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O ffprint from “M arine Biology”
International Jou rn a l on Life in Oceans and Coastal Waters, Vol. 12, N o. 3, February 1972, Pages 189— 193
© by Spring er-Verlag 1972 • Printed in Germany
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Are some bacteria toxic for marine algae ?
B. R.
B e rla n d ,
D. J .
B o n in
and
S.
Y.
M a e s trin i
Station m arine d ’E ndoum e; Marseille, F rance
Abstract
I t was observed in vitro th a t some bacteria from different
origins, including th e sea, are capable of inhibiting th e grow th
o f several m arine algae. However, th ere is too m uch difference
betw een in vitro experim ents a n d in situ environm ental con­
ditions to assum e th a t bacterial poisons m ay rule a g rea t deal
o f algae-bacteria relationships in th e sea as a whole. In th e
a u th o r’s opinion th e im portance of bacterial poison is lim ited
to special an d rare instances.
Introduction
T here is now a g re a t deal of evidence t h a t th e algal
and b acterial enviro n m en t in th e sea is n o t only ruled
b y a sim ple tro p h ic relationship; antibiosis m ay also
p la y a n im p o rta n t role in m arine ecology.
R ed -tid e algal tox in s h av e a ttra c te d a tte n tio n over
a long period because of th e ir toxic effects on fishes and
even m am m als, including m an (review b y S h i l o ,
1967). Some m arin e algae h av e also been proved
capable o f an tib io tic synthesis against b acteria
( S t e e m a n - N i e l s e n , 1955; S i e b u r t h , 1959; B u r k ­
h o l d e r e t ah, 1960; A u b e r t , 1965; D u f f e t al., 1966).
I t h a s long been recognised t h a t sea w ater possesses
bactericidal properties. R o s e n f e l d and Z o b e l l (1947)
suggest th a t th is m a y be due to th e existence of an
autoch th o n o u s bacterial m icroflora w hich produces
an tib io tic substances. K r a s i l ’n i k o v a (1964), B u r k ­
h o l d e r e t al. (1966) a n d G a u t h i e r
(1969, 1970)
re p o rt th e p ro d u ctio n o f an tib acterial substances by
m arine bacteria.
I n reg ard to organism s isolated from b o th fish and
w aste ponds, S h i l o (1967) an d S t e w a r t a n d B r o w n
(1969) p o in ted o u t th a t, in m ixed-plate cultures, th e
bacteria m ay lyse C y an o p h y ta or C hlorophyta cells.
I n o u r in vitro experim ents we observed th a t th e
substances released b y some b acteria in h ib it m arine
algae grow th u n til d e a th ensues.
Material
T he following algae species were used. Chloro­
phyceae: Chlamydomonas m agnusii1', Prasinophyceae:
1 D eterm ined b y Professor P. G a y r a l , Caen.
25
Marine Biology, Vol. 12
Prasinocladus m arinus2, Tetraselmis striata-, Bacilla­
riophyceae: Am phora sp ., Asterionella japonica, Chae­
toceros lauderi, H em iaulus sinensis, N itzschia acicula­
ris, Phaeodactylum tricornutum, Thalassiosira rotula-,
C hrysophyceae: Stichochrysis im mobilis1; X a n th o p h y ­
ceae: Heterothrix sp.3, M onallantus salina1.
All strain s were previously obtained in bacteriafree cultures an d routinely grow n in P r o v a s o l i ’s
(1968) E S solution. Tetraselmis striata and N itzschia
acicularis are P r o v a s o l i ’s collection strains (H askins
L aboratories, N ew -H aven, USA) obtained th ro u g h the
courtesy of D r. A u b e r t (C.E.R.B.O.M ., N ice, F rance);
th e o ther species were isolated from th e G ulf of
Marseilles.
Several of th e bacterial stra in s which hav e been
te ste d were obtained from th e I n s titu t P a ste u r (Paris,
F rance) by courtesy of D r. B i a n c h i (S tation m arine
d ’E ndoum e): Pseudomonas aeruginosa (C.I.P. strain
n um ber 6352), P . fluorescens (5690), P . m altophilia
(5490), P . putida (6323), Vibrio cholerae (6215). Dr.
B i a n c h i also provided stra in 558, which is a Pseudo­
m onas v ery closely related to Pseudomonas pseudoalcaligenes, an d strain 601, a brevibacterium ; b o th were
collected from deep m ud ( B i a n c h i , 1971). T w enty
stra in s were isolated from algae cultures as previously
described ( B e r l a n d e t ah, 1969); 8 Pseudomonas,
2 Vibrio, 4 Achromobacter an d 6 Flavobacteria were
used. T hree o th er strain s of th e sam e origin, as y e t
undeterm ined, were also used (reference num bers
00.27, 00.29, 00.30 of our collection). Two strains
(references 26.32 an d 26.51) were isolated off th e Gulf
o f Fos, near Marseille. These 5 strain s are presently
being investigated as to th e ir taxonom ic position.
Toxicity o f P s e u d o m o n a s a e r u g in o s a to
T e tr a s e lm is s t r i a t a
P seudom onads are freq u en tly reported to cause
diseases in anim als and p lan ts ( S h a c k l e t o n , 1968;
S i n d e n an d D u r b i n , 1969; H e c k l e y , 1970); we a t ­
te m p te d to discover if th e y are also toxic to m arine
algae. Pseudomonas aeruginosa was chosen, both
2 D eterm ined b y D r. M. J . C h r e t i e n n o t , Marseille.
3 D eterm ined b y D r. P. B o u r r e l l y , Paris.
190
B . R . B erland
e t al.: A re b acte ria tox ic for alg ae ?
because o f its w ide occurrence (it is possibly ru n off
w ith sewage to th e m arin e environm ent), and because
i t is a to x ic pigm ent-synthetizer, w hich indicates
w hen to x ic substances appear. The m otile chlorophyta
Tetraselmis striata w as used as a te s t stra in (m otility
indicates th e physiological s ta te of th e cells), and
cu ltiv ated in E S solution p repared from su p ern atan ts
o f Pseudomonas aeruginosa cultures, centrifuged a t
different step s o f developm ent.
M ethods
M ar. Biol.
Fluorescent tube
'S lit entrance into the
regular 1800 Spectralux
recording SAFAS spectro­
photometer
Po o
Outside light
source
i—
Fluorescent tube
Focusing - beam
condensor
(1)
T he b acteria were grow n a t 18 °C in enriched
sea w ater containing b acto peptone : 2 g ; beef e x tra c t :
Fig. 2. Growth recording equipm ent. E x tra light source located
0.5 g ; y e a st e x tra c t: 0.5 g ; 80% sea w ater: 11; p H :
outside th e usual a p p aratu s; lig h t beam focussed on slit
7.5 to 7,8; sterilizatio n : autoclaving (20 m ii a t 120 °C).
entrance b y accu rate lens condensor. The equipm ent was
b u ilt for taking one sam ple only crossing once th e light beam
w ith o u t stopping th e carrying wheel. Magnetic stirrers are
located on wheel ju s t below cylindric cuvettes; th e y stop when
optical density is recorded. The tw o fluorescent tu b es do not
d istu rb th e m easurem ents
0.350
.S' 0 .3 0 0
our SAFAS recording spectrophotom eter (Fig. 2)
allow ed us to su rv ey th e 14 cultures sim ultaneously,
while being sh ak en by m agnetic stirrers an d lighted
w ith Sylvania-G rolux fluorescent tubes. T he blank
m edium of reference cultures was prepared as follows :
E S solution + 2 g/1 bacto p e p to n e -)-0.5 g/1 beef
e x tra c t + 0.5 g/1 y e a st e x tra c t; p H : 7.7, sterilization:
filtering; T . striata grows quicker in th is reference
m edium th a n in th e original ES solution.
0.250
- -
0.200
g
0.150
“O
S 0.100
°
0.050
Time (days)
R esults
Fig. 1. Pseudomonas aeruginosa. Growth curve, expressed in
optical density/cm p ath len g th o f cultures
This m edium allows pig m en t synthesis of Pseudomonas
aeruginosa-, i t ap pears w hen th e optical density (OD)
of th e cu ltu re is aro u n d 0.075/cm p ath -len g th (Fig. 1).
T he cultu re vessel w as a 40 m m p ath -len g th spectrophotom etric tu b e , containing 100 m l medium . Inocula
were young (24 h old). G row th was followed by m easur­
ing th e OD o f th e culture, using a SAFAS spectro­
p h o to m eter; readings w ere m ade a t 600 nm .
(2) T he cultures were centrifuged and cells rem oved.
T hen E S n u trie n ts were added to th e su p ern atan ts in
order to p rep are a P r o v a s o l i ’s E S solution for algae
cultures. Sterilization w as conducted b y filtering
th ro u g h a Sw innex M illipore filter holder, containing
one GS 0.22 ¡a M illipore filter. One hundred ml filtrateE S solution w ere pou red into a sterile 40 m m -diam eter spectro p h o to m etric cuvette.
(3) A bacteria-free cultu re o f Tetraselmis striata
was in oculated in to th e tub es and in cu b ated a t 18 °C.
T he algal gro w th w as followed b y m easuring four tim es
a d ay th e OD a t 600 n m . A special arran g em en t of
O ptical d en sity d a ta of Tetraselmis striata cultures
are p lo tte d in F ig . 3. Obviously, algal grow th is en­
hanced by a slig h t bacterial population filtrate (OD =
0.065 an d 0.072) ; grow th rate is faster and population
d ensity higher th a n in th e reference culture. L ater
(when OD reaches 0.092 an d 0.113) th e lag phase
increases, while th e m axim al density rem ains more or
less equal. W hen OD values are higher th a n 0.137, the
T . striata cells are killed. I t should be n o te d th a t
inhibition appears a t th e beginning of synthesis of the
green fluorescent pigm ent of Pseudomonas aeruginosa.
As fa r as th e grow th curves are parallel, we express the
developm ent of T . striata as follows :
-p.
,
, __
^
M aximal density
Tim e needed to reach m axim al density *
R esults were expressed as per cent of reference culture
grow th (Fig. 4). T hey clearly indicate th a t T . striata is
strongly inhibited by P. aeruginosa; algal grow th is
nil, since th e bacteria has reached th e h a lf w ay point
of its logarithm ic phase.
T he trem endous inhibitory effect of Pseudomonas
aeruginosa leads us to ask: are other bacteria poison­
ous for algae ? W e a tte m p te d to clarify th is problem
Vol. 12, N o. 3, 1972
B. R . B e r l a n d e t a í.: A re bacteria toxic for algae ?
191
0 .4 0 0
Reference culture
0 .3 5 0
O.D. 0 .0 6 5
and 0 .0 7 2
0 .3 0 0
~
0 .2 5 0
O.D . 0.113
O.D. 0 . 0 9 2
0.200
O.D. 0 .1 3 7
0 .1 5 0
0.100
0 .0 5 0
O.D. 0.171 0 .2 8 7
0 .2 1 4 0 .3 6 3
0 .2 5 0
Tim e (days)
Fig. 3. Tetraselmis striata. Growth in ES solutions prepared from Pseudomonas aeruginosa cultures, centrifuged a t different steps
of grow th. A lgal an d bacterial developm ents are expressed in optical density/cm path len g th of culture. O .D.: optical density
using several b acterial strain s o f different origin, a n d
b acteria-free cultures o f m arine algae.
125
Screening experiments
M ethods
T he tech n iq u e described earlier was used, b u t
modified as follows :
(1) B a c te ria were grow n in a sim ple m edium :
B acto p e p to n e : l g ; y e a st e x tra c t: 0.5 g ; 80% sea
w ater: 11; p H : 7.5— 7.8; sterilization: autoclaving
(20 m n, 120 °C). C ultures w ere incubated in th e sam e
40 m m d iam eter tu b e u n til ju s t before th e y reached
th e end o f logarithm ic grow th. A previous stu d y h a d
given th e average grow th curve for each stra in used.
(2) E S -filtra te solutions w ere poured in to 18 x
180 m m te s t tu b es, containing 10 m l m edium .
(3) A fter inoculation b y bacteria-free cultures, te s t
tu b es w ere in cu b ated a t 18 °C, under a continuous
fight p ro v id ed b y Sy 1vania-G rolux fluorescent tubes.
25*
100
75
50
25
0
0 .0 5 0 0 .1 0 0 0.125 0 .2 0 0 0.2 50
Optical density / cm pathlength
Fig. 4. Tetraselmis striata developm ent as a function of optical
density o f Pseudomonas aeruginosa cultures
192
B . R . B erland
e t al.: A re bacteria toxic for alg ae ?
A fter a period o f a t least a week, results were re a d by
d irect observation, using a 4° scale as follows: com ­
plete in h ib itio n (I) — th e experim ental tu b es are
com pletely clear; inhibition (si) — th e re is evidence
t h a t te s t cultures are less deeply coloured th a n ref­
erence cu ltu re ; no inhibition (0) — te s t a n d reference
cultures are sim ilar; grow th enhancem ent (GE) of
algae. T he b lan k m edium was p repared as follows:
E S solution + 1 g/1 peptone + 0.5 g/1 y e a st e x tra c t;
p H : 7.7; sterilization: filtering.
R esu lts
All d a ta o f th e large-scale survey will be published
la te r, b u t th e p rim ary results, sum m arized in T able 1,
clearly in d icate t h a t several b acteria are capable of
strong in h ib itio n ag ain st algae. Asterionella japonica
a n d Chaetoceros lauderi ap p ear to be dam aged b y all
th e strain s tested . R egarding th e bacteria, some strains
bad ly dam age alm ost all th e algae, especially Pseudo­
monas aeruginosa, P . marinoglutinosa a n d stra in ref­
erence 00.30.
M ar. Biol.
T here is, how ever, no in d icatio n a t p re sen t th a t
bacterial toxins a r e capable o f playing a role in th e
algae-bacteria relationships in th e sea as a whole.
Summary
1. I n vitro exp erim en ts in d icate th a t th e pigm ented
poison of th e b a c te riu m Pseudomonas aeruginosa is a
strong grow th in h ib ito r of th e alga Tetraselmis striata.
2. Several b a c te ria stra in s from different origins
are recognized t o have th e sam e to x ic ity against
various m arine algae.
3. T aking in to account th e v ery g re a t differences
betw een in vitro experim ents and in situ environ­
m en tal conditions, it is n o t a t p resent possible to state
t h a t bacterial poisons really play a role in th e algaebacteria relationships in th e sea.
Acknowledgements. Our th an k s are due to D r. P. B o u r (Museum, P aris), D r. M. J . C h r e t i e n n o t (Station
m arine d’Endoum e) a n d Professor P. G a y r a l (U niversity of
Caen) for determ ining several m icro-algae; to D r. M. A u b e r t
(C.E.R.B.O.M., N ice/ and D r. A. B i a n c h i (Station marine
d ’Endoum e) for providing several strains tested.
relly
Discussion and conclusion
Literature cited
The in vitro experim ents reveal an obvious inhibi­
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I n so fa r as th e re is no q u a n tita tiv e sim ilarity be­
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tions, b acterial poisons m ay only be o f some im ­
po rtan ce w hen concentrated, as, for exam ple, in
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Prasinocladus m arinus
Tetraselmis striata
Am phora sp.
Asterionella japonica
Chaetoceros lauderi
H em iaulus sinensis
N itzschia acicularis
Phaeodactylum tricornutum
Thalassiosira rotula
Stichochrysis immobilis
Heterothrix sp.
Monallantus salina
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2
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11
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