Marine Pollution Bulletin, Vol. 34, No. 10, pp.
816-820, 1997
Pergamon
PII: S0025-326X(96)00086--0
Epiphytic Bacteria in a Copper-enriched
Environment in Northern Chile
CARLOS RIQUELME*, ALEJANDRO ROJAS*, VERONICA FLORESt and J U A N A. CORREAtJ~
*Facultad de Recursos del Mar, Universidad de Antofagasta, Casilla 170, Antofagasta, Chile
tDepartamento de Ecologia, Facultad de Ciencias Biol6gicas, Pontificia Universidad Cat6lica de Chile, Casilla l14-D,
Santiago, Chile
~Address for c o r r e s p o n d e n c e
This study was designed to characterize the bacterial
community epiphytic on the green alga Enteromorpha
compressa in terms of density, generic composition and
degree of copper tolerance. Algal hosts were collected in
Caleta Palito, a rocky beach in northern Chile which has
suffered the impact of waste disposal from the copper
mine El Salvador during the last 20 years. Comparisons
between epiphytic bacteria from Caleta Palito and those
from Caleta Zenteno, a control site with no history of
copper enrichment, showed that differences in generic
composition and density were very minor. However, a
larger number of isolates from Caleta Palito showed
higher copper tolerance. Our study demonstrates the high
copper tolerance of bacteria epiphytic on the dominant
algal taxa in Caleta Palito and suggests the potential role
of bacterial films in lessening the negative impact of
copper on algal hosts inhabiting copper-polluted environ-
ments. © 1996 Elsevier Science Ltd
Bacteria flourish in almost any marine habitat, and are
important structural components participating in a
number of biogeochemical processes like the nitrogen,
sulfur and carbon cycles (Austin, 1988a). Bacteria are
known for establishing close interactions with marine
animals and plants. Epiphytic bacteria (i.e. those which
colonize marine algae) have received attention from the
perspective of deleterious organisms that can eventually
cause diseases (Fujita, 1990), facilitate the establishment
of other fouling organisms (Wahl, 1989), participate in
settlement induction of invertebrate larvae (Johnson et
al., 1991) or as producers of growth-promoting
substances (Provasoli & Pintner, 1980).
Bacteria use copper as an essential micronutrient and,
similarly to all living organisms, respond differently to
abnormally high copper concentrations in the sea. It is
know that bacterial films can bind heavy metals (Gadd
& White, 1993) and a number of bacteria have been
reported as copper-tolerant organisms able to accumu-
late the metal in the cell (reviewed by Brown et al., 1992;
Silver & Ji, 1994). To our knowledge, however, no study
has dealt specifically with epiphytic bacteria. Based on
816
© 1996 Elsevier Science Ltd
All fights reserved. Printed in Great Britain
0025-326X/96 $15.00 + 0.00
their accumulation capabilities and under conditions of
heavy epiphytism (i.e. formation of bacterial films on
algal hosts), bacteria may act as filters or barrier to
diminish the amount of copper that actually reaches the
host. Thus, a possibility exists that algal species,
considered as copper tolerant organisms and used in
biomonitoring programmes, may display such responses
at least in part because of their epiph),tic bacterial flora.
Species belonging to the chlorophycean genus
Enteromorpha are known as copper-tolerant algae and
are used world-wide as bioindicators of copper
enrichments due to their capacity to accumulate the
metal (Evans, 1981; Seeliger & Cordazzo, 1982; Reed &
Moffat, 1983; Ho, 1987; Correa et al., 1996a). In this
context, in an effort to understand the interactions
between copper and marine organisms, we have
concentrated our studies in Chafiaral Bay (26°15'S,
69°39'W) and surroundings, northern Chile. For
decades Chafiaral Bay received the solid and liquid
wastes from the copper mine E1 Salvador. In 1990 the
company built a sedimentation dam where most
particulate residues accumulated, releasing mainly
liquid wastes known as 'aguas claras' or clear waters.
Prior to the construction of the sedimentation dam, the
site of direct discharge was changed from Chafiaral Bay
to Caleta Palito, a rocky beach 8 km north from
Chafiaral. As a result of the direct tailings, marine flora
and fauna in the area suffered severely, and the few
studies available reported major losses in species
diversity (Castilla & Nealler, 1987; Castilla, 1996).
Later, the intertidal fringe became dominated by
Enteromorpha compressa, a cosmopolitan green alga
recognized as a copper-tolerant organism (Reed &
Moffat, 1983; Correa et al., 1996a). Today, even
though the solids do not reach the sea, the values of
dissolved copper range from 14 to 29 ~tgl-1 (Correa et
al., 1996a,b) and are higher than those reported for non-
contaminated seawater (Davies & Bennett, 1985).
Furthermore, E. compressa remains as the dominant
organism on the rocks.
Based on the above information and on earlier
unpublished observations revealing that E. compressa
from Caleta Palito hosted an abundant bacterial flora,
Volume M/Number lO/October 1997
two main objectives were selected for this study. One
was to characterize the bacterial microflora epiphytic on
E. compressa from Caleta Palito in terms of density,
generic composition and degree of copper tolerance,
and the other was to compare the situation in Caleta
Palito with that in Caleta Zenteno, located 65 km south
from Chafiaral Bay. Caleta Zenteno, used as a control
locality in our previous studies (Correa et al., 1996a,b),
has values of dissolved copper of 3.1 ~g 1-1.
Materials and Methods
All samples of E. compressa were collected simulta-
neously at Caleta Palito and Caleta Zenteno on May
1995. Individual specimens of E. compressa were rinsed
repeatedly with sterile seawater immediately after
collection. Using autoclaved tweezers, individual
fronds were put into autoclaved plastic tubes, stored
in a cooler at 4°C and transported to the laboratory.
Once in the laboratory, triplicate samples were rinsed
several times in marine saline solution (Austin, 1988b).
Algae were blotted, weighed and homogenized in a
Stomacher 80 for 3 min (Baker, 1988). Subsequently,
homogenized samples were plated on thiosulfate-citrate-
bile sucrose agar (TCBS, Oxoid) and marine agax Zobell
2216 (Difco) for 2 and 7 days, respectively, following
the procedure outlined by Riquelme et al. (1995).
Bacterial counts were recorded at the end of each
incubation period and the different morphological types
of colonies were isolated and characterized according to
Muroga et al. (1989).
Simultaneously to the sampling of E. compressa for
bacteria isolations, samples were obtained for direct
quantification of bacteria on the surface of the alga.
This was done by fixation of tissue fragments in 2%
glutaraldehyde in seawater for 24 h, followed by
dehydration in an acetone series and critical point
drying. Observations were done in a Jeol JSM-25 S-I 1
scanning electron microscope (SEM) operated at 25 kV.
Densities of bacteria were estimated by using a 100-
point grid attached to the screen of the microscope. A
total of 24 individual plants from each locality were
screened under the microscope (6 fronds from 4 sites in
each locality). Five fields were screened in each
fragment, and counts were performed at 3000x.
When appropriate, data comparisons by G test (Zar,
1984) were done.
All strains isolated from E. compressa were tested for
copper tolerance. Trials were done by plating aliquots
of the individual strains on marine agar Zobell 2216
(Difco) prepared with deionized water and copper
additions of 1.5, 15, 150, 1500 and 150001~gl - l from
a CuC12 standard solution (Titrisol, Merck). Plates were
incubated at 20°C for 7 days.
In an attempt to detect possible sites of copper
accumulation, bacterial isolates that displayed copper
tolerance were processed for transmission electron
microscopy (TEM). Bacteria were either pelleted or
collected from agar plates and fixed in 2% glutar-
aldehyde in 0.451am filtered seawater, followed by
postfixation in 1% osmium tetroxide. Dehydration
was done in an ethanol series, followed by infiltration
and embedding in Spurr's resin. Sections were stained
with lead citrate and uranyl acetate and observed in a
Philips 300 TEM operated at 80 kV. The same strains
grown in absence of copper were used as controls.
Results and Discussion
Our SEM observations showed that the mean cover
value for epiphytic bacteria on E. compressa from
Caleta Palito was 67.9% (Fig. 1). Values for epiphytic
bacteria on E. compressa from Caleta Zenteno was
similar (65.8%; Fig. 1) and differences between localities
were not significant (G test, p > 0.05). During the SEM
observations, we detected a dominance by bacilli.
The counts of heterotrophic cultivable bacteria
oscillated around 106 CFU g - l (Fig. 2), with differences
between Caleta Palito and Caleta Zenteno being
statistically not significant (G test, p > 0.05). Identifica-
tion of these bacteria showed that Pseudomonas,
Flavobacterium and Moraxella were dominant and
together comprised 95.7% and 91.3% in Caleta Palito
and Caleta Zenteno, respectively (Table 1). Members of
the genus Vibrio were not detected among the cultivable
bacterial flora of E. compressa, nor did they grow in the
specific TCBS culture medium.
All epiphytic bacteria developed well in copper
additions of 1.5 to 1500t~gl -l. At concentrations of
ABUNDANCEOF BACTERIAEPIPHYTIC
ON Enteromorpha compressa
,°l
A 7 f N= 120 I
=g
ul
O
O
~2
Caleta Palito Caleta Zenteno
LOCALITY
Fig. 1 Mean cover values of epiphytic bacteria counted directly on
Enteromorpha compressa. Bars represent 1 standard deviation.
817
 Marine Pollution Bulletin

CULTURABLEHETEROTROPHICBACTERIA

am N= 8
6
T

tt 4
O
O a
O
--I 2

0
CALETA PALITO CALETAZENI"ENO

LOCALITY
Fig. 2 Mean values for epiphyticculturable bacteria on marine agar
Zobell 2216. Bars represent 1 standard deviation.

15000~tgl - l copper 87.5% of isolates from Caleta

Palito still developed well, whereas only 57% of the
isolates from Caleta Zenteno displayed copper toler-
ance. Furthermore, the examination under the TEM of
copper tolerant isolates of Pseudomonas exposed to high
copper concentrations showed the presence of dark,
finely granular intracellular deposits (Fig. 3), which Fig. 3 Transmission electron micrographs of copper-tolerant isolate
were not observed in cells of the same strain but grown CP 85 exposed to normal culture medium (a) and to copper
addition of 15000~tg1-1 (b). Scale bar: 0.2[am.
in absence of copper addition.
In spite of the 20 years of copper enrichment in
Caleta Palito, it seems apparent from our results that
only minor differences have developed between the et al. (1996a) confirmed that diversity of algae and
epiphytic bacterial communities from Caleta Palito and invertebrates remains low in Caleta Palito, with E,
Caleta Zenteno. Theoretically, at least two scenarios compressa as the single dominant species. This scenario,
could have developed as liquid and solid wastes from El however, is not supported by our results, which showed
Salvador began pouring into Caleta Palito in 1975. The that densities of total and cultivable bacteria and the
first scenario is one where epiphytic bacterial assem- generic composition of cultivable epiphytic bacteria
blages reacted similarly to algal and invertebrate from Caleta Palito are almost identical to those from
communities, that is decreasing both densities and Caleta Zenteno, an area with no history of copper
diversity, with few copper-tolerant taxa becoming enrichment (Correa et al., 1996a). A second scenario
dominant and prevailing today. Recent reports by can be hypothesized where a differential growth of the
Vermeer & Castilla (1991); Castilla (1996) and Correa copper-tolerant segment of each population within a
bacterial community is induced by copper enrichments.
According to this, neither density nor generic composi-
TABLE 1 tion would necessarily suffer major changes. Tolerance
Relative abundanceof various genera among the isolatesof culturable to copper seems to be inducible in exceedingly short
heterotrophic epiphyticbacteria. time periods, and values of 2 days (Vaccaro et aL, 1977)
and 1-2 days (Tubbing et al., 1995) have been reported.
Locality Alternatively, copper tolerance at community level has
Caleta Palito Caleta Zenteno been explained as selective growth of bacteria carrying
Genus N (%) N (%) plasmids or chromosomal mutations (Foster, 1983;
Pseudomonas 20 (43.6) 18 (39.0) Zelibor et al., 1987), a possibility which can not be
Flavobacterium 18 (39.l) 17 (37.0) discarded in our case.
Moraxella 6 (13.0) 7 (15.2) The second scenario is supported by our results on
N.D. 2 (4.3) 4 (8.7)
copper tolerance, which showed more copper-tolerant
N.D.: Not determinedtaxa. isolates from Caleta Palito than from Caleta Zenteno,

818
Volume 34/Number 10/October 1997
even t h o u g h the bacterial genera were the same and
densities were very similar in b o t h localities. The
m e c h a n i s m for this e n h a n c e d c o p p e r tolerance is n o t
k n o w n , a l t h o u g h the occurrence o f electron dense
particles inside bacterial cells exposed to high c o p p e r
c o n c e n t r a t i o n suggests that tolerance by accumulation
m a y be taking place. This finding is consistent with
reports where similar types o f deposits have been
described in o t h e r type o f marine organisms, mainly
protists and c y a n o b a c t e r i a g r o w n in the presence o f
heavy metals (Silverberg et al., 1976; S i c k o - G o a d &
Stoermer, 1979; Daniel & Chamberlain, 1981; Pedersrn
et al., 1981; Lignell et al., 1982; Pettersson et al., 1985;
Smith et al., 1986; G a d d & White, 1993; W o n g et al.,
1994).
The original suggestion that epiphytic bacteria could
serve as 'biofilters' (see L o b b a n et aL, 1985), lessening
the direct impact o f c o p p e r on the algal host, is
indirectly supported by o u r study. W e n o w k n o w that
bacteria growing o n E. compressa are copper-tolerant
organisms a n d it is likely that such tolerance is due to
intracellular accumulation. In fact, preliminary results
in our l a b o r a t o r y have shown that the values o f c o p p e r
in E. compressa samples w i t h o u t the bacteria cover
(mechanically removed) decreased significantly in
c o m p a r i s o n to samples whose bacterial cover was not
r e m o v e d prior to the chemical analysis (Correa et al.,
unpublished results). This is understandable considering
the high cover values for bacteria recorded in our S E M
study. F u r t h e r m o r e , and in addition to accumulation
which is likely an active a n d specific mechanism, we
have to consider the occurrence o f 'passive biofiltration'
in the bacterial epiphytic film. This p h e n o m e n o n is
determined by c o p p e r complexation to either bacterial
cell wall or bacterial exudates, all o f which have proved
to be efficient metal complexing agents ( G a d d & White,
1993).
There are certainly n u m e r o u s aspects in the area o f
marine pollution which remain to be addressed. O u r
study provides evidence that marine epiphytic bacteria,
organisms n o t considered in previous studies on heavy
metal pollution, are also responsive to c o p p e r enrich-
ments and therefore, stresses the need for integrative,
multidisciplinary a p p r o a c h e s for a better understanding
o f their role as potential biofilters that enhance the
tolerance o f algal hosts.
This study was supported by research grants CIMM/ICA 119504 to
J.C. and 119505 to C.R.
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