Journal of Microbiology,

Biotechnology and Zbakh et al. 2012 : 2 (1) 219-228

Food Sciences

SHORT COMUNICATION

ANTIBACTERIAL ACTIVITY OF BENTHIC MARINE ALGAE EXTRACTS FROM

THE MEDITERRANEAN COAST OF MOROCCO

Hanaâ Zbakh* 1,2, Houda Chiheb1, Hassan Bouziane1, Virginia Motilva Sánchez2 and
Hassane Riadi1

1
Laboratory of Diversity and Conservation of Biological Systems, Applied Algology-
Mycology Team, Faculty of Sciences – Abdelmalek Essaâdi University, 93002 Tetouan.
Morocco.
2
Department of Pharmacology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla,
Spain.

*Corresponding author: (H. Zbakh) [email protected], Tel: +212 5 39 99 75 00

Fax: +212 5 39 99 45 00

ABSTRACT

Marine organisms are potentially prolific sources of highly bioactive secondary

metabolites that might represent useful leads in the development of new pharmaceutical
agents. The Moroccan marine biodiversity including macroalgae remains partially unexplored
in term of their potential bioactivities. Antibacterial activity of methanolic extracts from 20
species of macroalgae (9 Chlorophyta, 3 Phaeophyta and 8 Rhodophyta) collected from
Moroccan Mediterranean coasts was evaluated against Escherichia coli, Staphylococcus
aureus and Enterococcus faecalis. The extracts of the studied Rhodophyceae inhibited
considerably the growth of the three tested bacterial strains and gave inhibition zones between
20 and 24 mm. The results indicate that these species of seaweed present a significant
capacity of antibacterial activities, which makes them interesting for screening for natural
products.

Keywords: Antibacterial activity, Macroalgae, Moroccan Mediterranean coast, Algal extract

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INTRODUCTION

Traditional and modern medicines have relatively exhausted most of their resources in
land plants. However, the marine environment by dint of its biological and chemical diversity
can be a source of new types of agents against cancer and infectious diseases (Bazes et al.,
2006; Chew et al., 2007; Mayer et al., 2007). During the last decades, numerous novel
compounds have been isolated from marine organisms and many of these substances have
been demonstrated to possess interesting biological activities (Duarte et al., 2001; Faulkner,
2002; Ely et al., 2004; Dubber and Harder, 2008).
Marine macroalgae are the most interesting algae group because of their broad
spectrum of biological activities such as antimicrobial (Chiheb et al., 2009, Bouhlal et
al.,2010), antiviral (Bouhlal et al., 2010, Bouhlal et al., 2011, Kim and Karadeniz, 2011),
antifungal (de Felício et al., 2010), anti-allergic (Na et al., 2005), anticoagulant (Dayong et
al., 2008), anticancer (Kim et al., 2011), antifouling (Bhadury and Wright, 2004) and
antioxidant activities (Devi et al., 2011). They produce a wide variety of chemically active
metabolites in their surroundings as an aid to protect themselves against other settling
organisms (Bhadury and Wright, 2004). There are numerous reports of macroalgae derived
chemical compounds that have a broad range of biological activities, some of which have
been used in pharmaceutical industries.
The algal richness of Moroccan coasts is undeniable in terms of diversity and quantity
(Kazzaz and Riadi, 2000). The antimicrobial potential of macroalgae from Mediterranean
Moroccan coasts remains partially unexplored. Many chemically unique compounds of
marine algae with antimicrobial activity have been isolated and a number of them are under
investigation and/or are being developed as new pharmaceuticals such as brominated phenols,
sterols, terpenoids, polysaccharides, peptides, proteins, acrylic acid, terpenes, chlorophyllides,
phenols and heterocyclic carbons etc. (Bhacuni et al. 2005; Li et al., 2007; Bouhlal et al.,
2011; Priyadharshini et al., 2011). The present study was undertaken to examine the
antibacterial effect of methanolic crude extracts of 20 species of marine benthic algae (9
Chlorophyceae, 3 Phaeophyceae and 8 Rhodophyceae), collected from the Mediterranean
Moroccan coasts, against pathogenic bacteria Escherichia coli, Staphylococcus aureus and
Enterococcus faecalis.

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MATERIAL AND METHODS

Algal material

The algae were collected between 1 and 4 m of depth, conserved on ice until use.
Sampling was conducted between May 2005 and June 2008 on the coast of Ksar Seghir
(35°50´52.58"N 5°33´39.04"0), Martil (35°37´10.22"N 5°16´15.79"0) and the lagoon of
Nador (33º37’00.23’’N 3º43’59.82"0). Taxonomic identification of species was performed
with the aid of standard literature and determination keys. Voucher specimens of all species
identified are deposited in the herbarium of our laboratory (Applied Algology-Mycology
Laboratory, Department of Biology, Faculty of Science, Abdelmalek Essaâdi University,
93002 Tetouan, Morocco).

Chemical extraction

The algae samples were rinsed with sterile seawater to remove sand and epiphytes and
washed with water. Then, they were dried on air ambient and finally in an oven (35-40 °C).
The dried seaweeds were crushed by an electric grinder and the obtained powder was then
stored at -12 °C until the extraction step. The powder of dried seaweeds (5 g) was extracted
with methanol (200 ml) for 8 h at 65°C using the Soxhlet apparatus. The obtained extracts
were concentrated under vacuum using a rotary evaporator. The residues were then diluted in
2 ml of pure methanol (Sreenivasa-Rao and Parekh, 1981).

Antibacterial activity test

Antibacterial tests of algal extracts were performed in vitro using the disc diffusion
method (Sreenivasa-Rao and Parekh, 1981) in Petri dishes. Sterile disks (BBLTM) of 6 mm in
diameter were impregnated with 25 µl of seaweeds extract, deposited on the surface of agar
medium (Mueller-Hinton Agar, pH 7.4 ± 0.2 at 25 °C) previously inoculated with bacteria
strains and incubated at 37 °C for 24 h (Ballantine et al., 1987). The results are expressed by
measuring in millimeter the diameters of the inhibition halos of bacterial growth around the
disk. Methanol (100%) without seaweed extract was used as negative control. All tests were
performed in triplicate, and clear halos greater than 10 mm were considered as positive results
(Lima-Filho et al., 2002).

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Bacteria strains

Bacteria strains used in this study are: E. coli ATCC 25922 (Gram-), S. aureus ATCC
25923 (Gram+) and E. faecalis ATCC 29212 (Gram+). They were obtained from the
Department of Microbiology, Faculty of Pharmacy - University of Granada, Spain (ATCC:
American Type Culture Collection).

RESULTS AND DISCUSSION

The antibacterial activity of crude methanol extracts of 20 species of Moroccan marine

algae is shown in the Table 1. The antibacterial activity is ranked from no activity (-:
inhibition diameter <10 mm), low (+: inhibition diameter between 10 and 15 mm), moderate
(++: inhibition diameter between 15 and 20 mm) and high activity (+++: diameter inhibition ≥
20 mm).
Among the algae tested, seventeen species have shown antibacterial activity, the
highest activity was shown by crude extracts of Ulva lactuca, Gracilaria bursa-pastoris and
Chaetomorpha linum. The majority of the algal extracts were active against at least one or
two microorganisms. Fifteen extracts (75%) were moderately to highly active against S.
aureus, seven (35%) against E. coli and finally two extracts (10%) inhibited E. faecalis.
Methanol extracts of Ulva rigida, Enteromorpha compressa, musciformis Hypnea, Caulerpa
prolifera, Asparagopsis armata and Cladostephus spongiosus showed inhibitory activity
against all strains tested.
Among the nine species tested of Chlorophyceae, the extract of Caulerpa prolifera has
the larger diameters of inhibition against S. aureus (23 mm), E. coli (16 mm) and E. faecalis
(13 mm). However, extracts of Ulva rigida (Fig 1), Enteromorpha compressa and Caulerpa
prolifera were the only ones to present inhibitory activity against E. faecalis. Their respective
inhibition diameters are 15 mm, 13 mm and 12 mm.
The results obtained with extracts of Ulva rigida and Caulerpa prolifera from the
Mediterranean Moroccan coasts are similar to those obtained with the same species from the
Canary Islands against Gram+ bacteria (González et al., 2001). Similar results were also
obtained with extracts of ethanol, ethanol/methanol and hexane of Caulerpa prolifera from
Mexico and the Caribbean (Ballantine et al., 1987; Freile-Pelegrin and Morales, 2004).

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Table 1 Antibacterial activity of marine algae extracts from different localities of the
Mediterranean Moroccan coasts

strains
Algae locality
Eco. Ent Sta
Chlorophyceae
Ulva rigida Ksar Seghir + ++ +
Ulva lactuca Nador + - +
U. olivascens Nador ++ - ++
Enteromorpha compressa Ksar Seghir +++ + +
E. linza Nador + - ++
E. intestinalis Nador + - ++
Chaetomorpha linum Nador + - +
Caulerpa prolifera Nador ++ + +++
Codium dichotomum Martil - - +++
Phaeophyceae
Cystoseira humilis Nador + nt ++
C. compressa Nador - - +++
Cladostephus spongiosus Martil + + +++
Rhodophyceae
Gymnogongrus patens Ksar Seghir +++ - ++
Plocamium coccineum Ksar Seghir + - ++
Asparagopsis armata Ksar Seghir + + ++
Centroceras clavulatum Nador ++ - ++
Gracilaria confervoïdes Nador + - ++
G. bursa- pastoris Nador + - +
Hypnea musciformis Nador +++ +++ +++
Alsidium corallinum Nador ++ - ++
Legend: -: no activity; +: 10mm< inhibition diameter <15 mm; ++: 15mm< inhibition diameter <20mm; +++:
inhibition diameter ≥ 20mm). Strains: Eco: Escherichia coli ATCC 25922, Sta: Staphylococcus aureus
ATCC25923, Ent: Enterococcus faecalis ATCC 29213, nt: not tested.

Extract of Cladostephus spongiosus showed a broad inhibitory activity against E. coli

(13 mm), E. faecalis (12 mm) and S. aureus (24 mm). Negative responses of Cystoseira
compressa, C. humilis and Cladostephus spongiosus extracts are consistent with those
reported by González et al. (2001) concerning samples from the Canary Islands.
The extract of Hypnea musciformis (Rhodophyceae) inhibited considerably the growth
of the three strains tested and gave inhibition zones between 20 and 24 mm. In the case of the
extract of Asparagopsis Armata, the inhibition diameters were smaller than those of Hypnea

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musciformis and varied between 12 and 15 mm. S. aureus appeared more sensitive than the
other two strains with larger inhibition zones.

Figure 1 Inhibition zones of Ulva rigida methanolic extract against Enterococcus faecalis in
triplicate and negative control

Nonetheless, E. Faecalis was highly resistant against the majority of the algal extracts
used. It was reported that Gram+ bacteria are more efficiently inhibited by algal extracts than
Gram- bacteria (Sreenivasa-Rao and Parekh, 1981; Pesando and Caram, 1984). Studies on
Hypnea musciformis from the Indian coast showed that methanol-dichloromethane extracts
are more effective against Gram+ bacteria strains than Gram- bacterial strains (Selvin and
Lipton, 2004).

CONCLUSION

The Mediterranean coasts are a source of great biological diversity with an almost
unexplored potential to provide significant therapeutic, as well as nutritional, benefits for
humans. The investigation and exploitation of the potential of marine algae will have
significant health implications for current and future generations, not only for local people
inhabiting in the Mediterranean border countries but also for people from all around the
world.

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From all these results, we can conclude that macroalgae from Mediterranean
Moroccan coasts represent a potential source of bioactive compounds and must be studied for
the production of natural antibiotics. Biochemical analysis are being undertaken to determine
the structure and nature of compounds responsible of the bioactivity of the extracts with high
antibacterial activity. Not only the presence of a particular compound which makes these
organisms, interesting but also their huge diversity and the possibility of not only harvesting
them but also of growing them at different conditions, leading to an enrichment of some
bioactive compounds.

Acknowledgments: Authors greatly thank the Prof. Jose Martinez Lopez from the Faculty of
Pharmacy (University of Grenada, Spain) for providing gratefully the bacteria strains and the
Dr. Conxi Rodriguez–Prieto from the Department of Environmental Sciences of the Faculty
of Sciences (University of Girona, Spain) for his help in discussions of results and for
providing documentation on the systematic of phycoflora.

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