J Appl Phycol (2009) 21:429–442
DOI 10.1007/s10811-008-9388-3
Effects of bacillamide and newly synthesized derivatives
on the growth of cyanobacteria and microalgae cultures
C. Churro & E. Alverca & F. Sam-Bento & S. Paulino &
V. C. Figueira & A. J. Bento & S. Prabhakar &
A. M. Lobo & A. J. Calado & P. Pereira
Received: 11 August 2008 / Revised and accepted: 14 October 2008 / Published online: 26 November 2008
# Springer Science + Business Media B.V. 2008
Abstract The antialgal activity of newly synthesized
bacillamides against several cyanobacteria and microalgae
isolates was screened using a rapid 96-well microplate
bioassay. Cultures were exposed to serial dilutions of each
bacillamide derivative (0–160 μg mL−1) in the microplate
wells and daily optical measurements were used to estimate
growth over a 216 h period. Inhibition values (%) were
calculated from the estimated growth curves and inhibitory
concentrations (IC50-216 h) were obtained from the
sigmoidal inhibition curves fitted by regression analysis.
The effects of bacillamides on cell morphology and
ultrastructure were also analysed by light and transmission
electron microscopy. In general, the toxic cyanobacteria
Microcystis aeruginosa, Aphanizomenon gracile, Anabaena
circinalis and Anabaenopsis circularis were much more
sensitive to bacillamides then the chlorophytes Ankistro-
desmus falcatus and Scenedesmus obliquus. However, clear
signs of morphological and ultrastructural changes induced
by bacillamide were observed on both cyanobacteria and
chlorophytes. Other cyanobacteria, namely the nostocalean
C. Churro : E. Alverca : F. Sam-Bento : S. Paulino :
P. Pereira (*)
Instituto Nacional de Saúde Dr Ricardo Jorge,
Av. Padre Cruz,
Lisbon 1649-016, Portugal
e-mail: paulo.pereira@insa.min-saude.pt
V. C. Figueira : A. J. Bento : S. Prabhakar : A. M. Lobo
Departamento de Química, REQUIMTE, Faculdade
de Ciências e Tecnologia da Universidade Nova de Lisboa,
Monte da Caparica 2829-516, Portugal
C. Churro : A. J. Calado
Departamento de Biologia, Universidade de Aveiro,
Campus Universitário, Santiago,
Aveiro 3810-193, Portugal
Nodularia spumigena and the oscillatorialeans Leptolyng-
bya sp. and Planktothrix rubescens, exhibit higher toler-
ances to bacillamides, similar to that shown by different
eukaryotic microalgae. Diatoms, on the other hand, proved
to be quite as sensitive to most bacillamides as the most
affected cyanobacteria. The properties of 5-iodo-Bacillamide
(algicide or algistatic) were further investigated. This
compound acted as an algistactic agent against eukaryotic
algae and, depending on its concentration, acted as either an
algicide or algistactic agent against most of the cyanobac-
teria tested. Although bacillamides cannot be considered as
broad spectrum cyanobacterial algicides, different bacilla-
mides might be of use in selectively controlling the growth
of particular species of cyanobacteria.
Keywords Algicide . Algistatic . Bacillamide .
Cyanobacteria . IC50
Introduction
Massive developments of phytoplankton in marine and
freshwater environments are a major concern from an
environmental and a human health perspective. Toxic
phytoplankton blooms are responsible for wildlife and
human health hazards, causing economic losses on fisher-
ies, aquaculture and recreational activities (Codd 1999;
Anderson et al. 2002; Hallegraeff 2003). In freshwater
systems, cyanobacteria are the most problematic bloom-
forming organisms. They may produce hepatotoxins
(microcystins, nodularins and cylindrospermopsins), neuro-
toxins (anatoxins and saxitoxins), cytotoxins and irritant
compounds [lipopolysaccharides (LPS)] (Codd 1999; Long
and Carmichael 2003; Apeldoorn et al. 2007). Some of
these toxins can also act as cancer promoters (Falconer et
430
al. 1999; Kuiper-Goodman et al. 1999). Cyanobacterial
blooms significantly deteriorate the quality of drinking
water and cause problems in water supply storage facilities,
increasing the cost of water treatment (Hrudey et al. 1999).
Much attention has been given in recent decades to
strategies for cyanobacteria bloom control and manage-
ment. These include various modes of artificial aeration
(Heo and Kim 2004), transport of hypolimnetic water, and
sediment dredging (Chorus and Mur 1999). Most of these
environmentally friendly treatments, while often being
effective, require time and expensive machinery to produce
satisfactory results.
Other approaches for bloom control rely on the use of
synthetic algicides. Chemical treatments with, for example,
potassium permanganate, chlorine, simazine, atrazine,
endothall, diquat, paraquat, diuron, and sodium hypochlo-
rite, and flocculants such as aluminium sulphate, copper
sulphate, copper chelates, ferric salts, and ferric aluminium
sulphate have been applied in lake waters. Most of those
compounds have been commonly used as algicides because
they are cheap and easy to apply (Hrudey et al. 1999;
Schrader et al. 1997, 2003). However, their efficiency is
sometimes unsatisfactory, because fish kills sometimes
occur following treatments and a variety of aquatic
organisms, including the whole phytoplankton community,
can be affected, causing significant water quality deteriora-
tion (Hrudey et al. 1999; Oliveira-Filho et al. 2004; Li and
Hu 2005). Furthermore, toxic by-product deposits may
accumulate in the sediments and repeated treatments may
induce the appearance of unpleasant resistant cyanobacteria
species (Garcia-Villada et al. 2004; Roussel et al. 2007).
Recently, much effort has been focused on the identifi-
cation of organisms capable of selective inhibition of
phytoplankton growth. These include submerged macro-
phytes (Körner and Nicklisch 2002; Mulderij et al. 2005;
Hilt et al. 2006; Erhard and Gross 2006), higher plants
(Zhou et al. 2007), macroalgae (Jeong et al. 2000; Nan et
al. 2004; Jin et al. 2005), cyanobacteria (Chauhan et al.
1992; Bagchi et al. 1993; Suikkanen et al. 2004) and other
microalgae (Wu et al. 1998), fungi (Safferman and Morris
1962; Redhead and Wright 1978), bacteria (Imai et al 1998;
Mayali and Doucette 2002; Jasti et al. 2005) and viruses
(Nagasaki et al. 1999; Baudoux and Brussard 2005).
However, opinions concerning the benefits resulting from
the introduction of new organisms in aquatic ecosystems
differ widely (Boyd and Massaut 1999; Verschuere et al.
2000). Addition of living organisms to natural environ-
ments or biomanipulation of parts of the food web can
disrupt the entire ecosystem with disastrous consequences
(Jeong et al. 2000; Verschuere et al. 2000).
Isolation of the inhibitory compounds produced by these
organisms can be a more effective and safe way to control
blooms, but this is not always an easy task. Rice straw and
J Appl Phycol (2009) 21:429–442
barley straw, for example, have proven to inhibit algal
growth in laboratory and field experiments (Everall and
Lees 1995; Park et al. 2006), but the isolation of the
inhibitory compounds from barley has been unsatisfactory,
since the inhibitory effect is likely to be a synergistic effect
of various compounds (Ball et al. 2001; Ferrier et al. 2005).
In some cases, the metabolites exhibiting growth-
inhibition activity towards phytoplankton organisms have
been discovered, their chemical structure elucidated and
biological activity tested. Examples include L-2-azetidine-
carboxylic acid (AZC; Kim et al. 2006), rutacridone
epoxide (Meepagala et al. 2005), anthraquinones (Schrader
et al. 2000, 2003), dicthol B acetate (Nagle et al. 2003),
lysine and its analogs (Hehmann et al. 2002; Kaya et al.
2005), harmane and norharmane (Kodani et al. 2002; Volk
2005, 2006; Volk and Furkert 2006; Volk and Mundt 2007),
β-cyanoalanine (Yoshikawa et al. 2000), fischellins (Gross
et al. 1991; Etchegaray et al. 2004), 12-epi-hapalindole
(Etchegaray et al. 2004) and nostcarboline (Blom et al.
2006). Some of these compounds have been reported to
have a strong potential for environmentally friendly control
of harmful algae and cyanobacteria based on the assump-
tion that natural compounds generally degrade faster and
have less probability to cause perturbations in natural
environments than synthetic algicides (Boyd and Massaut
1999). However, some of these compounds have also been
shown to produce strong undesirable side effects. Examples
include rutacridone epoxide, which has a direct-acting
mutagen effect (Meepagala et al. 2005); aaptamine, which
is cytotoxic to mammalian cells (Nagle et al. 2003);
norharmane, which showed a co-mutagenic and cytotoxic
activity in eukaryotic cells (Volk 2005; Volk and Mundt
2007); and anthraquinones that have a direct toxicity to fish
(Schrader et al. 1998). These substances are of little use as
agents to control blooms.
Recently, Jeong et al. (2003) reported that bacillamide, a
novel algicide isolated from the marine bacterium Bacillus
sp. SY-1, has a selective anti-growth activity against the
harmful dinoflagellate Cochlodinium polykrikoides. As a
result, the authors have raised the possibility of using this
compound as a natural algicide against this species in
natural environments. Bacillamide is obviously derived
from tryptophan by decarboxylation, the resulting amine
having further reacted with 2-acetylthiazole-4-carboxylic
acid. The latter compound was discovered in nature in 1990
and is widely distributed within eukaryotes, archaebacteria
and eubacteria. Its possible role as an as yet unrecognized
cofactor has been suggested (White 1990). More recently,
two different bacillamides were isolated, one from the
bacterium Microspora aerata from the Antarctic and the
other from Bacillus endophyticus from a Bahamian hyper-
saline microbial mat (Ivanova et al. 2007; Socha et al.
2007). The possible widespread distribution of bacilla-
J Appl Phycol (2009) 21:429–442 431

selectively controlling the growth of particular species of

cyanobacteria.

Materials and methods

The compounds tested for biological activity were

Fig. 1 Compounds screened for growth-inhibitory activity towards
cyanobacteria and microalgae. Bacillamide R=H, 5-methoxy-bacilla-
mide R=OCH3, 5-bromo-bacillamide R=Br, 5-chloro-bacillamide
R=Cl, 5-fluoro-bacillamide R=F, 5-iodo-bacillamide R=I
mides, coupled with their selective natural toxicity towards
algal bloom-forming species, highlights them as promising
compounds to use as selective algicides.
Apparently, natural bacillamide showed neither algicidal
effect nor growth inhibition towards bloom-forming cya-
nobateria (Jeong et al. 2003). Recent studies, however, have
shown that certain amino acids may be selective in causing
growth inhibition to cyanobacteria, while others do not
affect growth (Hehmann et al. 2002). Differential responses
have been attributed to different permeabilities and uptake
processes in these species to the different compounds
(Hehmann et al. 2002).
In this work, different bacillamide derivatives, obtained
from a similar synthetic pathway by making use of different
tryptamine derivatives, were tested for selective toxicity
towards different cyanobacteria and eukaryotic algae. The
aim of this study was to determine whether any of the
different bacillamide synthetic analogues might be of use in
bacillamide, 5-fluoro-bacillamide, 5-chloro-bacillamide, 5-
methoxy-bacillamide, 5-bromo-bacillamide and 5-iodo-
bacillamide (Fig. 1). All these compounds were obtained
in a pure state as white/yellow crystalline powders.
Bacillamide was synthesized according to the method
described by Figueira et al. (2005) for bacillamide. Stock
solutions for use in the bioassays were obtained by diluting
16 mg of each bacillamide-compound in 50 mL culture
medium (final concentration 320 μg mL−1). All stock
solutions were sonicated on ice for 3 min in 10-s pulses and
sterilized by filtering through sterile 0.22 μm-pore Milli-
pore membrane filters. The pH of the stock solutions was
checked using a digital pH meter. Stock solutions were
maintained at 4 °C and warmed up to room temperature
before use.
Test organisms and culture conditions
Species belonging to different phytoplankton groups were
bioassayed, aiming to represent a wide range of taxonomy,
morphology, physiology and ecological relevance. Isolates
were selected based on their availability in our laboratory
culture collection and their ability to produce rapid and
uniform growth under assay conditions. Isolate information

Table 1 Isolates used in the growth inhibition bioassays

Taxonomic group Species Origin Habitat

Cyanophyceae Microcystis aeruginosa Magos reservoir, Santarém 1998 Portugal Freshwater

Aphanizomenon gracile Crato reservoir, Portalegre 1996 Portugal
Anabaena circinalis Montargil reservoir, Ponte de Sor 2000 Portugal
Anabaena sp. Vitonogales, Guadiana river, 1999 Spain
Anabaenopsis circularis Nafarros pound, Sintra, 2003 Portugal
Nodularia spumigena North Sea, Skagerrak, 2000 Denmark Marine/Brackish
Leptolyngbya sp. Hydrothermal pound, Portugal Freshwater
Planktothrix rubescens Beliche reservoir, Algarve 2005 Portugal
a
Chlorophyta Ankistrodesmus falcatus
a
Scnedesmus obliquus
b
Chlamydomonas sp. Marine
b
Tetraselmis suecica
b
Bacillariophyceae Phaeodactylum tricornutum
Cyclotella sp. S. Domingos reservoir Peniche, 2007 Portugal Freshwater
b
Haptophyceae Diacronema sp. Marine
b
Eustigmatophyceae Nannochloropsis sp.
a
Kindly provided by V. Vasconcelos (CIIMAR- University of Porto, Portugal)
b
Isolated from monitoring samples from the west coast of Portugal
432
(including source) is given in Table 1. All isolates were
maintained as steady-state stock cultures in a culture
chamber programmed for a 16:8 h light:dark cycle with a
light intensity of 28 μmol photons m−2 s−1, and a constant
temperature of 20±1 °C. Inocula from stock cultures
growing on 20 mL T-flasks were regularly transferred to
new medium in order to provide a continuous source of
cells growing at a fairly constant rate. Freshwater and
marine algae were cultivated in Z8 medium (Skulberg
and Skulberg 1990) and in f/2 medium at 27 psu (Guillard
and Ryther 1962), respectively. Freshwater diatoms were
grown in Z8 medium supplemented with silica. Nodularia
spumigena was cultivated in f/2 medium with the salinity
adjusted to 15 psu.
Growth inhibition bioassays
A rapid 96-well microplate bioassay developed by Schrader
et al. (1997) for screening selective algicide compounds
against microalgae was used in this study with slight
modifications. Aliquots (100 μL) of late exponential
growing stock cultures were added at three different culture
dilutions to obtain three different cell-concentrations to
microplate wells previously filled with serial dilutions of
each tested compound from the stock solutions. The total
assay volume in each well was 200 μL. Final yields of
bacillamides in the wells ranged from 5 to 160 μg mL−1.
This range of concentrations was determined by prelimi-
nary range-finding assays. Wells with no tested com-
pounds added were used as controls. Three replicates were
used for each experimental and control condition. The
plates were sealed with perforated adhesive tape to reduce
evaporation and allow gas exchange. Sealed plates were
placed in the culture chamber under the same light and
temperature conditions described for stock-culture main-
tenance. Optical densities of each well were measured
daily for 9 days at 450 nm using a microplate absorbance
reader. Plates were also examined under the inverted
microscope to check for contamination and to verify the
activity of motile organisms (flagellates and gliding
cyanobacteria). Standard curves relating spectrophotomet-
ric absorbance readings at 450 nm with cell counting were
obtained for each cultured organism. Counting was
performed in a Sedgwick-Rafter chamber. A strong
correlation between optical densities and cell densities of
the algae tested was confirmed for all the organisms tested
(r2 values always >0.97).
Mean values and the coefficient of variation (standard
deviation/mean) of optical density measurements from
triplicates were calculated and used to estimate culture
growth over a 216-h period. Growth inhibition values (%)
were calculated after integrating the areas of the growth
curves obtained for each of the experimental and control
J Appl Phycol (2009) 21:429–442
conditions by using the equation Ii(%)=100×(Ac−Ai)/Ac,
where Ii is the percentage growth inhibition obtained with
the concentration i, Ai is the integral area of the growth
curve obtained with the concentration i, and Ac is the
integral area of the growth curve obtained for the control.
The IC50 values (i.e., the concentrations of tested com-
pounds that inhibited growth by 50% relative to the
controls) were obtained from the sigmoidal inhibition
curves fitted by probit regression analysis (SPSS for
Windows v.1.3). The dose-response equations were χ2-
tested for 95% confidence.
Algicidal versus algistatic properties of the compounds
To differentiate between algistatic and algicidal properties
of the tested compounds, samples of 25 μL were removed
from apparently inhibited cultures, and added to new
microplate wells previously filled with 175 μL fresh culture
medium. The new plates were then incubated in the culture
chamber under the light and temperature conditions
described above. Optical measurements were taken in time
lapses of 72 h for 216 h. When growth was observed, the
concentration of the tested compound to which the
organism had been previously exposed was considered to
be algistatic. When no growth appeared on the subculture,
the concentration of the tested compound was considered to
be algicidal (Fitzgerald 1964).
Cell ultrastructural analysis
Two isolates were selected for ultrastructural analysis:
Aphanizomenon gracile and Ankistrodesmus falcatus. Cells
were treated with bacillamide [0 (control), 40 and
80 μg mL−1] in 24-well tissue culture plates for 144 h.
Plates were inspected daily under the inverted microscope
and samples (3 mL) were taken at 72 and 144 h of
incubation for transmission electron microscopy (TEM)
observation. Cell material was prefixed in 2.5% glutaralde-
hyde in culture medium for 30 min at 20 °C. Cells were
then transferred to 2.5% glutaraldehyde in 0.1 M cacodylate
buffer and 2.5 mM CaCl2 overnight at 4°C. Post-fixation
was in 1% buffered osmium tetroxide for 3 h and cells were
then washed three times in 0.1 M cacodylate buffer for
10 min. To increase cell contrast, cells were incubated with
1% uranyl acetate for 1 h in the dark and rinsed with
distilled water for 10 min. Cells were dehydrated in a
graded ethanol series (30, 50, 70, 95%, 20 min each, and
absolute ethanol 3×20 min) at 4°C, with a final step of
propylene oxide for 10 min. After dehydration, cells were
embedded in Spurr’s low-viscosity epoxy resin overnight,
placed in Beem capsules and polymerized at 60°C for 24 h.
Sections were post-stained with 2% uranyl acetate (40 min
in the dark) and lead citrate (5 min). Sections were
J Appl Phycol (2009) 21:429–442 433

examined with a Philips Morgagni 268D transmission
electron microscope.
Results
Most strains adapted well to the growing conditions in the
microplate. Control treatments showed a clear exponential
growth phase starting from the 1st or 2nd day of the test
period and reaching considerable densities at the end of the
216 h period. Some strains exhibited a typical sigmoid
growth curve reaching the start of the stationary growth
phase at the end of the experiment, while others showed a
continuous steady growth throughout the whole period
(data not shown).
For the most affected organisms, differences between the
test treatments and the appropriate control treatment started
to be noticed within the first days of incubation. Inhibition
of growth in those organisms showed an apparent dose-
dependent relationship.
Table 2 lists the IC50-216 h values obtained for each
bacillamide derivative towards the different isolates
screened in this study. Among cyanobacteria, IC50 values
varied substantially, i.e., different species showed different
sensitivities to the same compound. Bacillamide was two to
five times more toxic to M. aeruginosa than to most of the
other cyanobacteria screened. On the other hand, 5-
methoxy-bacillamide was less toxic than bacillamide for
all the isolates screened except for Anabaenopsis circinalis
which seems to be affected more by 5-methoxy-bacillamide
than by bacillamide. 5-Bromo-, 5-fluoro-, 5-chloro- and 5-
iodo- bacillamides were, in turn, far more toxic to the
freshwater Nostocales (Anabaena spp., Aphanizomenon
gracile and Anabaenopsis circularis) than to the Chrooc-
cocales (M. aeruginosa) and the Oscillatoriales (P. rubes-
cens and Leptolyngbya sp.). These data indicate that none
of the compounds tested can be considered as a broad-
spectrum cyanobacterial algicide.
By comparing the IC50-216 h values obtained for
cyanobacteria with those obtained for the eukaryotic
microalgae, it can be seen that microalgae were often
equally, or even more, affected than cyanobacteria by most
compounds screened. Within freshwater organisms, the
diatom Cyclotella sp. proved just as sensitive as the most
affected cyanobacteria to all the bacillamide analogs
screened. Thus, bacillamides treatments that might affect
the growth of sensitive cyanobacteria will also, most
probably affect the growth of centric diatoms. On the other
hand, the freshwater chlorophytes (S. obliquus and Ankis-
trodesmus falcatus) seem to be much less affected by most
bacillamide analogs (as shown by higher IC50 values) than
several bloom-forming cyanobacteria. In fact, bacillamide
was far more toxic to the cyanobacteria M. aeruginosa,
Anabaena circinalis and Aphanizomenon gracile than to the
freshwater chlorophytes. 5-Methoxy-bacillamide also
proved to affect the growth of Anabaena circinalis (IC50-
216 h=36.9 μg mL−1) while not affecting the growth of S.
obliquus and Ankistrodesmus falcatus even at the highest

Table 2 IC50-216 h of bacillamides for each of the cyanobacteria and microalgae screened

BAC CH3O-BAC Br-BAC F-BAC Cl-BAC I-BAC

Cyanobacteria M. aeruginosa 29.0 NOEb 93.1 80.5 118.1 101.4

Anabaena circinalis 69.2 36.9 52.3 39.5 28.4 21.5
Anabaena sp. 117.0 115.5 81.8 61.1 55.9 32.1
Aphanizomenon gracile 57.6 85.2 25.6 42.9 20.8 19.7
Anabaenopsis circularis 120.8 NOE 34.9 41.5 43.7 37.2
N. spumigenaa 116.8 NTc NT 89.0 124.6 110.8
P. rubescens NOE NOE 146.5 128.2 150.1 122.6
Leptolyngbya sp. 140.1 NOE 131.0 145.4 99.1 85.5
Eukaryotic microalgae S. obliquus NOE NOE 106.1 NOE 110.6 47.0
Ankistrodesmus falcatus 101.7 NOE 143.0 NOE NOE 100.0
Cyclotella sp. 58.2 NT NT 22.6 42.8 42.5
P. tricornutuma 70.3 NOE NOE 134.3 56.1 108.6
Chlamydomonas sp.a NOE 39.3 154.2 NOE 54.8 73.8
T. suecica* NOE NOE NOE NOE 85.0 102.2
Diacronema sp.a 133.6 42.7 153.9 71.8 20.8 62.6
Nannochloropsis sp.a NOE 104.2 NOE NOE 153.8 93.4

BAC Bacillamide, CH3O-BAC 5-methoxy-bacillamide, Br-BAC 5-bromo-bacillamide, F-BAC 5-fluoro-bacillamide, Cl-BAC 5-chloro-bacillamide,
I-BAC 5-iodo-bacillamide
a
Marine species
b
No observed effect
c
Not tested
434 J Appl Phycol (2009) 21:429–442

concentration screened (160 μg mL−1). 5-Bromo-, 5-fluoro-, Fig. 2 Growth curves of cultures grown in 5-iodo-bacillamide-free b
culture medium after being exposed to 5-iodo-bacillamide for 216 h.
and 5-chloro-bacillamides were, in turn, far more toxic to ● Control, ■ 5 μg mL−1, * 10 μg mL−1, ♦ 20 μg mL−1, ○
the freshwater Nostocales (Anabaena spp., Anabaenopsis 40 μg mL−1, △ 80 μg mL−1, □ 160 μg mL−1. The Y axes are for
circularis and Aphanizomenon gracile) than to both the absorbance units and the X axes are for time (h). Data represent mean
freshwater chlorophytes. Thus, when applied at appropriate values from triplicates; variation coefficients never exceeded 0.2
(20%)
concentrations, these compounds can selectively inhibit the
growth of different cyanobacteria without affecting growth
of freshwater chlorophytes. algistatic or algicidal, cultures previously exposed to 5-
Within marine and brackish organisms, the IC50-216 h iodo-bacillamide were subsequently transferred to new,
values found for each bacillamide analog towards the compound-free, culture medium and incubated for another
cyanobacterium N. spumigena were often higher than those 216 h. When no growth appeared on the subcultures, the
obtained for the marine eukaryotic microalgae (Table 2). concentration of the chemical and treatment time was
Treatments with any of those bacillamides in marine/ considered algicidal. When growth was observed on pre-
brackish environments are therefore expected to affect the viously inhibited cultures, the concentration of the chemical
growth of most eukaryotic algae more effectively than the was considered algistatic. Figure 2 presents the growth
growth of N. spumigena. curves of 5-iodo-bacillamide-exposed cultures obtained
As seen in Table 3, the IC50-216 h values of bacillamide after being transferred to new compound-free culture
obtained towards the different cyanobacteria and micro- medium. For the eukaryotic microalgae, all 5-iodo-bacilla-
algae were directly related to the amount of cells in the mide treatments that inhibited growth proved to have
culture inoculums. Similar results were obtained for the algistatic properties, i.e., growth was resumed in all
other bacillamide analogues screened in this work (data not apparently inhibited cultures. For most cyanobacteria, 5-
shown), showing that the amount of chemical needed to iodo-bacillamide acted either as an algicide or as an
prevent growth depended on the initial cell density of the algistatic, depending on the concentration added. Treat-
test organism. ments with 160 μg mL−1 proved algicidal to all cyanobac-
5-Iodo-bacillamide clearly affected the growth of all teria except for M. aeruginosa. For Anabaena circinalis,
cyanobacteria and eukaryotic algae screened in this assay. 5-iodo-bacillamide acted as an algicide when concentra-
Based on IC50-216 h values, this compound was slightly tions were at 20 μg mL−1 or above, whereas for Anabaena
more toxic to most cyanobacteria than the other tested sp. and Aphanizomenon gracile, 5-iodo-bacillamide acted
bacillamides (Table 2). In order to determine whether the as an algicide at 40 μg mL−1 or above. Treatments below
effects of 5-iodo-bacillamide on culture growth were such concentrations were either algistatic to those cyano-
bacteria or showed no inhibitory effect on cyanobacteria
growth.
Table 3 Effect of the inoculum concentration on the IC50-216h values Figure 3 shows photomicrographs of unstained living
of bacillamide towards different cyanobacteria and microalgae
cells of Aphanizomenon gracile (a–c) and Anabaena
IC50-216h Inoculum dilution factor circinalis (d–f) taken from control (a, d) and bacillamide-
treated cultures (b,c,e,f) after a 72-h exposure period. Cells
No dilution 1:2 1:4
exposed to increasing amounts of bacillamide showed a
M.aeruginosa 28.99 22.24 18.13 gradual dose-dependent bleached appearance, lacking the
Anabaena circinalis 114.85 69.18 61.86 numerous gas vesicles and cytoplasmic inclusions clearly
Anabaena sp. 115.07 116.98 102.26 visible in the control treatments. Heterocysts could be seen
Aphanizomenon gracille 57.62 58.62 60.66 in both treated and untreated cultures, though showing a
Anabaenopsis circularis 129.20 133.98 120.82 more translucid appearance in the exposed cultures. These
N. spumigena 116.84 100.86 37.05
effects were even more pronounced (at lower doses) after a
P. rubescens NOEa NOE NOE
Leptolyngbya sp. NOE 140.05 123.77
216-h exposure period and were shared among the other
S. obliquus NOE NOE NOE screened cyanobacteria (data not shown). In the case of P.
Ankistrodesmus falcatus 139.25 111.38 101.67 rubescens, the oscillatory capability of movement seen in
Cyclotella sp. 58.15 22.13 12.43 the controls was lost after treatments with bacillamide.
P. tricornutum 74.54 70.29 50.56 Among the chlorophytes, the most evident morpholog-
Chlamydomonas sp. NOE NOE NOE ical effects induced by bacillamide treatments occurred in
T. suecica NOE NOE NOE Ankistrodesmus falcatus (Fig. 4). Unexposed cells appeared
Diacronema sp. 133.63 126.52 106.85
elongated, with a prominent chloroplast (Fig. 4a), while
Nannochloropsis sp. NOE NOE NOE
bacillamide-treated cells became consistently deformed and
a
No observed effect swollen (Fig. 4b).
J Appl Phycol (2009) 21:429–442 435
436
Fig. 3 Phase-contrast micrographs of Aphanizomenon gracile (a–c) and Anabaena circinalis (d–f) cultures, exposed to 0 μg mL−1 (a, d),
40 μg mL−1 (b, e), and 80 μg mL−1 (c, f) bacillamide, for 72 h. Bars 10 μm
Within diatoms, the effects of bacillamide treatments on
cell morphology were less visible. There was an apparent
fragility of treated Cyclotella sp. cells, which lysed easily
after being exposed to the microscope incident light for a
few seconds. No visible effects were observed between
control cells and exposed cells of the other algal groups
tested (data not shown).
The effects of bacillamide on Aphanizomenon gracile at
the ultrastructural level were analyzed by transmission
electron microscopy (Fig. 5a–e). Control cells showed the
typical cyanobacterial ultrastructure (Fig. 5a) with a three-
layered Gram-negative cell wall, absence of nucleus and
membrane-bound organelles, and photosynthetic lamellae
composed of single thylakoids. The cells presented numer-
ous gas vacuoles occupying a large part of the cytoplasm,
and cellular inclusions such as carboxysomes (polyhedral
bodies), cyanophycean granules, lipid droplets and poly-
phosphate bodies. No differences were observed in control
cells at 72 and 144 h of incubation (data not shown). In
Fig. 4 Phase-contrast
micrographs of Ankistrodesmus
falcatus from control (a) and
bacillamide-treated (b) cultures.
Bar 10 μm
J Appl Phycol (2009) 21:429–442
cells treated with 40 μg mL−1 of bacillamide for 72 h
(Fig. 5b), there was a drastic reduction of gas vacuoles, an
increase in intrathylakoidal spaces and a slight distortion
of the cell wall. After a 144 h exposure period (Fig. 5c) a
more severe cell wall distortion was observed, as well as
an increase in lipid droplets near the thylakoid membranes
and the absence of gas vacuoles and intrathylakoidal
spaces. Heterocysts from bacillamide-treated cultures fre-
quently showed large vacuoles localized in the middle of
the cell (Fig. 5e), which were absent in unexposed cells
(Fig. 5d).
The bacillamide-induced ultrastructural changes in the
chlorophyte Ankistrodesmus falcatus are illustrated in
Fig. 6. Control cells showed a prominent chlorophycean
chloroplast with thylakoids grouped in lamellae, intra-
plastidial starch grains and a three-layer cell wall structure
with a thicker layer in the middle. The cytoplasm presented
the typical eukaryotic organelles, like mitochondria, dense
primary lysosomes and lipid droplets (Fig. 6a). No differ-
J Appl Phycol (2009) 21:429–442 437

Fig. 5a–e Transmission electron micrographs of Aphanizomenon
gracile. a Control cells after 72 h incubation, showing the typical
cyanobacterial ultrastructure. Prominent gas vacuoles (GV) occupy a
large part of the cytoplasm. Note also the presence of numerous
cytoplasmic inclusion bodies: PB polyhedral bodies, Ph polyphosphate
bodies, CY cyanophycin granules, Lp lipid droplets. Thylakoid
membranes (Th) distribute at the cell periphery. b Cells exposed to
40 μg mL−1 bacillamide for 72 h show an evident increase in
intrathylakoidal space and reduction in the number of gas vacuoles. c
Distortion of cell wall (arrows) in cells exposed to 40 μg mL−1
bacillamide for 144 h. Note also the increase in lipid droplets that
localize near the thylakoids and the absence of gas vacuoles. d, e A.
gracile heterocysts displaying an elaborated membrane system (M),
pore channel to the adjacent cell (P) and multilayered envelope (E). d
Control cell after 72 h incubation. e Heterocyst treated with
80 μg mL−1 for 72 h. Bar1 μm

Fig. 6a–i Transmission electron

micrographs of Ankistrodesmus
falcatus. a Control A. falcatus
cells after 72 h incubation,
showing a prominent chloroplast
(Chl) with grana thylakoids
(Thy) and starch grains (S).
Some lipid droplets (Lp) are
present in the cytoplasm. b Cells
treated with 40 μg mL−1 bacil-
lamide after 144 h of exposure,
displaying numerous cytoplas-
mic lysosome-like structures
(Lys). gb Golgi body, m mito-
chondria, n nucleus. c Cells
treated with 80 μg mL-1 after
72 h of exposure, with numer-
ous lipid droplets dispersed in
the cytoplasm. Note a slight
cell wall swelling (arrowheads).
i After 144 h of exposure to
80 μg mL−1 bacillamide, cells
are clearly swollen
(arrowheads). Bar 1 μm
438
ences could be observed between control cells (72 and
144 h incubation time) and cells exposed to 40 μg mL−1
bacillamide for 72 h (data not shown). However, cells
treated with higher bacillamide concentrations and/or
longer exposure times showed a dose-dependent increase
in the number of lipid droplets, which in some cases
appeared as large cytoplasmic globules (Fig. 6b–d). Cells
exposed to bacillamide for 144 h (independent of the dose),
showed numerous cytoplasmic granular lysosome-like
structures, ultrastructurally different from the electro-dense
primary lysosomes present in control cells (Fig. 6b,d). A
slight swelling of the cells observed after exposure to
80 μg mL−1 bacillamide for 72 h (Fig. 6c) became more
pronounced when the exposure time was doubled (Fig. 6d).
Discussion
In order to screen the effects of different compounds at
several concentrations in a range of different cyanobacteria
and microalgae cultures, we applied a 96-well microplate
bioassay previously developed by Schrader et al. (1997).
This bioassay allows several conditions to be screened
simultaneously in a single culture plate, with results being
obtainable within a few days and the selectivity of the
compounds easily determined. The method proved to be
much less space- and time-consuming than classic methods
performed in tubes or flasks and based on direct cell counts.
However, there were some drawbacks that should be noted.
For example, the method was not adequate for testing some
species, namely the motile dinoflagellates (Amphidinium
carterae, Gymnodinium catenatum and Alexandrium minu-
tum), which did not grow in the plate wells. Some problems
were also noted with filamentous species such as Plankto-
thrix rubescens, Anabaena circinalis and Nodularia spumi-
gena, which tended to clump in the plate wells after the first
2 days, causing irregular absorbance readings. This prob-
lem was overcome to some extent by plate shaking before
optical readings. However, some strains of Microcystis
aeruginosa had to be excluded from the screening tests as
they formed dense indissoluble colonies. For all the
organisms used in the experiments a good linear relation-
ship between cell number and optical density was obtained.
Bacillamides were toxic to different species of cyano-
bacteria in a dose-dependent manner. The Oscillatoriales
were in general less sensitive to most bacillamide analogues
than the Chroococcales and the Nostocales. Such different
sensitivities are perhaps related to the different growth
habits of the Oscillatoriales screened. As mentioned above,
both P. rubescens and Leptolyngbya sp. tended to aggre-
gate, forming bundles of dense, tightly packed filaments. It
is conceivable that the filaments on the outer surface of the
bundles may have protected the cells in the inner part of the
J Appl Phycol (2009) 21:429–442
colonies from the action of bacillamides. Nodularia
spumigena also showed a higher tolerance to most
bacillamides then the other Nostocales, perhaps through a
protective effect of the abundant mucilage layer.
Different sensitivities among the different types of
cyanobacteria indicate that bacillamides cannot be consid-
ered as broad spectrum cyanobacterial algicides. However,
different bacillamide analogues have potential for use in
selectively controlling the growth of particular species of
cyanobacteria. Bacillamide itself was far more toxic to
Microcystis aeruginosa, Anabaena circinalis and Aphani-
zomenon gracile than to the chlorophytes Scenedesmus
obliquus and Ankistrodesmus falcatus. Thus, bacillamide
treatments of up to 80 μg mL−1 are expected to suppress the
growth of these cyanobacteria without affecting growth of
the green algae. 5-Bromo- and 5-chloro-bacillamides, when
applied at the same concentrations, also prevented the
growth of the cyanobacteria Anabaena circinalis, Aphani-
zomenon gracile and Anabaenopsis circularis, with no
significant consequences to the above-mentioned chloro-
phytes. 5-Flouro- and 5-iodo-bacillamides showed a much
wider-spectrum activity against cyanobacteria, although the
latter affected also the chlorophytes and thus showed a
much lower selectivity in its toxic action. On the other
hand, 5-methoxy-bacillamide (CH3O-bacillamide) proved
to be quite specific towards Anabaena circinalis at
concentrations apparently non-toxic to any of the other
freshwater organisms screened.
Taken together, these results indicate that decisions
regarding the type of bacillamide to be used for the
selective control of freshwater cyanobacteria should depend
on the relative composition of the phytoplankton commu-
nity in each particular situation. In cases where M.
aeruginosa is found as the most abundant species, treat-
ments with either bacillamide or 5-fluoro-bacillamide
would be more adequate if co-occurring chlorophytes are
not to be affected. To control Aphanizomenon gracile,
treatments with either 5-methoxy-, 5-bromo- or 5-chloro-
bacillamides would be preferable given their higher
differential selectivities towards this species relative to
freshwater chlorophytes. Concerning Anabaena spp., more
satisfying results would be expected from applying 5-
chloro- or 5-fluoro-bacillamides, although these cyanobac-
teria showed some intra-generic variation in their sensitivity
to bacillamides.
Regardless of the particular species of cyanobacteria
focused on, special attention should also be paid to diatoms
as these proved to be nearly as sensitive to most
bacillamide analogues as the most affected cyanobacteria.
In temperate regions, diatoms and cyanobacteria usually
tend to colonize eutrophic and hypertrophic freshwater
systems in a seasonal succession. Cyanobacteria often
dominate the summer phytoplankton and, as winter
J Appl Phycol (2009) 21:429–442
approaches, the increasing turbulence and decreasing light
leads to their replacement by diatoms in association with
rapidly growing small flagellates (Chorus and Bartram
1999). These can, in turn, be followed by green algae in late
spring and early summer and then by species that cannot
easily be eaten by zooplankton, such as dinoflagellates,
desmids and large yellow-green algae in autumn. In cases
where such a pattern of succession is followed, the use of
bacillamides in the early stages of the cyanobacteria-
dominant season are not expected to affect diatom
populations to a great extent. However, if seasonable
differences and other environmental factors are not great
enough to induce phytoplankton succession, the use of
bacillamide in mixed communities of cyanobacteria and
diatoms will probably affect both.
Within marine species, Nodularia spumigena was
affected equally by bacillamide, 5-chloro-bacillamide 5-
fluoro-bacillamide and 5-iodo-bacillamide. However, the
IC50-216 h obtained for this cyanobacterium where quite
high (89–124 μg mL−1) and, when compared to other
marine algae, the screened bacillamides showed differential
selective values too low to be pursued for the practical
control of harmful blooms of this particular species.
To predict the effectiveness of a chemical for controlling
a particular problem, it is essential to know whether the
chemical acts as an algicide or as an algistatic. Differenti-
ation between these types of action will provide valuable
information on whether the chemical must be in constant
contact with the algae to prevent further growth (algistatic)
or if, after a sufficient treatment time, the algae will have
absorbed enough chemical to eventually die. Results
obtained in this work showed bacillamides to act as
algistatic agents against eukaryotic algae and, depending
upon the concentrations added, to act either as algicide or
algistatic agents against most cyanobacteria. The need to
know what type of action bacillamides will have at a
particular concentration towards a particular species might
be important when a method of application must be
selected. Occasional “shock” treatments with high bacilla-
mide concentrations should be sufficient to eliminate
undesirable cyanobacteria while temporarily affecting eu-
karyotic algae. Being algistatic to those algae, growth
would be expected to resume after the amount of chemical
drops below its algistatic concentration. Such “shock”
treatments, when applied to well established blooms of
toxic cyanobacteria, could possibly lead to massive cell
lysis, with the consequent risk of toxin release to the water.
When applied before cyanobacteria communities turn into
massive proliferations, such treatments will probably
prevent the development of blooms, and have no such
consequences.
The amounts of bacillamides required to inhibit the
growth of most organisms tested was related to the amount
439
of cells initially present in the inocula. This must be kept in
mind when considering the amounts of chemicals to be
applied to control an algal problem, and emphasizes the
importance of preventive measures in controlling the
growth of cyanobacteria before they turn into a major
problem in the aquatic medium.
In this study, the initial cell densities, as measured by
optical density readings, were kept as similar as possible for
all species to correctly compare both the sensitivities to
each test compound and the toxicities of the different
compounds towards each test culture. The concentrations of
each bacillamide required to inhibit growth of the most
sensitive species were significantly higher than those
reported in the literature for other natural compounds with
algicidal properties. Studies involving the use of aaptamine
(Nagle et al. 2003), ethyl-2-methylacetoacetate (Li and Hu
2005), palmiolate (Chung et al. 2007), AZC (Kim et al.
2006), rutacridone epoxide (Meepagala et al. 2005),
anthraquinone and its analogues (Schrader et al. 2000,
2003), dichtol B acetate (Nagle et al. 2003), harmane
and norharmane (Kodani et al. 2002), β-cyanoalanine
(Yoshikawa et al. 2000), gramicidin (Reim et al. 1974),
4,4′-dihydroxybiphenyl (Volk 2006), fisherellins (Etchegaray
et al. 2004), 12-epi-hapalindole (Etchegaray et al. 2004),
kasumigamide (Ishida and Murakami 2000), nostocarbolines
(Blom et al. 2006) and many others, report much higher
toxicities of these natural compounds against cyanobacteria.
Some of the compounds listed have not yet been tested for
their selectivity on a wide, representative range of other
organisms. Others were shown to have noxious, undesirable
side effects such as high mutagenic and/or cytotoxic
activities on fish and mammalian cells (Nagle et al. 2003;
Meepagala et al. 2005; Volk 2005; Volk and Mundt 2007;
Schrader et al. 1998). Synthetic production of bacillamides
has been accomplished only very recently and no studies on
toxicity towards other organisms have yet been made.
Nevertheless, the high concentration of bacillamides needed
to prevent cyanobacterial growth (IC 50 -216 h = 30–
100 mg L−1) may limit the usefulness of these compounds
for large water bodies.
The major effect of bacillamide on the ultrastructure of
cyanobacteria cells was the reduction and collapse of gas
vesicles and the distortion of cell shape. The observed
interference with gas vacuole integrity may increase the
sedimentation rate of cyanobacteria, preventing them from
becoming dominant. Similar results were obtained by
Hehmann et al. (2002) while studying the effects of lysine
on the growth of M. aeruginosa. These latter authors
observed that this amino acid affected the cell membranes
and caused gas vacuoles to disappear, suggesting that those
effects resulted from the feedback inhibition of biosynthetic
enzymes. Although we can only speculate about the
mechanisms underlying the toxic action of bacillamides
440
on cyanobacteria, it is possible to admit that growth
inhibition by these compounds might be the result of
analogous mechanisms. In fact, the rapid recovery of
growth of previously inhibited cultures after being trans-
ferred to fresh culture medium suggests that the observed
effects could be due to feedback inhibition of metabolic
pathways, which, in turn, could resume activity upon
removal of the inhibiting compound. Interestingly, Ankis-
trodesmus cells exposed to bacillamides showed an increase
in the number of lysosome-like particles when compared to
unexposed cells (especially with the 144 h treatment). The
presence of such structures in cells subjected to long-term
treatment suggests an attempt by the organism to detoxify
or metabolize the toxic agent. Recovery should then be
possible as long as the concentration and the exposure time
to the compound are sufficiently low to allow cells to repair
and recover from damage.
Acknowledgments The authors would like to thank the Center of
Marine and Environmental Research at Porto University (Portugal)
and the Phycology Department at the University of Copenhagen
(Denmark) for kindly providing some of the isolates used in this work.
This project was supported by Fundação para a Ciência e Tecnologia
(FCT) through Project POCI/AMB/60531/2004 (Portugal). Two of us
(V. C. F. and A. J. B.) are also thankful to FCT for research grants.
References
Anderson D, Glibert P, Burkholder J (2002) Harmful algal blooms and
eutrophication: nutrient sources, composition, and consequences.
Estuaries 25:704–726. doi:10.1007/BF02804901
Apeldoorn M, Egmond H, Speijers G, Bakker G (2007) Toxins of
cyanobacteria. Mol Nutr Food Res 51:7–60. doi:10.1002/mnfr.
200600185
Bagchi S, Chauhan V, Marwah B (1993) Effect of an antibiotic from
Oscillatoria late-virens on growth, photosynthesis, and toxicity
of Microcystis aeruginosa. Curr Microbiol 26:223–228.
doi:10.1007/BF01577380
Ball A, Williams M, Vincent D, Robinson J (2001) Algal growth
control by a barley straw extract. Bioresour Technol 77:177–181.
doi:10.1016/S0960-8524(00)00148-6
Baudoux A, Brussard C (2005) Characterization of different viruses
infecting the marine harmful algal bloom species Phaeocystis
globosa. Virology 341:80–90. doi:10.1016/j.virol.2005.07.002
Blom J, Brutsch T, Barbaras D, Bethuel Y, Locher H, Hubschwerlen
C, Gademann K (2006) Potent algicides based on the cyanobac-
terial alkaloid nostocarboline. Org Lett 8:737–740. doi:10.1021/
ol052968b
Boyd C, Massaut L (1999) Risks associated with the use of chemicals
in pond aquaculture. Aquacult Eng 20:113–132. doi:10.1016/
S0144-8609(99)00010-2
Chauhan B, Marwah J, Baghi S (1992) Effect of an antibiotic from
Oscillatoria sp. on phytoplankters, higher plants and mice. New
Phytol 120:251–257. doi:10.1111/j.1469-8137.1992.tb05661.x
Chorus I, Bartram J (1999) Introduction. In: Chorus I, Bartram J (eds)
Toxic cyanobacteria in water: a guide to their public health con-
sequences, monitoring and management. WHO/Spon, London,
pp 1–14
J Appl Phycol (2009) 21:429–442
Chorus I, Mur L (1999) Preventive measures. In: Chorus I, Bartram J
(eds) Toxic cyanobacteria in water: a guide to their public health
consequences, monitoring and management. WHO/Spon, London,
pp 235–273
Chung I, Ali M, Ahmad A, Chung S, Kim J, Sultana S, Kim J, Min S,
Seo B (2007) Steroidal constituents of Rice (Oriza sativa) hulls
with algicidal and herbicidal activity against blue-green algae and
duckweed. Phytochem Anal 18:133–145. doi:10.1002/pca.961
Codd GA (1999) Cyanobacterial toxins: their occurrence in aquatic
environments and significance to health. Bull Inst Oceanogr
Monaco 19:483–500
Erhard D, Gross EM (2006) Allelopathic activity of Elodea
Canadensis and Elodea nuttallii against epiphytes and phyto-
plankton. Aquat Bot 85:203–211. doi:10.1016/j.aquabot.
2006.04.002
Etchegaray A, Rabello E, Dieckmann R, Moon DH, Fiore MF, Döhren
H, Tsai SM, Neilan BA (2004) Algicide production by the
filamentous cyanobacterium Fischerella sp. CENA19. J Appl
Phycol 16:237–243. doi:10.1023/B:JAPH.0000048509.77816.5e
Everall N, Lees D (1995) The use of barley-straw to control general
and blue-green algal growth in a Derbyshire reservoir. Water Res
30:269–276
Falconer I, Bartram J, Chorus I, Kuiper-Goodman T, Utkilen H, Burch
M, Codd G (1999) Safe levels and safe practices. In: Chorus I,
Bartram J (eds) Toxic cyanobacteria in water: a guide to their
public health consequences, monitoring and management. WHO/
Spon, London, pp 155–178
Ferrier M, Butler B, Terlizzi D, Lacouture R (2005) The effects of
barley straw (Hordeum vulgare) on the growth of freshwater
algae. Bioresour Technol 96:1788–1795. doi:10.1016/j.biortech.
2005.01.021
Figueira V, Prabhakar S, Lobo A (2005) Synthesis of the algicide
bacillamide. Arkivoc Online J Org Chem xiv:14–19
Fitzgerald G (1964) Factors in testing the application of algicides.
Appl Microbiol 12:247–253
García-Villada L, Rico M, Altamirano M, Sánchez-Martín L, López-
Rodas V, Costas E (2004) Occurrence of copper resistant mutants
in the toxic cyanobacteria Microcystis aeruginosa: characteriza-
tion and future implications in the use of copper sulphate as
algicide. Water Res 38:2207–2213. doi:10.1016/j.watres.
2004.01.036
Gross E, Wolk C, Jüttner F (1991) Fischerellin, a new allelochemical
from the freshwater cyanobacteria Fischerella muscicola. J
Phycol 27:686–697. doi:10.1111/j.0022-3646.1991.00686.x
Guillard R, Ryther J (1962) Studies of marine planktonic diatoms I.
Cyclotella nana Hustedt, and Detonula confervacea (Cleve)
Gran. Can J Microbiol 8:229–237
Hallegraeff GM (2003) Harmful algal blooms: a global overview. In:
Hallegraeff GM, Anderson DM,Cembella AD (eds) Manual on
harmful marine microalgae. UNESCO, Venice, pp 25–49
Hehmann A, Kaya K, Watanabe M (2002) Selective control of
Microcystis using an amino acid-a laboratory assay. J Appl
Phycol 14:85–89. doi:10.1023/A:1019546829940
Heo W, Kim B (2004) The effect of artificial destratification on
phytoplankton in a reservoir. Hydrobiologia 524:229–239.
doi:10.1023/B:HYDR.0000036142.74589.a4
Hilt S, Ghobrial M, Gross E (2006) In situ allelopathic potential of
Myriophyllum verticillatum (Haloragaceae) against selected phy-
toplankton species. J Phycol 42:1189–1198. doi:10.1111/j.1529-
8817.2006.00286.x
Hrudey S, Burch M, Drikas M, Gregory R (1999) Remedial measures.
In: In Chorus I, Bartram J (eds) Toxic cyanobacteria in water: a
guide to their public health consequences, monitoring and
management. WHO/Spon, London, pp 275–312
Imai I, Kim M, Nagasaki K, Itakura S, Ishida Y (1998) Relationships
between dynamics of red tide-causing raphidophycean flagellates
J Appl Phycol (2009) 21:429–442
and algicidal micro-organisms in the coastal Sea of Japan. Phycol
Res 46:139–146. doi:10.1111/j.1440-1835.1998.tb00106.x
Ishida K, Murakami M (2000) Kasumigamide, an antialgal peptide
from the cyanobacterium Microcystis aeruginosa. J Org Chem
65:5898–5900. doi:10.1021/jo991918f
Ivanova V, Kolarova M, Aleksieva K (2007) Microbiaeratin, a new
natural indole alkaloid from a Microbispora aerata strain,
isolated from Livingston Island, Antarctica. Prep Biochem
Biotechnol 37:161–168. doi:10.1080/10826060701199122
Jasti S, Sieracki ME, Poulton NJ, Giewat MW, Rooney-Varga JN
(2005) Phylogenetic diversity and specificity of bacteria closely
associated with Alexandrium spp. and other phytoplankton. Appl
Environ Microbiol 71:3483–3494. doi:10.1128/AEM.71.7.3483-
3494.2005
Jeong J, Jin H, Sohn C, Suh K, Hong Y (2000) Algicidal activity of
the seaweed Corallina pilulifera against red tide microalgae. J
Appl Phycol 12:37–43. doi:10.1023/A:1008139129057
Jeong S, Ishida K, Ito Y, Okada S, Murakami M (2003) Bacillamide—
a novel algicide from the marine bacterium, Bacillus sp. SY-1,
against the harmful dinoflagellate, Cochlodinium polykrikoides.
Tetrahedron Lett 44:8005–8007. doi:10.1016/j.tetlet.2003.08.115
Jin Q, Dong S, Wang C (2005) Allelopathic growth inhibition of
Prorocentrum micans (Dinophyta) by Ulva pertusa and Ulva
linza (Chlorophyta) in laboratory cultures. Eur J Phycol 40:31–
37. doi:10.1080/09670260400019741
Kaya K, Liu Y, Shen Y, Xiao B, Sano T (2005) Selective control of
toxic Microcystis water blooms using lysine and Malonic acid: an
enclosure experiment. Environ Toxicol 20:170–178. doi:10.1002/
tox.20092
Kim J, Kim J, Lee S, Lee B, Cho K (2006) Biological activity of L-2-
azetidinecarboxylic acid, isolated from Polygonatum odoratum
var. pluriflorum, against several algae. Aquat Bot 85:1–6.
doi:10.1016/j.aquabot.2006.01.003
Kodani S, Imoto A, Mitsutani A, Murakami M (2002) Isolation and
identification of the antialgal compound, harmane (1-methyl-β-
carboline), produced by the algicidal bacterium, Pseudomonas
sp. K44-1. J Appl Phycol 14:109–114. doi:10.1023/A:1019
533414018
Körner S, Nicklisch A (2002) Allelopathic growth inhibition of
selected phytoplankton species by submerged macrophytes. J
Phycol 38:862–871. doi:10.1046/j.1529-8817.2002.t01-1-02001.x
Kuiper-Goodman T, Falconer I, Fitzgerald J (1999) Human health
aspects. In: In Chorus I, Bartram J (eds) Toxic cyanobacteria in
water: aguide to their public health consequences, monitoring
and management. WHO/ Spon, London, pp 113–153
Li F, Hu H (2005) Isolation and characterization of a novel antialgal
allelochemical from Phragmites communis. Appl Environ Micro-
biol 71:6545–6553. doi:10.1128/AEM.71.11.6545-6553.2005
Long BM, Carmichael W (2003) Marine cyanobacterial toxins. In:
Hallegraeff GM, Anderson DM, Cembella AD (eds) Manual on
harmful marine microalgae. UNESCO, Venice, pp 279–296
Mayali X, Doucette G (2002) Microbial community interations and
population dynamics of an algicidal bacterium active against
Karenia brevis (Dinophyceae). Harmful Algae 1:277–293.
doi:10.1016/S1568-9883(02)00032-X
Meepagala K, Schader K, Wedge D, Duke S (2005) Algicidal and
antifungal compounds from the roots of Ruta graveolens and
synthesis of their analogs. Phytochemistry 66:2689–2695.
doi:10.1016/j.phytochem.2005.09.019
Mulderij G, Mooij W, Smolders A, Donk E (2005) Allelopathic
inhibition of phytoplankton by exudates from Stratiotes aloides.
Aquat Bot 82:284–296. doi:10.1016/j.aquabot.2005.04.001
Nagasaki K, Tarutani K, Yamaguchi M (1999) Growth characteristics
of Heterosigma akashio virus and its possible use as a
microbiological agent for red tide control. Appl Environ Micro-
biol 65:898–902
441
Nagle D, Sultana G, Schrader K, Hossain C, Stanikunaite R, Hamann
M, Rajbandari I (2003) Secondary metabolites from plants and
marine organisms as selective anti-cyanobacterial agents. In:
Rimando A, Schrader K (eds) Off-flavour in aquaculture. ACS
Symposium Series 848, Oxford University Press, New York,
pp 179–194
Nan C, Zhang H, Zhao G (2004) Allelopathic interactions between the
macroalga Ulva pertusa and eight microalgal species. J Sea Res
52:259–268. doi:10.1016/j.seares.2004.04.001
Oliveira-Filho E, Lopes R, Paumgartten F (2004) Comparative studies
on the susceptibility of freshwater species to copper-based
pesticides. Chemosphere 56:369–374. doi:10.1016/j.chemo
sphere.2004.04.026
Park M, Han M, Ahn C, Kim H, Yoon B, Oh H (2006) Growth
inhibition of bloom-forming cyanobacterium Microcystis aerugi-
nosa by rice straw extract. Lett Appl Microbiol 43:307–312.
doi:10.1111/j.1472-765X.2006.01951.x
Redhead K, Wright SJL (1978) Isolation and properties of fungi that
lyses blue-green algae. Appl Environ Microbiol 35:962–969
Reim R, Shane M, Cannon R (1974) The characterization of Bacillus
capable of blue-green algae bactericidal activity. Can J Microbiol
20:981–986
Roussel H, Ten-Hage L, Joachim S, Cohu R, Gauthier L, Bonzon J
(2007) A long-term copper exposure on freshwater ecosystems
using lotic mesocosms: primary producer community responses.
Aquat Toxicol 81:168–182. doi:10.1016/j.aquatox.2006.12.006
Safferman R, Morris M (1962) Evaluation of natural products for
algicidal properties. Appl Microbiol 10:289–292
Schrader KK, Regt MQ, Tucker CS, Duke SO (1997) A rapid
bioassay for selective algicides. Weed Technol 11:767–774
Schrader KMQ, Tidwell PD, Tucker CS, Duke SO (1998) Selective
growth inhibition of the musty-odour-producing Cyanobacterium
Oscillatoria cf. chalybea by natural compounds. Bull Environ
Contam Toxicol 60:651–658. doi:10.1007/s001289900676
Schrader K, Dayan F, Allen S, Regt M, Tucker C, Paul R (2000) 9,10-
Anthraquinone reduces the photosynthetic efficiency of Oscil-
latoria perornata and modifies cellular inclusions. Int J Plant Sci
161:265–270. doi:10.1086/314255
Schrader K, Nanayakkara N, Tucker C, Rimando A, Ganzera M,
Schaneberg B (2003) Novel derivates of 9,10-Anthraquinone are
selective against the musty-odour cyanobacterium Oscillatoria
perornata. Appl Environ Microbiol 69(9):5319–5327.
doi:10.1128/AEM.69.9.5319-5327.2003
Skulberg R, Skulberg O (1990) Forskning med algekulturer NIVAs
kultursampling av alger. NIVA, Norway
Socha A, Long R, Rowley D (2007) Bacillamides from a hypersaline
microbial mat bacterium. J Nat Prod 70:1793–1795. doi:10.1021/
np070126a
Suikkanen S, Fistarol G, Granéli E (2004) Allelopathic effects of the
Baltic cyanobacteria Nodularia spumigena, Aphanizomenon flos-
aquae and Anabaena lemmermannii on algal monocultures. J
Exp Mar Biol Ecol 308:85–101. doi:10.1016/j.jembe.2004.
02.012
Verschuere L, Rombaut G, Sorgeloos P, Verstraete W (2000) Probiotic
bacteria as biological control agents in aquaculture. Microbiol
Mol Biol Rev 64:655–671. doi:10.1128/MMBR.64.4.655-
671.2000
Volk R (2005) Screening of microalgal culture media for the presence
of algicidal compounds and isolation and identification of two
bioactive metabolites excreted by the cyanobacteria Nostoc
insulare and Nodularia harveyana. J Appl Phycol 17:339–347.
doi:10.1007/s10811-005-7292-7
Volk R (2006) Antialgal activity of several cyanobacterial exometabo-
lites. J Appl Phycol 18:145–151. doi:10.1007/s10811-006-9085-z
Volk R, Furkert F (2006) Antialgal, antibacterial and antifungal
activity of two metabolites produced and excreted by cyanobac-
442
teria during growth. Microbiol Res 161:180–186. doi:10.1016/j.
micres.2005.08.005
Volk R, Mundt (2007) Cytotoxic and non-cytotoxic exometabolites of
the cyanobacterium Nostoc insulare. J Appl Phycol 19:55–62.
doi:10.1007/s10811-006-9110-2
White R (1990) Widespread occurrence of 2-acetylthiazole-4-carbox-
ylic acid in biological material. Cell Mol Life Sci 46:274–276
Wu J, Kuo-Huang L, Lee J (1998) Algicidal effect of Peridinium
bipes on Microcystis aeruginosa. Curr Microbiol 37:257–261.
doi:10.1007/s002849900375
J Appl Phycol (2009) 21:429–442
Yoshikawa K, Adachi K, Nishijima M, Takadera T, Tamaki S,
Harada K, Mochida K, Sano H (2000) β- Cyanoalanine
production by the marine bacterium on cyanide-free medium
and its specific inhibitory activity toward cyanobacteria. Appl
Environ Microbiol 66:718–722. doi:10.1128/AEM.66.2.718-
722.2000
Zhou L, Zheng T, Wang X, Ye J, Tian Y, Hong H (2007) Effect of five
chinese traditional medicines on the biological activity of a red
tide causing alga—Alexandrium tamarense. Harmful Algae
6:354–360. doi:10.1016/j.hal.2006.10.002