World Journal of Microbiology & Biotechnology 2005 21: 4550

DOI: 10.1007/s11274-004-1555-3

Springer 2005

Production of antibacterials from the freshwater alga Euglena viridis (Ehren)

B.K. Das*, J. Pradhan, P. Pattnaik, B.R. Samantaray and S.K. Samal
Aquatic Animal Health Division, Central Institute of Freshwater Aquaculture (CIFA), P.O. Kausalyagnaga,
Bhubaneswar 751002, Orissa, India
*Author for correspondence: Tel.: +91-674-2465446, Fax: +91-674-2465407, E-mail: basantadas@yahoo.com
Received 28 January 2004; accepted 20 May 2004

Keywords: Aeromonas hydrophila, bio control, Euglena viridis, organic extract, Pseudomonas, Vibrio

Summary
The antibacterial properties of Euglena viridis, collected from a freshwater pond at the Central Institute of
Freshwater Aquaculture (CIFA), Bhubaneshwar, India, were tested against various strains of virulent pathogens
viz. Pseudomonas putida (PP1, PP2), P. aeruginosa (PA1, PA2, PA3, PA4), P. uorescens (PF1, PF2, PF3, PF4),
Aeromonas hydrophila (AH30, AH31, AH32, AH34), Edwardsiella tarda, Vibrio alginolyticus (VA1), V. anguillarum
(VN1, VN2 & VN3), V. uvialis (VF1), V. parahemolyticus (VP1) and V. harveyi (VH1) and Escherichia coli (O115,
O1, O156, O164, O111 & O109). Four organic extracts viz. methanolic, ethanolic, acetone and acetone/ethanol of
the E. viridis showed moderate to high antibacterial activity to all the bacterial pathogens. Rotavapor extraction
products showed higher sensitivity in comparison to cold and hot extractions.
Introduction
Freshwater microalgae comprise a vast group of photosynthetic, heterotrophic organisms, which have an
extraordinary potential for cultivation as energy crops.
They are able to produce a wide range of commercially
interesting byproducts such as fats, oils, sugars and
functionally bioactive compounds. Discovering new
therapeutic molecules is becoming increasingly important as more and more bacteria become resistant to the
commonly used antibiotics. Traditionally used in Asiatic
medicines, algae, since the second half of the 20th
century, have been screened for their biological activities. Thus, antibacterial eects have been noticed in all
the algal classes and notably in diatoms, the major
component of the phytoplankton (Du et al. 1966;
Cooper et al. 1983; Reichelt & Borowitska 1984; Viso
et al. 1987). However, most of these antibiotic activities
have only been tested against human pathogens and the
active molecule(s) have rarely been puried. The antibacterial activities of marine algae have been well
documented (Vora & Babu 1994; Padmakumar 1997;
Naviner et al. 1999). However, a few studies have been
initiated in freshwater algae (Moore et al. 1996).
The present preliminary investigation was carried out
to examine the production of antimicrobial/bioactive
compounds from freshwater Euglenoids by using microbially based assay systems. In this preliminary work
we have investigated the eects of organic extracts of
Euglena viridis (Ehren) against dierent strains of sh
and shellsh pathogens, e.g. Aeromonas hydrophila,
Pseudomonas putida, P. aeruginosa, P. uorescens,

Edwardsiella tarda, Vibrio alginolyticus, v. anguillarum,

V. harveyi, V. uvialis, and V. parahaemolyticus and
animal isolates of Escherichia coli.

Materials and methods

Euglena viridis blooms were collected from the ponds
(0.22 ha) of the Central Institute of Freshwater Aquaculture (CIFA), Kausalyaganga, Bhubaneswar, India in
month of September 2002 with the help of a plankton net
made of bolting silk cloth. The samples were simultaneously (4%) preserved in formalin for identication and
identied as E. viridis as per the protocols cited in the
Records of the Botanical Survey of India (Biswas 1949).
Collected samples were washed three times in sterile
ltered water in order to remove suspended particles
adhered to it and nally centrifuged at 1000 g using
macro rotor (Sorvall CE, UK). The pellets were harvested and 4.0 kg of E. viridis (w/w) was dried under
room temperature for 23 days. The dried weight of the
algae was taken and found to be 2.46 kg. Then it was
powdered. Five grams of dry euglena powder were taken
for each solvent extraction. Eight samples were taken, to
which dierent solvents were added.
Solvent extraction
Chloroform, methanol, ethanol, acetone and acetic acid
were used for solvent extraction. Three types of extraction, i.e. cold extraction, hot extraction and Rotavapor
extraction were followed.

46
Cold extraction
Eight samples were prepared by adding dry euglena
powder (5 g each) and 25 ml of chloroform (EC1),
methanol (EC2), acetic acid (EC3), acetone (EC4),
ethanol (EC5), chloroform/methanol (EC6), acetic
acid/acetone (EC7) and acetone/ethanol (EC8) separately respectively. They were incubated for 7296 h at
room temperature (30 C).
Hot extraction
Eight samples were prepared by adding dry euglena
powder (5 g each) and 25 ml of chloroform, methanol,
ethanol, acetone, acetic acid, chloroform/methanol,
acetic acid/acetone, acetone/ethanol, separately respectively. Then it was kept for 7296 h in a hot water bath
(Remi, India) at 60 C.

B.K. Das et al.

diam., Hi Media, India). After solvent evaporation the
discs were placed on nutrient agar (Hi Media, India) test
plates. The plates were incubated for 48 h at 37 C. In
the case of Rotavapor extracts 160 lg/15 ll concentration was applied to paper discs. Discs with solvent
(15 ll) used for dissolution were taken as control after
evaporation of the solvent. The zone of inhibition of
bacteria around the disc (average of three experiments)
was measured, and the assay was scored positive (+) if it
was <5 mm, double positive (++) if 5 mm, triple
positive (+++) if 10 mm and negative ()) if there was
no inhibition of microbial growth. The antibacterial
activities of Euglena extracts were compared with
inhibition zones around three commercial antibacterial
discs, i.e. Fluconazole (Fu), Cotrimazole (Cc) and
Cephaloxin (Cp) (Hi Media, India) that were used as
references.

Rotavapor extraction

Results

Fifty grams of dry euglena powder was added to 1.5 l

ethanol and incubated for 24 h at room temperature.
After incubation it was ltered using Whatman lter
paper no. 40, was made solvent-free by using a Rotavapor (Buchli rotary evaporator-11) rotary evaporation.
The same procedure was followed for methanolic and
aqueous/ethanol extraction. All the crude extracts were
concentrated under vacuum at 35 C and stored in
darkness at 4 C.

Antibacterial sensitivity of Pseudomonas species

Microorganisms
A. hydrophila (AH30, AH31, AH32 and AH34), P.
putida (PP1, PP2), P. uorescens (PF1, PF2, PF3 &
PF4), P. aeruginosa (PA1, PA2, PA3, PA4), V. alginolyticus (VA1), V. anguillarum (VN1, VN2, VN3), V.
uvialis (VF1), V. parahemolyticus (VP1), V. harveyi
(VH1), E. coli (O115, O1, O156, O164, O111 & O109)
and E. tarda which were maintained in the Aquatic
Animal Health Division, CIFA, Bhubaneswar, were
taken for the antibacterial sensitivity study. Pure cultures of dierent bacterial strains were taken and
inoculated into brain heart infusion (BHI) broth (Hi
Media, India) and incubated at 37 C for 18 h. All the
crude extracts of Euglena were tested against the
bacteria listed in Table 1(ad).
Antibacterial sensitivity assay
Algal extract activities were tested by the agar disc
diusion method (Chabbert 1963). 12 ml of nutrient
agar was introduced into sterile petri dishes (9 cm diam.)
and the agar were cooled prior to introducing the
bacteria and that the agar was then allowed to set prior
to use. Then the medium was inoculated with 50 ll of
18 h broth culture of respective bacteria (107 bacteria/
ml) in to each plate. Each extract 160 lg/15 ll concentration was applied to sterile lter paper discs (6 mm in

Hot and cold extracts of EC4 showed identical zones

(12 mm) of activity against P. putida (PP1, PP2). In
contrast EC5 showed a mild zone (8 mm) of inhibition
in both cold and hot extracts to P. putida (PP1) and was
highly sensitive (+++) to both cold and hot extracts.
Hot extracts (EH2) showed higher antibacterial activity
to both the strains of P. putida than cold extracts of
EC2. However both the hot and cold extracts of EC8
showed high sensitivity to P. putida (PP1 & PP2)
(Table 1a).
While comparing the hot extracts and cold extracts
with Rotavapor extracts, it was found that methanolic
and ethanolic extracts in the Rotavapor produced a
higher zone of inhibition than that of cold and hot
extracts. P. putida (PP1 & PP2) strains produced a
greater zone in Rotavapor extracts than that of EC2 and
EC8.
The activity of various extracts against P. aeruginosa
and P. uorescens is indicated in Figure 1. Organic
extracts (EC2, EC4, EC5 and EC8) produced zones of
inhibition 1015 mm to four strains of P. aeruginosa
(PA1, PA2, PA3 & PA4) except EC2 extract to strain
PA1 and PA4 which produced zones of 4 and 6 mm
respectively to both cold and hot extracts. However,
Rotavapor extracts produced zones of inhibition between 11 and 18 mm (Figure 2).
P. uorescens strain (PF1) showed a moderate zone of
inhibition (++) to EC2, EC4, EC5, EC6, EC7 and EC8
(Table 1a), whereas PF3 strain showed the greatest zone
of inhibition (+++) to EC2, EC5 and EC8 and a
moderate zone of inhibition (++) to EC4 and a mild
zone of inhibition to EC2, EC6 and EC7. P. uorescens
(PF3, PF4) strains showed the greatest zone to EC8
moderate to EC4 and EC5. In case of EC2 extracts P.
uorescens, PF3 strain showed a high zone of inhibition
as compared to PF4, which showed mild zone (++) of

Antibacterial properties of Euglena Viridis

47

Table 1 Antibacterial activity of various extracts.

Sl. no.Dierent extracts

Pseudomonas putida

Pseudomonas aeruginosa

PP1

PA1

PP2

Pseudomonas uorescens

PA2

PA3

PA4

PF1

PF2

PF3

PF4

(a) Euglena against Pseudomonas putida, P. aeruginosa and P. uorescens

1
Chloroform
)
)
)
)
2
Methanol
++
+++
+
+++
3
Acetic acid
+
+
+
+
4
Acetone
+++
+++
+++
+++
5
Ethanolic
++
++
+++
+++
6
Chloroform/methanol
++
)
)
)
7
Acetic acid/acetone
+
+
+
+
8
Acetone/ethanol
+++
+++
+++
+++
9
Fluconazole(Fu)10 mcg, +++
++
+++
+++
10
Clotrimazole(Cc), 10 mcg +++
+++
+++
+++
11
Cephalaxin(Cp), 30 mcg ++
+++
++
+++

)
+++
+
+++
+++
)
)
+++
+++
+++
++

)
++
+
+++
+++
)
+
+++
+++
++
++

)
++
+
++
++
++
++
++
++
+++
++

)
+++
+
++
+++
+
+
+++
++
+++
++

)
+++
+
++
++
++
+
+++
+++
+++
++

+
++
+
++
++
+
)
+++
+++
+++
+++

Aeromonas hydrophila
AH30

E. tarda
AH31

(b) Euglena against Aeromonas hydrophila (AH30-AH34) and E. tarda

1
Chloroform
)
)
2
Methanol
+++
+++
3
Acetic acid
+
+
4
Acetone
+++
+++
5
Ethanolic
+++
+++
6
Chloroform/methanol
+
+
7
Acetic acid/acetone
+
+
8
Acetone/ethanol
++
+++
9
Fluconazole(Fu), 10 mcg
+++
+++
10
Clotrimazole(Cc), 10 mcg
+++
+++
11
Cephalaxin(Cp), 30 mcg
+++
+++

(c) Euglena against Vibrio (VA1,

1
Chloroform
2
Methanol
3
Acetic acid
4
Acetone
5
Ethanolic
6
Chloroform/methanol
7
Acetic acid/acetone
8
Acetone/ethanol

V. alginolyticus

V. anguillarum

VA1

VN1

VN1, VN2, VN3, VF1, VP1, VH1)

)
)
+++
+++
)
+
++
+++
+++
++
+
+
+
+
+++
+++
O115

O1

(d) Euglena against E. coli (O115, O1, O111, O109, O164, 156)
1
Chloroform
)
)
2
Methanol
+++
+++
3
Acetic acid
+
+
4
Acetone
+++
++
5
Ethanolic
+++
+++
6
Chloroform/methanol
)
)
7
Acetic acid/acetone
+
+
8
Acetone/ethanol
+++
+++

AH32

AH34

)
+++
+
+++
+++
+
)
+++
++
+++
+++

)
+++
)
++
+++
+
+
+++
+++
++
+++

+
+++
+
+++
++
+
+
+++
+++
++
+++

V. uvialis

V. parahemolyticus V. harveyi

VN2

VN3

VF1

VP1

VH1

)
+++
)
+++
+++
+
+
+++

)
++
+
+++
++
)
)
+++

)
++
+
+++
+++
)
+
+++

)
+++
+
+++
+++
)
+
+

++
+++
+
+++
+++
)
+
+++

O111

O109

O164

O156

)
++
)
+++
++
)
+
+++

+
+++
)
++
++
)
)
+++

+
+++
)
++
++
++
)
+++

)
+++
+
+
+++
+++
+
++

Zone size = 0 or <0=), up to 5=+, 510 = ++ and 1020 = +++.

inhibition. There is no dierence between the zones of

inhibition for cold, hot and Rotavapor extracts against
P. uorescens while comparing the conventional antibiotics such as Fluconazole (Fu), Cotrimazole (Cc) and
Cephaloxin (Cp).

Antibacterial sensitivity of A. hydrophila and E. tarda

The results of the antibacterial sensitivity of A. hydrophila are listed in Table 1b. It is clearly evident that EC1
did not show any positive eect on any of the strains of

48

B.K. Das et al.

14
12

Zone size in mm

10
8
6
4
2
0
Chloroform

Methanol

Acetic acid

Acetone

Ethanol

Chlo/meth

Acetic/acetone Aceto/ethanol

Extracts

PF1

PF2

PF3

PF4

Figure 1. Antibacterial sensitivity of extracts of Euglena against Pseudomonas uorescens (PF1, PF2, PF3, PF4).

A. hydrophila (AH30, AH31, AH32 and AH34). E. tarda

showed mild antibacterial sensitivity to EC1 extract. Out
of all extracts of E. viridis, EC2, EC4, EC5 and EC8
showed moderate to high antibacterial activities against
all the strains of A. hydrophila and E. tarda.
All the strains of A. hydrophila (AH30, AH31, AH32
& AH34) and E. tarda showed high sensitive to EC2,
EC4, EC5 & EC8 organic extracts except AH34 strain to
EC4 & AH30 strain to EC8 and E. tarda to EC5 which
was moderately sensitive. Among all the A. hydrophila
strains, the widest zone was produced by AH34 strain to
EC2 & EC4 extracts (13 mm each). However, Rotavapor extracts (EtOH & MeOH) produce the highest zone
of inhibitions (1922 mm) to all the extracts to all the
strains of A. hydrophila and 1315 mm to E. tarda.

Antibacterial sensitivity of Vibrio species

Vibrio alginolyticus (VA1) showed wide zones of inhibition (1013 mm, +++) to both the cold and hot
extracts of EC2, EC4, EC5 & EC8. In case of Rotavapor
extracts the zone of inhibition was 18 mm to methanolic
extracts and 20 mm to ethanolic extracts against V.
alginolyticus. All the strains of V. anguillarum (VN1,
VN2 & VN3) showed a wide zone (+++) of inhibition
against EC2, EC4, EC5 and EC8 except VN3 strains of
V. anguillarum to EC2 and EC5, which showed moderate zone of inhibition (++). V. uvialis (VF1) showed a
high zone of inhibition (except EC2) to EC4, EC5 and
EC8. V. parahaemolyticus (VP1) and V. harveyi (VH1)
showed a high zone of inhibition to EC2, EC4 and EC5

25

Zone size in mm

20

15

10

0
PP1

PP2

PA1

PA2

PA3

PA4

PF1

PF2

PF3

PF4 AH30 AH31 AH32 AH34 VA1

VN1

VN2

VN3

VF1

VP1

O115

O1

O156 O164 O111 O109

Extracts
Ethanol Methanol

Figure 2. Antibacterial activity of Rotavapor extracts of Euglena against P. putida (PP1, PP2), P. aeruginosa (PA1PA4), P. uorescens (PF1
PF4), A. hydrophila (AH30AH34), Vibrio (VA1, VN1,VN2, VF1,VP1, VH1) and E. coli (O115, O1, O156, O164,O111, O109).

Antibacterial properties of Euglena Viridis

of E. viridis. In case of extract EC8, V. harveyi showed a
zone of inhibition (11 mm) and V. anguillarum showed
negative activity. However, in most cases Rotavapor
extraction produced larger zones of inhibition, than that
of methanolic and ethanolic cold and hot extracts
(Table 1c).
Antibacterial sensitivity of E. coli
The antibacterial responses of E. coli to various organic
extracts of Euglena are represented in Table 1d. The
antibacterial response was found to be similar to other
bacteria. However, EC1 showed mild positive response
to O109 and O164. In contrast O1, O109, O164, O156
and O115 serotypes of E. coli showed a wide zone of
inhibition to EC2 & EC8 and showed moderate zone of
inhibition to EC4 & EC5 except O156. All the strains
of E. coli showed high sensitivity to EtOH & MeOH
Rotavapor extracts of E. viridis.

49
As Euglena causes blooms in the freshwater aquaculture system in tropical countries and if the blooms
persist for a long time, it is detrimental to sh growth. It
is high time to develop some of the processed product
for this bloom which could be eective in controlling
sh microbial diseases as well as enteric pathogens. The
results obtained in the present experiment could not be
compared with other workers, as literature on antibacterial study of this alga, on antibacterial pathogens is
not available.

Acknowledgements
The authors are grateful to the Indian Council Agricultural Research (ICAR), New Delhi for providing the
nancial assistance and the Director, Central Institute
Of Freshwater Aquaculture (CIFA), Bhubaneswar for
providing facilities for the experiment.

Discussion
References
This study conrms that Euglena viridis has antibacterial
properties; however the result should be further
strengthened by individual fractionation through silica
gel column chromatography. It was noticed that EC2,
EC4, EC5 & EC8 extracts showed more antibacterial
activity against all the strains of Pseudomonas species, A.
hydrophila, E. tarda, Vibrio species and serotypes of E.
coli. The antibacterial activity of the algal extracts was
very encouraging for all the aquatic bacteria and E. coli,
which was isolated from animal sources tested here.
Bacterial activity of unsaturated and saturated long
chain fatty acids has been reported (Neiman 1954;
Galbraith & Miller 1973ac). These authors have shown
that fatty acids of chain length more than 10 carbon
atoms induced lysis of bacterial protoplasts. Thus many
authors have found antibacterial activities of microalgae
due to fatty acids (Du et al. 1966; Cooper et al. 1983;
Findlay & Patil 1984; Viso et al. 1987; Kellam et al.
1988).
So, due to the increase of therapeutic resistance to
conventional antibiotics (Hjeltnes et al. 1987; Aoki 1992;
Nash et al. 1992) and due to its antibacterial activity
there appears to be a signicant role for E. viridis in the
control of sh and shellsh diseases. However, aqueous
extracts did not show potential in controlling the
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The present study has not characterized what components of the extracts are responsible for antibacterial
activity. However, the process has been initiated in our
laboratory by fractionating the individual components
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Antibacterial properties of Euglena Viridis

51