021). By releasing al-
lelochemicals, it was observed that some submerged macrophytes in-
hibited the growth of cyanobacteria by allelopathy (Huang et al., 2020;
Tazart et al., 2021; Zhu et al., 2021). Moreover, these plant species are
important to maintain the quality of lakes since they compete with
phytoplankton for light and nutrients, reduce suspended sediments,
and contribute to water purification (Li et al., 2021). So, these tech-
niques can be considered an efficient nature-based solution (NBS) that
can corroborate to mitigate the impact of harmful algae blooms (Kita-
mura et al., 2021; Zhu et al., 2021).
Myriophyllum aquaticum (Vell.) Verdc is a submerged macrophyte
that presents fast growth in a eutrophic environment, phytoremediator
potential, and can regulate ecosystems by allelopathic effects (Cheng
et al., 2008). M. aquaticum was reported as a promising species to con-
trol cyanobacteria (Cheng et al., 2008; Wang et al., 2017a), mainly by
inhibiting cell growth. Thus, this work aimed to evaluate the inhibitory
activity of M. aquaticum on Microcystis aeruginosa through co-expo-
sure experiments, as well as to investigate the effects on photosynthetic
pigments and the impact on microcystin-LR concentration. The pres-
ent work hypothesizes that M. aquaticum can have effects on cyano-
bacteria cell growth, mainly by affecting photosynthetic pigments and
the capacity to reclaim microcystin-LR from the medium. In addition,
we hypothesized that inhibition processes are caused by allelopathic
mechanisms of macrophytes and these effects are independent of other
competition processes (space and nutrients, as orthophosphate).
Material and Methods
Culture of cyanobacteria and plants
The Microcystis aeruginosa strain (code BB005, isolated from
harmful bloom and provided by the Botany Department of Universi-
dade Federal de São Carlos, Brazil) was cultivated in the ASM-1 medi-
um (Gorham et al., 1964 with adaptations of Almeida et al., 2016) with
inoculum at an initial concentration of 1.6x105 cell mL-1. The cultures
were maintained at 25 ± 3°C under luminosity of 36.81 ± 2.58 μmol of
photons.s-1.m² -1and photoperiod of 12:12 h (light: dark).
The Myriophyllum aquaticum (12 ± 2 cm of height) plants were pur-
chased from specialized suppliers and maintained for two months in
an aquarium of 20 L (approximately 15 plants per aquarium) contain-
ing dechlorinated water and coarse gravel sediment. The plants were
disinfected and grown at 25°C under luminosity of 36.81 ± 2.58 μmol
of photons.s-1.m² -1 and photoperiod of 12:12 (light: dark).
Experimental conditions
The experiments were performed on flasks (1,000 mL) containing
1 L of ASM-1 sterile medium. Three types of treatments were defined:
M. aquaticum + M. aeruginosa (MMa), M. aeruginosa (Control), and
artificial plants + M. aeruginosa (AMa).
In each flask, the MMa and AMa treatments, two M. aquaticum
with 40 ± 2 cm in length of the submerged part and plastic plants, re-
spectively, were used. The length of natural and artificial plants was
standardized. In addition, the choice of artificial plants was based on
the morphology that was most similar to M. aquaticum. All the treat-
ments were inoculated with M. aeruginosa inoculum, corresponding
to an initial density of 1x106 cells mL-1. The systems were kept under
Kitamura, R.S.A. et al.
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luminosity and photoperiod at the same culture conditions described
early, for seven days. For each treatment, three replicates were used.
Cyanobacteria growth
M. aeruginosa growth was evaluated by cellular counting in a Neu-
bauer chamber, withdrawing aliquots (10 μL) every 24 hours. The inhibi-
tion rate (IR) was calculated according to Equation 1 (Cheng et al., 2008).
IR = [1 – N0 x N
-1) x 100 (1)
Where:
N = cell density in the MMa or AMa flask;
No= cell density in Control.
Orthophosphate analysis
The influence of this limiting nutrient on M. aeruginosa growth was
evaluated by the quantification of total orthophosphate by the method
of digestion using potassium persulfate (Valderrama, 1981). A calibra-
tion curve was prepared from monobasic potassium phosphate solu-
tions at concentrations of 0.025; 0.05; 0.1; 0.15; 0.2; 0.25; 0.5; 1; 2; 4,
and 8 mg mL-1 (linear regression: y = 0.0506x + 0.2213; r = 0.9819).
To compare the values of orthophosphate in a non-consumable system
and establish the influence of macrophyte alone, the analyses were also
carried out in flaks with an artificial plant (AP) and with M. aquaticum
only, without M. aeruginosa (M).
Photosynthetic pigments
Chlorophyll-α and phycobiliprotein concentrations were evaluated
after the period of exposure. For chlorophyll-a analysis, aliquots (6 mL)
were withdrawn from the samples, and they were centrifuged at 10,500
rpm (Hitachi) for 4 minutes. The supernatant was discarded, and 2 mL of
acetone 80% (v/v) was added to the cellular pellet, homogenized, and re-
frigerated in the dark for 24 hours to extract the pigments. The liquid was
homogenized and centrifuged again before spectrophotometric analysis
(UV-1800, Shimadzu) at 646 and 663 nm. The chlorophyll-α concentra-
tion was calculated by Equation 2 (Lichtenthaler and Wellburn, 1983).
Chlorophyll (mg L-1) = 12.21(A663) – 2.81(A646) (2)
Where:
A663 = absorbance at 633 nm;
A646 = absorbance at 646 nm.
The phycobiliprotein analysis was performed using aliquots (10
mL) of the liquid sample that were withdrawn, and two cycles of freez-
ing/thawing were carried out for cell lysis and release of pigments in
the medium. After that, the samples were centrifuged, and the super-
natant liquid was spectrophotometrically analyzed at 565 nm for phy-
coerythrin (PE), at 620 nm for phycocyanin (PC), and at 650 nm foro
allophycocyanin (APC). The quantification was performed according
to Equations 3 to 6 (Chapman and Kremer, 1988).
PC (mg mL-1) = [A620 − (0.72 x A650)] / 6.29 (3)
APC (mg mL-1) = [A650 − (0.191 x A620)] 5.79 (4)
PE (mg mL-1) = {A565−[(2.41 x PC) − (1.41 x APC)]}/13.02 (5)
Total Phycobiliproteins, TP (mg mL-1) = PC + APC + PE (6)
Where:
A620 = absorbance value at 620 nm;
A650 = absorbance value at 650 nm;
A665 = absorbance value at 665 nm.
Microcystin-LR analysis
The analyses were performed in a liquid chromatographic system
(Prominence, Shimadzu) equipped with a quaternary pump (LC-2AT),
degasser unit (DGU-20A), oven (CTO-20A), diode array detector (SPD-
M20A), and a controller unit (CBM-20A) operated by the LC Solutions
software. The separation was obtained on an analytical column with a
polymeric octadecyl stationary phase (Xterra, Waters, 150 x 3 mm d.i.,
3.4 μm of particle size). The quantification of microcystin-LR was con-
ducted at 238 nm by linear regression (y = 322.481 x – 32832; r = 0.9998) of
a validated method developed in our research group (Torres et al., 2020).
The samples (50 mL) were withdrawn on the last experimental day
and submitted to three cycles of freezing-thawing for the cellular dis-
ruption and release of their content in the liquid medium. Then, the
samples (40 mL) were centrifuged (8,000 rpm for 20 minutes at 10°C)
to remove cell debris, and the supernatant was used in the pre-concen-
tration procedure by solid-phase extraction (SPE). The C18 cartridges
(1,000 mg, Applied Separations) were conditioned with 10 mL of meth-
anol (HPLC grade, J.T. Baker) and 10 mL of ultrapure water (Mega UP,
Megapurity), followed by 10 mL of the sample. The cartridge was dried
for one minute. The cleanup was carried out by applying 10 mL of wa-
ter and the elution with 10 mL of methanol. The eluate was completely
evaporated and dissolved in 1 mL of water.
To evaluate whether the presence of M. aquaticum reduced the
concentration of microcystin-LR by inhibiting growth or it directly
affected the production of cyanotoxin, the concentration of microcys-
tin-LR per cell was calculated. For this, the Equation 7 was used:
(7)
Where:
MCLR = the concentration of microcystin-LR on the last experimen-
tal day;
cell No. = the cell concentration obtained on the last experimental day.
Use of Myriophyllum aquaticum to inhibit Microcystis aeruginosa growth and remove microcystin-LR
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Data analysis
Data were expressed as the mean of three independent replicates.
Data were submitted to Shapiro-Wilk normality, Levene’s test for ho-
mogeneity, and evaluated by one-way ANOVA. The means were then
compared by the Tukey test (P < 0.05). The factors which contribute
the most to the inhibition rate of M. aeruginosa were analyzed by the
Pearson correlation analysis with photosynthetic pigments and micro-
cystin-LR. The analyses were carried out with software R, version 3.5.0
(R Core Team, 2018), and a significant level p < 0.05 was considered.
Results and Discussion
Inhibitory effects of Myriophyllum aquaticum
on Microcystis aeruginosa growth
The presence of M. aquaticum significantly interfered with M. aeru-
ginosa cellular growth when compared to the Control during the entire
exposure duration (F (2,6) = 1225.9; p < 0.001, Figure 1A; Figure 2) with a
marked decline in cellular concentration from the third day. There was
no observed significant difference between the Control and the treat-
ments using artificial plants (AMa), demonstrating that there was no
influence of space competition in the observed effect. Expressing these
results in percent inhibition values, the rates obtained reached 100%
on the seventh experimental day for MMa (F(2,6) = 551.3404, p < 0.001,
Figure 1B). On the other hand, when considering AMa, no inhibition of
M. aeruginosa was observed. Qian et al. (2018) observed inhibitory ef-
fects on Microcystis aeruginosa growth in a co-culture with a submerged
macrophyte (Pontederia cordata), as observed in our work, in which we
reached 100% inhibitory effects. These results can corroborate the in-
fluence of submerged macrophytes to inhibit cyanobacteria cell growth.
The interference of light and space with the inhibition effects was
evaluated using artificial plants in independent flasks with the same
experimental conditions and no effects on cyanobacteria growth were
observed (Figures 1A and 2). The concentration of orthophosphate
was measured to verify the influence of nutrients on the observed ef-
fect. Ferreira et al. (2018) showed that orthophosphate is an essential
and limiting nutrient to cyanobacteria growth. However, we do not
see the nutritional limitation of plants on cyanobacteria growth since
they were not different between the Control group and M. aquaticum
(MMa) in orthophosphate concentrations (Figure 3). Moreover, there
was no correlation between total orthophosphate and the rate of cell
density growth (r = 0.3239; p = 0.3967).
Effects on photosynthetic pigments of Microcistys aeruginosa
Chlorophyll-a (Figure 4) and the accessory pigments (phycoeryth-
rin, phycocyanin, and allophycocyanin) were significantly reduced
(p < 0.0001, Table 1) in M. aeruginosa grown in the presence of mac-
rophytes. A strong correlation was observed between cellular growth
and pigments (r = 0.999; p < 0.0001), with decreasing cell growth be-
ing reflected in reduced pigment concentrations. No significant effects
(p > 0.05) of artificial plants were observed on M. aeruginosa pigments.
Wang et al. (2017a) observed that growth inhibition of M. aerugi-
nosa and Anabaena flos-aquae strains was associated with allelochem-
icals being released by M. aquaticum. Similarly, Cheng et al. (2008)
reported that M. aquaticum could secrete some allelochemicals in the
medium, which reduces M. aeruginosa growth. The principal effects of
allelochemicals secreted by M. aeruginosa were the inhibition of super-
oxide dismutase enzyme activity (leading to oxidative stress), in addition
to chlorophyll-α reduction, as also observed in our study (Figure 3).
The main bioactive substances reported in the Myriophyllum genus
are phenolic compounds (Zhu et al., 2010; Zhu et al., 2021). These mac-
rophytes can produce allelochemicals as (+)-Catechin, caffeic acid,
ellagic acid, gallic acid, nonanoic acid, pyrogallol, tellimagrandin II,
Control: only M. aeruginosa; AMa: artificial plants + M. aeruginosa; MMa: M. aquaticum + M. aeruginosa. The bars represent means ± SD of three replicates; *a sig-
nificant difference considering p < 0.05 by ANOVA followed by the Tukey test.
Figure 1 – Inhibition effect on Microcystis aeruginosa cellular growth during exposure to Myriophyllum aquaticum. (A): Effect on cell density; (B): percentual
inhibition.
Kitamura, R.S.A. et al.
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AMa: artificial plants + M. aeruginosa; MMa: M. aquaticum + M. aeruginosa;
AP: artificial plants; M: M. aquaticum.
Figure 2 – Representative image of the bioassay on the last experimental day.
AMa: artificial plants + M. aeruginosa; MMa: M. aquaticum + M. aeruginosa;
AP: artificial plants; M: M. aquaticum. The bars represent the means ± SD of
three replicates.
Figure 3 – Orthophosphate concentration in the culture medium on the last
experimental day.
Control: only M. aeruginosa; AMa: artificial plants + M. aeruginosa; MMa: ma-
crophytes + M. aeruginosa. The bars represent the means ± SD of three replica-
tes. Different letters indicated a significant difference considering p < 0.05 by the
ANOVA test followed by the Tukey test.
Figure 4 – Chlorophyll-α concentration after the exposure duration (7 days).
phenylpropanoid glucosides, and hydrolyzable tannins (Zhu et al.,
2021). Nakai et al. (2012) observed that M. spicatum can release five
polyphenols (catechin, eugeniin, gallic, pyrogallic, and ellagic acids)
and three fatty acids in culture media and these allelochemicals were
responsible for the inhibition of M. aeruginosa cell growth. The interac-
tive effects of polyphenols and fatty acids can contribute to the effective
allelopathic effects of this plant and increase the efficiency of cyano-
bacteria control (Gross et al., 1996). Leu et al. (2002) and Gross (2003)
also demonstrated that tellimagrandin II produced by Myriophyllum
spicatum has deleterious effects on cell growth and interferes with the
photosynthetic apparatus — which can be associated with decreased
photosynthetic pigments as observed in the present study (Table 1).
In cyanobacteria, the phycobilisomes concentrate the reactions
that convert solar energy into high-energy electrons in photosystem
II. They are composed of accessory pigments, mainly phycoerythrin,
phycocyanin, and allophycocyanin, as well as chlorophyll-α (McColl
2018). Interferences in these pigments can promote reduced photosyn-
thetic performance of cyanobacteria, which highlights the importance
of these analyses. Moreover, there is another piece of evidence of the
effects caused by the allelopathic substances released by macrophytes.
Table 1 – Concentration of accessory photosynthetic pigments of Microcystis
aeruginosa after a co-culture experiment (7 days) with Myriophyllum
aquaticum (n = 3)*.
Treatments
Parameters ANOVA
Phycocyanin
% of
reduction
Df F p
Control (a) 0.0251 ± 0.0014 c -
2 1,304.8 < 0.0001AMa (b) 0.0252 ± 0.0016 c -0.43
MMa (c) 0.0004 ± 0.00001a,b 98.05
Allophycocyanin
% of
reduction
Df F p
Control (a) 0.0341 ± 0.0017 c -
2 1,609.3 < 0.0001AMa (b) 0.0342 ± 0.0016 c -0.39
MMa (c) 0.0005 ± 0.00000 a,b 98.04
Phycoerythrin
% of
reduction
df F p
Control (a) 0.0139 ± 0.0007 c -
2 1,690.7 < 0.0001AMa (b) 0.0139 ± 0.0006 c -0.38
MMa (c) 0.0002 ± 0.00000 a,b 98.48
Total
phycobiliproteins
% of
reduction
df F p
Control (a) 0.0731 + 0.0035 c -
2 873.77 < 0.0001AMa (b) 0.0734 + 0.0035 c -0.40
MMa (c) 0.0012 + 0.00004 a,b 98.30
*Different letters indicate a significant difference (p < 0.05) by the ANOVA test
followed by the Tukey test; Control: only M. aeruginosa; AMa: artificial plants +
M. aeruginosa; MMa: M. aquaticum + M. aeruginosa.
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Table 2 – Total intracellular microcystin-LR and microcystin-LR produced per
cell concentration after the exposure duration (7 days; n = 3) of M. aeruginosa
to M. aquaticum*.
Treatments
Parameters ANOVA
Microcystin-LR
% of
reduction
Df F P
Total Microcystin-LR (μg.L-1)
Control (a) 0.073 ± 0.001c -
2 38.14 0.002AMa (b) 0.071 ± 0.01 c 3.329%
MMa (c) 0.015 ± 0.011a,b 78.990%
Microcystin-LR per cell (ng.L-1)
Control (a) 16.034 ± 0.552c -
AMa (b) 15.541 ± 2.808 c 2.710% 2 50.27 < 0.001
MMa (c) 0.015 ± 0.011a,b 99.904%
Different letters indicate a significant difference (p < 0.05) by the ANOVA test
followed by the Tukey test.
Cheng et al. (2008) used only the liquid media in which M. aquaticum
had been developed and observed similar effects of reduced photosyn-
thetic pigment concentrations.
Phenolic substances isolated from Myriophyllum spicatum, such
as tellimagrandin II, affected photosystem II when in contact with cy-
anobacteria, degrading accessory pigments and causing cell death by
inhibiting enzymes such as alkaline phosphatase (Leu et al., 2002).
Zhu et al. (2010), when applying catechins, gallic, and ellagic acids ex-
tracted from M. spicatum, verified the effect on photosystem II of M.
aeruginosa, reducing electron transduction. These phenolic compounds
can cause isolated and synergic effects, mainly pyrogallic and gallic acid.
The synergic effects can explain the macrophytes’ efficiency in inhib-
iting cyanobacteria growth, reducing photosynthetic concentrations
of the pigment, and affecting mainly non-photochemical quenching.
These parameters efficiently screen the effects of allelochemicals to in-
hibit cyanobacteria isolated or in combined modes (Huang et al., 2020).
Allelopathic application is a promising strategy for cyanobacteria
control, especially because this technique is inspired by natural phe-
nomena. The use of plants and isolated allelochemicals is recommend-
ed and studies show the effects as interfering with photosynthesis,
generating oxidative stress, and the possibility of causing other distur-
bances (Zhu et al., 2021).
Effects on the concentration of intracellular microcystin-LR
After seven days of exposure, intracellular microcystin-LR concen-
tration was significantly decreased in the growth media by the presence
of M. aquaticum (F
(2,6) = 38.14; p = 0.0002, Table 2). On the other hand,
it was not affected by the presence of artificial plants (F(2,6) = 38.19;
p > 0.05, Table 2). The same effects were observed for the concentration
of microcystin-LR produced per cell in the presence of M. aquaticum
(F(2,6) = 50.27; p < 0.0001, Table 2).
Besides inhibiting M. aeruginosa growth, total intracellular mi-
crocystin-LR removal (79%) was observed, as well as removal of the
microcystin-LR produced per cell (> 99%). In a review of the inter-
actions among compounds with allelopathic properties produced by
macrophytes and cyanobacteria, Mohamed (2017) explained that in
addition to controlling cell density, the macrophytes had the poten-
tial for phytoaccumulation and phytotransformation of cyanotoxins
with potential metabolization and reduced toxicity, indicating that
these species can be used to mitigate the impacts of harmful algal
blooms. Besides, it is known that methanolic extracts of M. aquat-
icum corroborate to reduce microcystin-LR concentration and M.
aeruginosa cell growth (Kitamura et al., 2021). In addition to the pos-
sible capacity of phytoremediation, the present results of microcys-
tin-LR produced per cell demonstrated that allelopathic mechanisms
could interfere in the production of cyanotoxins by cyanobacteria
and more studies should be conducted to understand the possible
mechanisms involved.
Pflugmacher et al. (2015) described the performance of a combina-
tion of live submerged macrophytes Ceratophyllum demersum, Elodea
canadenses, and Myriophyllum spicatum and they showed that micro-
cystins (LR, RR, and YR) were reduced from 67 to 85%. Calado et al.
(2019) observed that an experiment using Egeria densa, Ceratophyllum
demersum, and Myriophyllum aquaticum removed 100% of microcys-
tin-LR after three days. These studies contributed to the understanding
that microcystin-LR can be effectively removed and corroborated the
hypothesis that phytoremediation can occur in addition to allelopathic
effects, which makes the use of aquatic macrophytes in the control and
remediation of harmful algal blooms even more beneficial.
Finally, the present work indicated the allelopathic effects of Myrio-
phyllum aquaticum in inhibiting M. aeruginosa growth, causing effects
on the photosynthetic apparatus. It is efficient to reduce microcystin-LR
concentrations. These results can encourage the use of macrophytes in
water remediation treatments for harmful Microcystis blooms.
Conclusions
The inhibitory effect of Myriophyllum aquaticum on the cellular
growth of Microcystis aeruginosa was observed without interferences
on nutrients and space restriction, demonstrating that the observed in-
hibition did not occur by competition but by allelopathy mechanisms.
Concerning the reduction of photosynthetic pigments and microcys-
tin-LR (total and produced per cell), the results demonstrated that the
application of submerged macrophytes species is a powerful and prom-
ising tool to remediate harmful algal blooms and can be considered an
efficient nature-based solution.
Acknowledgements
The authors thank the Laboratory of Equipment and Environ-
mental Analyses of the Universidade Federal de Tecnologia do Paraná
(LAMEAA - UTFPR) for the chromatographic analysis.
Kitamura, R.S.A. et al.
440
RBCIAMB | v.57 | n.3 | Sep 2022 | 434-441 - ISSN 2176-9478
Contribution of authors:
KITAMURA, R. S. A.: Conceptualization; Formal Analysis; Investigation; Methodology; Validation; Project administration; Writing — Original Draft. SILVA,
A. R. S.: Formal Analysis; Writing — Original Draft. PAGIORO, T. A.: Supervision; Resources. MARTINS, L. R. R.: Supervision; Validation; Resources; Project
Administration; Writing — Original Draft.
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