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British Phycological Journal
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Antibacterial activity from marine
microalgae in laboratory culture
Stephen J. Kellam
a
& John M. Walker
a
a
Algae Biotechnology Group, Division of Biological Sciences ,
The Hatfield Polytechnic , PO Box 109, College Lane, Hatfield,
Hertfordshire, AL10 9AB, England
Published online: 23 Feb 2007.
To cite this article: Stephen J. Kellam & John M. Walker (1989) Antibacterial activity from
marine microalgae in laboratory culture, British Phycological Journal, 24:2, 191-194, DOI:
10.1080/00071618900650181
To link to this article: http://dx.doi.org/10.1080/00071618900650181
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Br. phycol. J. 24:191-194
1 June 1989
S HORT NOT E
Antibacterial Activity from Marine Microalgae in
Laboratory Culture
By STEPHEN J. KELLAM and JOnN M. WALKER
Algae Biotechnology Group, Division of Biological Sciences,
The Hatfield Polytechnic, PO Box 109, College Lane,
Hatfield, Hertjordshire ALIO 9AB, England
Culture supernatants and methanolic and hexane extracts of t32 marine microatgae have
been screened against six strains of bacteria. In vitro antibacterial activity was detected in 28
of the organic solvent extracts, primarily those of the hexane. The extracts were most effective
against Staphylococcus aureus and to a lesser extent Bacillus subtitis. No antimicrobial
activity was detected in the culture supernatants and no extract inhibited the growth of
Escherichia coli, Streptococcus faecalis, Klebsiella pneumoniae or Pseudomonas aeruginosa.
The pr oduct i on of ext racel l ul ar ant i bi ot i c
met abol i t es by mar i ne al gae has been well
st udi ed in recent years ( Hor ns ey & Hi de,
1974; Henr i quez et al . , 1979; Accori nt i ,
1983; Rei chel t & Bor owi t zka, 1984;
Caccamese et al., 1985; Cannel l , Ows i anka
& Wal ker , 1988; Kel l am et al., 1988). The
gr eat maj or i t y of these studies have been
made on macr oal gae (seaweeds) col l ect ed by
dr edgi ng or f r om t he seaf r ont . Macr oal gae
ar e less amenabl e t o l abor at or y cul t ure t han
mi cr oal gae, whi ch have gr eat pot ent i al f or
the i ndust ri al devel opment of bi oact i ve
met abol i t e pr oduct i on. I f al gae are t o be
used f or t he l arge scale pr oduct i on o f
phar macol ogi cal l y act i ve compounds , it will
be necessar y t o opt i mi ze pr oduct i on under
cont r ol l ed cul t ure condi t i ons, pe r f or m
gr owt h/ act i vi t y studies and t hen mas s
cul t ure t he i ndi vi dual species.
I n our l abor at or y, we have a p r o g r a mme
t o screen mi cr oal gae f or t he pr oduct i on of
bi oact i ve c ompounds of pot ent i al medi ci nal
val ue (Cannel l et aI., t987; Cannel l et al.,
t988). As par t o f this p r o g r a mme we have
screened 132 mar i ne mi cr oal gae f or t he
pr oduct i on of ant i bact er i al activities.
MATERI AL AND ME T HODS
All algae for the screening programme were
obtained from the Culture Centre of Algae and
Protozoa (CCAP), Cambridge (Asher &
Spalding, 1982). Algae were cultured in 250 ml
conical flasks containing 100ml of marine
medium (CCAP medium Ml l ) and agitated on
orbital shakers for periods ranging between 30
and 50d. The incubation temperature was
constant at 25C and illumination at 2500 lx with
a 16: 8 h light-dark cycle provided by warm-
white fluorescent strip lamps. Those cultures
showing obvious contamination or that did not
grow were discarded.
The cultures were harvested when the onset of
senescence was apparent. Those that showed no
visible changes were harvested after 50 d. The
cells were harvested by centrifugation at 5000 g
for 5 min. The aqueous supernatant was collected
and the algal pellet was extracted with 10 ml of
methanol (SLR grade) followed by 10rot of
hexane (HPLC grade), each time by shaking for
20 min. The culture filtrates and solvent extracts
were stored at - 18 C until screening.
The following bacteria were used as test
organisms: Gram-positive Staphylococcus aureus
[Hatfield Polytechnic (HP) culture B3], Bacillus
subtilis (HP culture B30; Ex: NCIB 8054) and
Streptococcusfaecatis (HP culture B80; Ex: NCIB
8256); Gram-negative bacteria Escherichia coli
(HP culture Bl l ; Ex: NCTC 9002), Ktebsielta
0007-1617/89/020191 + 04 $03.00/0 1989 British Phycological Society
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192 S. J . Kellam and J. M. Walker
pneumoni ae (HP culture B97; Ex: NCI B 7244)
and Pseudomonas aeruginosa ( HP culture B13;
Ex: NCI B 418). All were gr own in 100ml of
nut ri ent br ot h (Oxoi d CM3, 13 g 1 i) overni ght
at 37C in an orbi t al shaker.
Fi ft een ml aliquots of nut ri ent agar (Oxoi d
CM3, 28g 1 -~) cont ai ni ng 2, 3, 5-t ri phenyl
t et razol i um chl ori de (25 ~tg ml -~), were seeded
wi t h 25 ~tl of a 1 : l 0 dilution in sterile distilled
wat er of either the E. coli, S. aureus,
P. aeruginosa, or K. pneumoni ae culture, or 120 ~tl
of a 1 : 10 dilution of B. subtilis or S. f ae c al i s
culture. The seeded aliquots were i nt roduced i nt o
sterile Petri plates after gentle swirling and a
sterile 5 mm cork borer used to produce wells in
t he agar when coot.
A test sampl e of 40 ~tl was placed in each well.
On each pl at e cont rol wells were i ncl uded
cont ai ni ng the same vol ume of the appr opr i at e
sol vent used for ext ract i on or mari ne medi um.
The plates were pre-i ncubat ed at 4C for 1 h to
allow the diffusion of solutes into t he agar
surroundi ng t he well and were t hen i ncubat ed at
37C for 24 h.
Those ext ract s cont ai ni ng ant i bact eri al com-
ponent s pr oduced distinct, clear, circular zones of
i nhi bi t i on ar ound t he wells and t he radii of these
were measured.
TABLE I. Antibacterial activity of methanolic and hexane extracts of marine microatgae. Radii of zones of inhibition
given in mm with hole diameter subtracted
Test organism
S. aureus B. subtilis
Alga CCAP no. Methanol Hexane Methanol Hexane
Chiorophyta
Chlorophyceae
Chlamydomonas vectensis 11/90 - - 4-5
Chlorelta stigmatophora 211/20 1-5 6-5
Chlorella sp. 211/53 - - 3-0
Nannochloris maculata 25 I/3 - - 4-5
Nannochloris oculata 251/6 - - 7.0
Prasinophyceae
Tetraselmis chui 8/6 - - 4.5
Tetraselmis sp. 66/8 - - 3.5
Tetraselmis hazeni 66/7 - - 4-0
Tetraselmis impelagicus 66/32 - - 4.0
Tetraselmis marina 163/1B - - 2.5
Tetraselmis striata 66/5 - - 4,5
Tetraselmis suecica 66/4 - - 7-0
Tetraselmis suecica 66/22D - - 5.0
Tetraselmis tetrathele 66/IC - - 5-5
Cryptophyta
Cryptomonas calceformis 979/6 1"5 - -
Chrysophyta
Bacillariophyceae
Amphiprora hyalina 1003/1 2.0 6.0
Gyrosigma spenceri 1034/1 - - 3.5
Nitzschia closterium 1052/8B - - 6.5
Phaeodactylum tricornutum 1052/1A - - 6-0
Phaeodactylum tricornutum 1052/6 1.5 7.0
Chrysophyceae
Coccolithus pelagicus 913/2 - - 6,5
Cricosphaera elongata 961/3 .-- 8,0
Ochrosphaera neapolitana 932/1 2.0 6-5
Pavlova gyrans 940/4 1"5 4-5
Syncrypta gtomerifera 958/1 2,0 4- 5
Phaeophyta
Streblonema sp. 1337/I 1.5 - -
Rhodophyta
Porphyridium purpureum 1380/3 - - 4.0
4-0
5.5
5-0
5.5
4.5
2.5
3.0
6.5
4.0
4-0
5.5
7.0
5.5
6,5
5.0
7.5
5-5
4-0
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Antibacterial activity from marine mieroalgae 193
RESULTS
The algal ext ract s t hat demons t r at ed
ant i bact er i al act i vi t y are shown in Tabl e I.
No sol vent or mar i ne me di um cont r ol , nor
any cul t ure super nat ant , pr oduced zones of
i nhi bi t i on. The gr owt h of E. coli, S. f aecal i s ,
K. pne umoni ae or P. aerugi nosa was not
i nhi bi t ed by any ext ract .
DI S CUS S I ON
Mar i ne or gani sms are known t o cont ai n a
wide r ange of novel st r uct ur es ( Baker &
Mur phy, 1981) and c ompounds wi t h ant i -
mi cr obi al act i vi t y are i ncreasi ngl y bei ng
i sol at ed f r om mar i ne al gae ( Oht a, 1979;
Faul kner , 1986). The numbe r of species so
f ar exami ned is onl y a smal l per cent age o f
t he est i mat ed t ot al of 30,000 and it is likely
t hat new ant i bi ot i cs will be i sol at ed.
However , in t hose screening studies where
in vi vo ant i bact er i al t est i ng was subsequent l y
per f or med (e.g. Rei chel t & Bor owi t zka,
1984), t he gr eat maj or i t y of t he met abol i t es
det ect ed in vi t ro were f ound to be ineffective.
Of t he 132 mi cr oal gal species screened
here, 27 (20. 5%) showed significant in vi t ro
ant i bact er i al act i vi t y agai nst at least one
Gr am- pos i t i ve bact er i um. Thi s figure is
si mi l ar to t hose f r om pr evi ous studies, t he
gr eat maj or i t y of which have been upon
macr oal gae. Usi ng a var i et y of medi a and
gr owt h condi t i ons, the numbe r of species
f ound t o be pr oduci ng ant i bact er i al
activities woul d pr obabl y i ncrease.
A vari et y of sol vent s f or t he ext r act i on o f
t he al gal bi omass has been r epor t ed, hexane,
met hanol , et hanol and di chl or omet hane
bei ng the mos t frequent l y used. Li ke ot her
wor ker s (e.g. Mor eau, Pes ando & Ca r a m,
1984), we have f ound t hat hexane pr oduced
t he l ar ger zones o f i nhi bi t i on; this pr oba bl y
reflects the nonpot ar nat ur e of the act i ve
component s . S. aureus has pr oved t he mos t ,
and E. col i t he least, suscept i bl e bact er i um
t o i nhi bi t i on by algal ext ract s in ma n y
studies (e.g. Hor ns ey & Hi de, 1974; Rei chel t
& Bor owi t zka, 1984), i rrespect i ve of t he
sol vent used f or ext r act i on of t he al gal
bi omass. Hor ns ey & Hi de (1974) al so
r epor t ed t hat B. subt i l i s was i nhi bi t ed by the
s ame algal or gani c sol vent ext r act s but t o a
lesser extent, The expl anat i on f or this is
uncert ai n, par t i cul ar l y since the act i ve met a-
bolites are unkown.
Ant i bact er i al activities f r om Gyr os i gma
spenceri and Ni t z s c hi a sp. ( Auber t , Auber t &
Gaut i er , 1968) and Tet r as el mi s sp. (Duff,
Bruce & Anti, t 966) have been r epor t ed
previ ousl y, but t he act i ve c ompone nt s have
not been i sol at ed and char act er i zed.
Ant i bact er i al act i vi t y in our st udy was
seen pr edomi nant l y f r om the algal classes
Pr asi nophyceae and Baci l l ari ophyceae, wi t h
Tet r as ehni s species bei ng the mos t not abl e
f r om the f or mer class. These t wo classes
woul d t her et br e seem par t i cul ar l y wor t hy of
f ur t her i nvest i gat i on.
ACKNOWL E DGE ME NT S
This work was supported by the NAB Bio-
technology Initiative. We are particularly grateful
to Dr John Baker, Director of the CCAP,
Cambridge for access to his culture collection.
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