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Antibacterial activity in microalgae cultures

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Aquaculture Research, 2012, 43, 1520–1527
Antibacterial activity in microalgae cultures
Foteini Kokou1, Pavlos Makridis2,*, Maroudio Kentouri1 & Pascal Divanach2
1
Biology Department, University of Crete, Crete, Greece
2
Hellenic Center for Marine Research, Institute of Aquaculture, Crete, Greece
Correspondence: P Makridis, Hellenic Center for Marine Research, Institute of Aquaculture, PO Box 2214, Iraklion, Crete, Greece.
E-mail: makridis@hcmr.gr
*Present address: Biology Department, University of Patras, 26500, Rio, Patras, Greece
Abstract
Five cultures of microalgae (Chlorella minutissima,
Tetraselmis chui, Nannochloropsis sp., Arthrospira
platensis and Isochrysis sp.) with no culturable
bacteria were tested for their ability to inhibit the
growth of six Vibrio bacterial strains (V. parahaemo-
lyticus, V. anguillarum, V. splendidus, V. scophthalmi,
V. alginolyticus and V. lentus). The influence of light
on the antibacterial activity of the microalgae was
investigated. All microalgae cultures inhibited the
growth of bacteria compared with the control
treatments (P < 0.05), and their antibacterial
activity was not influenced by the presence or
absence of light. In the control groups, the num-
bers of bacteria increased exponentially during the
experimental period in the absence of microalgae
cells demonstrating that the bacterial cells were
able to utilize the growth medium of microalgae
cultures. The present results may explain the low
levels or absence of Vibrio strains in microalgae
cultures, and the positive effect of addition of
microalgae in rearing of fish larvae, and implicate
the production of antibacterial compounds by mic-
roalgae cells.
Keywords: fish, larvae, green water, fish patho-
gens, Vibrio
Introduction
Problems with diseases are often encountered in
aquaculture. Use of antibiotics has been at times
extensive and there is continuous research on the
field to limit the use of antibiotics, because their
prolonged use may result in propagation of resis-
tant bacterial strains. An alternative strategy to
1520
doi:10.1111/j.1365-2109.2011.02955.x
antibiotics is the use of natural compounds that
limit the growth of pathogenic bacteria.
Use of cultured microalgae is common in the
rearing of larvae of marine fish, crustaceans and
bivalves. It has been observed that the addition of
microalgae has a positive effect on the bacterial
load of larval rearing systems by decreasing the
numbers of opportunistic bacteria (Salvesen,
Skjermo & Vadstein 1999). Enhanced survival
rates of the larvae in tanks added microalgae are
observed in the rearing of many marine fish spe-
cies (Oie, Makridis, Reitan & Olsen 1997; Shields
2001). The positive effect of microalgae added to
larval rearing tanks has been attributed to the sta-
bilization of the nutritional value of live food
organisms added to the tanks (Makridis & Olsen
1999), as well as to non-nutritional factors such
as the stimulation of the digestive system and the
immune system of the larvae, and the effect of the
bacterial communities associated with the microal-
gae (Reitan, Rainuzzo, Oie & Olsen 1993; Cahu,
Zambonino Infante, Pιres, Quazuguel & Le Gall
1998; Salvesen, Reitan, Skjermo & Oie 2000). The
microalgae used in the aquaculture industry are
not axenic cultures, so their effect on the bacterial
communities of the rearing system could be attrib-
uted to: (i) the microbiota associated with microal-
gae cultures (Makridis, Alves Costa & Dinis 2006),
(ii) compounds produced by the microalgae cells
(Duff & Bruce 1966), and (iii) oxygen radicals pro-
duced during photosynthetic process (Marshall, de
Salas, Oda & Hallegraef 2005).
Microalgae produce secondary metabolites,
which are accumulated in the growth medium at
the end of the exponential and the stationary
phase of growth (Borowitzka 1995). A variety of
compounds have been suggested for having anti-
bacterial activity, such as fatty acids (Desbois,
© 2011 Blackwell Publishing Ltd
Aquaculture Research, 2012, 43, 1520–1527
Mearns-Spragg & Smith 2009), terpenoids, carbo-
hydrates (Duff & Bruce 1966), peptides, polysac-
charides and alkaloids (Borowitzka 1995). The
production of secondary metabolites is usually
higher when culture conditions are unfavourable
(Skulberg 2004).
The aim of this study was to determine the anti-
bacterial activity of cultures of five microalgae spe-
cies with no culturable bacteria against Vibrio
bacteria isolated in aquaculture.
Materials and methods
Microalgae cultures
All experiments were carried out at the Institute of
Aquaculture, Hellenic Center for Marine Research,
Crete, Greece. Cultures of five cultures of microal-
gae with no culturable bacteria were used: (i)
Chlorella minutissima (strain Foti and Novak, iso-
lated from Iraklio bay, Crete), (ii) Tetraselmis chui
isolated by Dr Luis M. Lubián, Instituto de Ciencias
Marina de Andalucı́a, Spain, (iii) Arthrospira platen-
sis (PCC7345) supplied by Institut Pasteur, France
(Collection Nationale de Cultures de Microorganis-
mes), (iv) Nannochloropsis sp. (CCAP 849/9) and
Isochrysis sp. (CCAP 927/14) supplied by Dunstaff-
nage Marine Laboratory, UK (Culture Collection of
Algae and Protozoa, CCAP).
Cultures of C. minutissima with no culturable
bacteria were prepared after inoculation of 2 mL
sterile 70% seawater with C. minutissima cells
grown on solid F/2 growth medium (Guillard &
Ryther 1962), and addition of 10 lg mL 1 broad-
spectrum antibiotic imipenem, while cultures of
T. chui with no culturable bacteria were obtained
after streaking to pure culture in solid F/2 med-
ium.
All microalgae were cultured at 23–25°C under
continuous illumination in F/2 medium, except A.
platensis, which was cultured in ASN-III [contain-
ing 25.0 g NaCl, 2.0 g MgCl2.6H2O, 0.5 g KCl,
0.75 g NaNO3, 0.02 g K2HPO4.3H2O, 3.5 g
MgSO4.7H2O, 0.5 g CaCl2.2H2O, 0.003 g citric
acid, 0.003 g ferric ammonium citrate, 0.0005 g
EDTA (disodium magnesium), 0.02 g Na2CO3,
0.00286 g H3BO3, 0.00181 g MnCl2.4H2O,
0.000222 g ZnSO4.7H2O, 0.0004 g Na2MoO4.
2H2O, 0.00008 g CuSO4.5H2O, 0.00005 g Co
(NO3).6H2O, made up to 1 L with deionized
water and adjusted to pH 7.5 after autoclaving
and cooling].
© 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 1520–1527
Antimicrobial activity in microalgae F Kokou et al.
Every second day, the density of the cultures
was measured using Mallasez haemocytometer,
appropriate microalgae medium was added, and
the cultures were tested for contamination after
plating 50 lL samples on tryptic soy agar added
2% (w/v) NaCl (TSAS) under sterile conditions.
The dishes were incubated for 14 days at 20–22°C
in the dark.
Bacterial strains
A bacterial strain (A-C) was isolated from homoge-
nized Artemia metanauplii, and another bacterial
strain (T-Y) from diseased gilthead sea bream (Spa-
rus aurata). After pure culture, phenotypical tests
were run according to Smibert and Krieg (1994) to
determine: Gram staining, fermentation, oxidase,
catalase test and susceptibility to vibriostatic agent
O/129 (2,4-diamino-6,7-diisopropylpteridine).
In addition, four bacterial strains were used for
the experiments, which were kindly provided
by Prof. Harry Birkbeck, University of Glasgow,
Scotland: Vibrio parahaemolyticus (NCIMB 1902),
Vibrio anguillarum (NCIMB 1197), Vibrio splendidus
(DMC–1), and Vibrio scophthalmi (NCIMB 13623).
A standard growth curve was obtained for each
bacterial strain after culture in tryptic soy broth
added 2% w/v NaCl (TSBS) for 24 h, measurement
of OD600 and spreading of 10-fold dilutions on
TSAS for the enumeration of colony-forming units
(CFU) after 2 days (Makridis, Fjellheim, Skjermo &
Vadstein 2000).
DNA isolation and molecular identification
The two bacterial strains isolated from Artemia
and S. aurata were identified by amplification of
part of 16S rDNA (Jensen, Bergh, Enger & Hjeltnes
2002). Total DNA of the bacterial strains was iso-
lated by cell breakage with heat-shock at 95°C for
5 min using a Peltier Thermal Cycler PTC-200
(MJ Research, Watertown, MA, USA). Cell debris
was removed by centrifugation at 1400 g for
10 min. The universal 16S rDNA primers 27f
(5′-AGAGTTTGATC(A:C)TGGCTCAG-3′) and 1492r
(5′-TACGG(C:T)TACCTTGTTACGACTT-3′) were used
to amplify 16S rDNA sequences (Jensen et al.
2002). PCR was performed in 0.2 mL PCR tubes.
The reaction mixtures had a final volume of 50 lL
and contained 1 lL of DNA template, 5 lL of 109
Taq buffer, MgCl2 at a concentration of 2.5,
200 lmol L 1 of each dNTP, 0.5 lmol L 1 of
1521
Antimicrobial activity in microalgae F Kokou et al.
each primer and 2.5 U of Taq polymerase. Reac-
tions were carried out in a PTC-200 thermal
cycler (MJ Research). All PCR reactions comprised
an initial denaturation step of 95°C for 5 min, 30
cycles of 92°C for 1 min, 58°C for 1 min, 72°C for
1 min, followed by a final extension of 5 min at
72°C. The PCR products, which had an expected
size of 1500 bp, were examined for purity and size
in 1% agarose gels, visualized by staining with
1 mg ethidium bromide and photographed using
white/UV transilluminator UVP.
The amplified rDNA products were purified
using the Nucleospin extract Kit (Macheray-Nagel,
Duren, Germany) and quantified using spectropho-
tometer (NanoDrop V3.30, ND-1000; NanoDrop
Technologies, Wilmington, DE, USA). Purified PCR
products were subjected to single sequencing using
both primers 27f and 1492r (Macrogen, Kumc-
hun-ku, Seoul, Korea). The sequenced fragments
were used as the basis of homology searches per-
formed in BLAST (Altschul, Gish, Miller, Myers &
Lipman 1990).
Experimental procedures
Cultures of five microalgae species with no cultur-
able bacteria, C. minutissima, Tetraselmis chui, A.
platensis (PCC7345), Nannochloropsis sp. (CCAP849/
9) and Isochrysis sp. (CCAP927/14) were used in
a first series of in vivo experiments. The ability of
these microalgae species to inhibit the growth of
six bacterial strains [Vibrio parahaemolyticus
(NCIMB 1902), Vibrio anguillarum (NCIMB 1197),
Vibrio splendidus (DMC–1), Vibrio scophthalmi,
V. alginolyticus and V. lentus] was tested by incuba-
tion of bacteria in cultures of microalgae (co-cul-
ture) with no culturable bacteria (Tendencia &
dela Pena 2003). Bacteria were cultured in TSBS
for 24 h at 20–22°C, and thereafter inoculated in
5 mL of cultures of microalgae at a final concen-
tration of 104 CFU mL 1. Cell concentration in
bacterial cultures was determined by the measure-
Table 1 Experimental conditions for each microalgae species used in the first and second series of experiments
Species Temperature (°C)
C. minutissima 34
T. chuii 26
A. platensis 28
Nannochloropsis sp. 30
Isochrysis sp. 30
1522
Aquaculture Research, 2012, 43, 1520–1527
ment of OD at 550 nm. The necessary volume
was directly added to 5 mL of sterile microalgae
medium. Due to increased dilution rate, the inocu-
lum was not treated further prior to addition. A
control treatment was included for each bacterial
strain and for each microalgae tested, where 5 mL
of sterilized microalgae medium were inoculated
with the bacterial culture. All experiments were
run in duplicates.
The bacterial strains were incubated with the
microalgae cultures for 5 days in natural photope-
riod and light conditions (1500–1700 lux during
midday). Samples were taken 0, 12, 24, 48, 76, 96
and 120 h after start, and 10-fold dilutions were
spread on TSAS dishes to estimate the number of
bacteria present in the samples. The experimental
conditions for each microalgae species are shown
in Table 1. The microalgae cell concentration was
measured at the beginning of each experiment.
The influence of light on the antibacterial activity
of the microalgae was tested in a second series of
experiments where the antibacterial activity of the
microalgae Isochrysis, Tetraselmis and Nannochlorop-
sis against the bacterial strains V. lentus, V. scop-
hthalmi, and V. alginolyticus was examined both in
the same light conditions as in the first series of
experiments, as well as in dark conditions. All other
experimental conditions were kept the same as pre-
viously described and a control treatment was
included for each microalgae species, where each
pathogen was grown in bacteria-free microalgae
medium in both dark and in light conditions. All
experiments were run with two replicates.
Statistical analysis
Differences in concentration of bacteria at each
point of time were analysed using the one-way
analysis of variance (Zar 1998). Statistical analyses
were run using the software program STATGRAPHICS
plus 5.0 (Statgraphics Corporation, Rockville, MD,
USA).
1st experiment (cells mL 1) 2nd experiment (cells mL 1)
76 9 106 Not applicable
1.4 9 106 1.2 9 106
Not countable Not applicable
27.8 9 106 26.5 9 106
7.8 9 106 7.4 9 106
© 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 1520–1527
Aquaculture Research, 2012, 43, 1520–1527 Antimicrobial activity in microalgae F Kokou et al.

deposited in GenBank database and given the

Results
Both bacterial strains isolated were Gram negative,
catalase and oxidase positive, fermentative, and
susceptible to action of O/129. Sequencing of the
16S rDNA genome was used to identify the bacte-
rial strains isolated. The bacterial strains A-C and
T-Y were identified showing a high homology
(99%) by the use of BLAST in NCBI with the
strains of Vibrio alginolyticus and Vibrio lentus,
respectively, and these results were in agreement
with the results of the biochemical characteriza-
tion of the same strains. The partial 16S rDNA
genes sequences of the two isolated strains were
accession numbers FJ870691 and FJ870692 for
strains A-C, and T-Y respectively.
Inoculation of bacteria in all microalgae cultures
inhibited growth of the bacteria compared with the
control treatments in the first series of experiments
(P < 0.05) (Fig. 1). Two bacterial strains, V. algino-
lyticus and V. anguillarum, showed a higher resis-
tance to the antibacterial activity of the microalgae,
and a slower decrease of their bacterial counts com-
pared with the other four bacterial strains (Fig. 1).
However, even in this case, the concentration of
these bacteria in the presence of microalgae was sig-
nificantly lower than in the control group.

Figure 1 Number of bacteria (CFU mL 1) (±SE) in first series of experiments in the presence of light after co-culture
with five microalgae species and control group.

© 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 1520–1527 1523

Antimicrobial activity in microalgae F Kokou et al. Aquaculture Research, 2012, 43, 1520–1527

Figure 2 Number of bacteria (CFU mL 1) (±SE) in the second series of experiments in the presence (graphs to the
left) and absence of light (graphs to the right) after co-culture with three microalgae species.

1524 © 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 1520–1527

Aquaculture Research, 2012, 43, 1520–1527
The other four bacterial strains, V. lentus,
V. splendidus. V. scophthalmi and V. parahaemolyti-
cus were influenced to a high degree by the pres-
ence of microalgae cells and their colonies could
not be detected at the end of the experiment in all
cases. In the presence of cells of C. minutissima,
colonies of these isolates were not detected after
48 h except of V. lentus, which was viable 96 h
after inoculation. In the presence of T. chui and
Isochrysis sp. no growth was shown in the dishes
of these four Vibrio strains 44 h after inoculation,
whereas in the case of Nannochloropsis sp. and A.
platensis no growth was shown in the dishes of
these four Vibrio strains 24 h after inoculation.
In the control groups, the numbers of bacteria
increased exponentially during the experimental
period in the absence of microalgae cells. Bacterial
counts stabilized at about two log scales higher
than the initial bacterial concentration for all bac-
terial strains except V. scophthalmi, demonstrating
that the bacterial cells were able to utilize the
growth medium of microalgae cultures.
In the second series of experiments, incubation
of bacteria in microalgae cultures showed similar
results as in the first experiment (Fig. 2). The con-
centration of bacteria was significantly lower in
the presence of microalgae compared with the
control treatments both under light and dark con-
ditions (P < 0.05). Vibrio alginolyticus was, as in
the first series of experiments, the most resistant
among the three bacterial isolates tested, but it
could not be detected 96 and 120 h after inocula-
tion. A difference was shown as well in the degree
of resistance of the other two strains to the anti-
bacterial action of microalgae compared with first
series of experiments, as V. lentus and V. scopthalmi
persisted the antibacterial action of microalgae for
a longer period of time. This was pronounced in
the case of V. lentus which was present in samples
taken 72 h after inoculation in all experiments in
the dark and the experiment with Nannochloropsis
sp. in the presence of light.
Discussion
Results of the present study showed that cultures
of microalgae strains C. minutissima, T. chuii,
A. platensis, Nannochloropsis sp. and Isochrysis sp.
in the absence of culturable bacteria showed anti-
bacterial activity against bacterial strains V. algino-
lyticus, V. lentus, V. splendidus, V. scophthalmi,
V. parahaemolyticus, and V. anguillarum, as they
© 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 1520–1527
Antimicrobial activity in microalgae F Kokou et al.
prevented successfully the proliferation of these
bacteria when co-cultivated with the microalgae.
As cultures of microalgae with no culturable bac-
teria were used in this study, it can be concluded
that the antibacterial activity was not due to asso-
ciated microbiota. In addition, antibacterial activity
was shown both in the presence and in the
absence of light. It can therefore be concluded that
the antibacterial activity was not caused by oxygen
radicals produced during photosynthesis by the
microalgae cells.
All microalgae strains showed high antibacterial
activity as the numbers of bacteria at the end of
the experimental period (96–120 h after inocula-
tion) were at least 1000 times lower than in the
control treatment for all bacterial isolates in both
experiments. Four of the bacterial strains were
more susceptible to the antibacterial activity of the
microalgae. Some of the bacterial strains were
below the detection level after 48 h. In the second
series of experiments, where bacteria were added
at lower initial cell concentration than in the first
series of experiments, no bacterial strain was
detectable in the two last sampling points (96 and
120 h after inoculation).
No bibliographic data exist on antibacterial
activity of Nannochloropsis sp. At the present study,
however, Nannochloropsis sp. revealed strong anti-
bacterial activity. The microalgae species Nanno-
chloropsis sp., Nannochloropsis oculata, Nannochloris
atomus and Isochrysis sp. have been shown to pro-
duce short chain fatty acids (Roncarati, Meluzzi,
Acciari, Tallarico & Melotti 2004), and it was
shown that production of short chain fatty acids
was dependent of growth phase of the cultures
being higher in cultures in stationary phase. These
compounds are involved in antibacterial activity in
other studies (Defoirdt, Boon, Sorgeloos, Verstraete
& Bossier 2007). Cultures of Isochrysis sp. showed
high antibacterial activity in both experiments as
it has been shown in an earlier study with human
pathogens (Rajeev, Prakash & Bhimba 2006).
Antibacterial activity of members of the Chlorella
genus has been shown in previous studies (Pratt,
Daniels, Eiler, Gunnison, Kumler, Oneto, Strait,
Spoehr, Hardin, Milner, Smith & Strain 1944; Ten-
dencia & dela Pena 2003), although the functional
mechanisms of this activity have not been yet
explained. Cultures of Chlorella sp. were co-culti-
vated with the pathogen V. harvei at 103 CFU
mL 1 concentration, and slowed down bacterial
growth for 72 h (Tendencia & dela Pena 2003).
1525
Antimicrobial activity in microalgae F Kokou et al.
These findings come in agreement with the results
of the present study, and lead us to the conclusion
that microalgae cells probably excrete substances
that inhibit the growth of bacterial strains. Anti-
bacterial action of C. vulgaris is possibly related
with a lipophilic substance, which is called chlorel-
lin, and is produced during the initial phase of
the culture and is released in microalgal culture
medium (Pratt et al. 1944). The antibacterial
activity of Arthrospira and Tetraselmis sp. has been
shown in earlier studies (Ozdemir, Karabay, Dalay
& Pazarbasi 2004; Regunathan & Wesley 2004).
Another possible way of action of antibacterial
activity of microalgae could also be related to dis-
ruption of quorum sensing. Vibrio bacteria are
quite sensitive to such mechanisms as shown in
earlier studies (Van Cam, Van Hao, Dierckens,
Defoirdt, Boon, Sorgeloos & Bossier 2009).
The bacterial strains V. anguillarum and V. algi-
nolyticus showed highest resistance to antibacterial
activity of microalgae. These bacterial strains are
commonly isolated in rearing systems of marine
fish larvae despite the addition of microalgae
(Munro, Barbour & Birkbeck 1994; Verner-Jeffreys,
Shields, Bricknell & Birkbeck 2003; Prol, Bruhn,
Pintado & Gram 2009). In the second series of
experiments, V. alginolyticus decreased to very low
levels. Bacteria well inoculated at a lower initial
concentration in second series of experiments
(about 103 bacteria mL 1) compared with the first
series of experiments 10 4 bacteria mL 1), and
this could explain the lower resistance to antibac-
terial activity of microalgae.
In the second series of experiments, the antibac-
terial activity was slightly different than in the first
series. This was partly explained by the fact that
initial concentration of bacteria was lower in the
second experiment. In addition, a factor that could
explain these differences is the fact that antibacte-
rial activity could be related to the growth phase
of the microalgae cultures. Although we added the
three microalgae species in similar concentration
in both experiments, the growth phase of the origi-
nal cultures could be different and this could have
influenced our results. Nevertheless, the main
result of this study which was the difference
between exponential growth in control groups and
stagnant phase or decrease of counts in the pres-
ence of microalgae cell was repeated in the second
experiment.
In each of these experiments a separate control
treatment was added, where high proliferation of
1526
Aquaculture Research, 2012, 43, 1520–1527
Vibrio was observed. Therefore, the growth of bac-
teria was not influenced by temperature. The pro-
liferation of Vibrio bacterial strains in culture
medium for microalgae indicates that in cases
where for various reasons microalgae show poor
growth, there is a danger for increased bacterial
load in the microalgae cultures. Such cultures
when added in larval rearing tanks may cause
high mortalities (Nicolas, Robic & Ansquer 1989).
In conclusion, all five microalgae species tested
showed antibacterial activity against six patho-
genic and opportunistic bacteria. These results
may explain the absence or low levels of Vibrio
strains in microalgae cultures. The mechanisms of
action of this antibacterial activity should be the
object of future research.
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