The inhibitory effect of biofilms produced by wild bacterial isolates to the larval settlement of the fouling ascidia Ciona intestinalis and Pyura praeputialis
Financial support: Chilean National Fund for Promotion of Scientific and Technological Development (FONDEF) project Nº DO1I1166.
Keywords: antifouling extracts, ascidian bioassays, bacterial biofilms, Ciona intestinalis, marine biofouling, natural antifoulants, Pyura praeputialis.
biofouling is a present and potentially increasing future problem
at molluscan culture centres. The problem is highly variable, exists
on different scales, and its negative impact on cultured organisms
and related economic losses at these centres has not been significantly
controlled. One approach to fouling control has been the incorporation
of natural substances into anti-fouling paints which inhibit the settlement
of common fouling organisms. The main objective of the present study
was the isolation of naturally occurring substances from marine bacteria
which were inhibitory to the settlement of Ciona intestinalis
and Pyura praeputialis, two tunicate species causing serious
fouling problems in scallop culture systems in
causes severe problems for structures submerged in the sea, such as
increases in mass, and corrosion of surfaces resulting in economic
losses. These effects are of particular consequence in the aquaculture
industry, and on structures in the sea such as the supports of oil
drilling platforms and ship hulls (Armstrong et al. 2000).
A common method for avoiding settlement of marine fouling organisms
on marine structures has been coating with antifouling paints, preferably
those containing tributyl-tin (TBT) or copper. These paints are highly
effective in controlling fouling on ships, with periods of up to seven
years without maintenance and representing savings of US$ 6.5 billion
per year. Considerations of environmental protection, however, now
prevent use of the TBT coatings on vessels of under
Environmentally acceptable antifouling substances are needed for incorporation into antifouling coatings, and these may include natural products isolated from certain marine organisms (Clare, 1996) which natural products isolated from certain marine organisms (Clare, 1996). Incorporation of naturally repellent products into anti-fouling paints has been tried by some researchers (Armstrong et al. 2000; Peppiatt et al. 2000). Some marine organisms such as corals, algae, sponges, and ascidians have been shown to produce antifouling substances which in nature maintain them free from undesirable encrusting organisms (Hentschel et al. 2001; Dobretsov and Qian 2002; Harder et al. 2003).
Some studies have shown that bacterial biofilms isolated from organisms having low degrees of surface macrofouling release compounds which repel other bacteria, acting in a protective role (Boyd et al. 1998; Boyd et al. 1999a; Boyd et al. 1999b; Burgess et al. 2003).Some bacteria present on the surfaces of crustacean larvae produce antimicrobial compounds which protect them from fungal infections (Gil-Turnes and Fenical, 1992). Biofilms of the bacteria Pseudoalteromonas tunicata, isolated from the surface of a tunicate, showed antifouling activity against larvae of Balanus amphitrite and C. intestinalis (James et al. 1996; Holmström and Kjelleberg, 1999).
intestinalis (white sea squirt) is an important cosmopolitan fouling
organism on both natural and artificial substrates (Connell,
2000; Mazouni et al. 2001). This tunicate produces
serious operational difficulties in diverse aquaculture centres, for
main objective of the present study was to search for indigenous marine
bacteria occurring in biofilms which exerted inhibitory activity on
the settlement of larvae of the tunicates common in the fouling of
A. purpuratus culture systems. A long term goal of this work
was to concentrate natural antifouling materials produced by bacteria
in a way that they could be employed in fouling prevention without
toxic side effects for the organisms in culture. The culture of A.
purpuratus has become very important in
intestinalis. Adult C. intestinalis were obtained from
the El Golfo Culture Center located at
praeputialis. Adult specimens were collected at low tide from
the rocky intertidal zone near
and artificial substrates sampled for naturally occurring film-forming
bacteria are listed in Table 1. These were rinsed
copiously with sterile seawater to remove debris and weakly adhered
microorganisms (Jiang et al. 1999). Samples of strongly
adherent epibiotic bacteria were obtained using a bacteriological
loop. Samples were spread on TCBS Agar (Oxoid Ltd.) and Zobell 2216
Marine Agar (Difco,
A preliminary characterization of the bacterial strains had inhibitory effects on the settlements of C. intestinalis larvae (Gram reaction and motility) was made using light microscopy. The biochemical tests catalase and O/F reaction with glucose (Gerhardt et al. 1994) were also carried out.
were prepared from the 73 bacterial strains isolated, as well as from
the two control species, using the methodology given by Maki
et al. (2000). Overnight bacterial cultures grown in the medium
VNSS, (Holmström et al. 1998) were centrifuged at
5520 x g for 15 min. The pellet was washed and resuspended in artificial
seawater (ASW, "Sea Salt", Sigma Co.), and re-centrifuged.
The pellet was then resuspended in 1/10 VNSS/ASW. The bacterial count
in this suspension was determined using DAPI (
settlement assays of C. intestinalis were done by introducing
15 larvae in a total of 3 ml seawater into all dishes containing living
and killed bacterial biofilms, plus controls without bacterial films.
The systems were incubated at
fractions were prepared from bacterial strains Ni1-LEM and HA, and
the positive controls Pt and Hm, all of which had been cultured to
the stationary phase in Marine Minimal Medium (MMM) (Maki et al. 2000)
with constant stirring at room temperature (
inert polymer PhytagelTM (Sigma
Chemical Company), was prepared by diluting
order to determine the effects of heat treatment on the bacterial
EP, the extracts were treated at
approximate molecular size bacterial antifouling compounds was determined
using dialysis by fractionation from bacterial strains Nil-LEM and
HA by dialysis against sterile distilled water for 12 hrs at
assay was carried out to determine if the EP factors inhibitory to
tunicate settlement were proteic or peptidic in nature. For this the
EP were separately treated at a final concentration of 200 µgmL-1
pronase E and carboxypeptidase G. The pronase E mixture was incubated
supernatants from pre-stationary phase cultures of the bacterial strain
Ni1-LEM were concentrated by vacuum evaporation at
One-way analyses of variance (ANOVA, α = 0.05) were used to evaluate the effects of both living and non-living biofilms, as well as the effects of the bacterial extracellular components, on the inhibition of C. intestinalis and P. praeputialis larvae settlement. The determination of the factors which were responsible for significant differences was carried out using a multiple comparison, test (Tukey-HSD).
A total of 73 film-forming bacterial strains were isolated (Table 1). The assays with living bacterial films showed that 20% of the strains had inhibitory effects on the settlement of C. intestinalis larvae; strong inhibition was indicated when 50% or more of the larvae present failed to settle in the plates. These bacterial strains isolated were gram-negative, fermentative, positive for motility and catalase.
Figure 1 shows the bacterial strains having the highest antifouling effects, as well as the positive (Hm, Pt) and negative controls. Significant differences were observed in averages of non-settled larvae between treatments and negative controls (ANOVA, p < 0.05).
The inhibitory ability of the biofilms was lost when they were tested in the non-living condition, as shown by Figure 2. No significant differences were found in the percentages of non-adhered larvae between (non-living) biofilmed surfaces and controls (ANOVA, p < 0.05).
The extracellular products incorporated into the Phytagel which had inhibitory effect was strain Ni1-LEM (16.8%). Significant differences (ANOVA, p < 0.05) occurred between the results obtained with strains Pt, Hm (positive controls) and Ni1-LEM, and the negative controls (Figure 3).
The EP from bacterial strains Pt, Hm and Ni1-LEM did not lose their ability to inhibit settlement of larval C. intestinalis and P. praeputialis after dilution and/or denaturation (Figure 4 and Figure 5). Here, significant differences were detected between the treatments and the negative controls (ANOVA, p < 0.05). The bacterial strains Pt, Hm, and Ni1-LEM lost their antifoulimg activity against C. intestinalis after exposure to protease, although peptidase had no effect. A contrasting result was obtained with strain HA (Figure 4) which lost its antifouling activity after exposure to peptidase, but not with protease. This could be indicate that EP of the strain HA are peptide origin.
Figure 5 shows that the EP of strains Pt, Hm, Ni1-LEM and HA lost their inhibitory effect against P. praeputialis, after treatment with proteolytic enzymes. Dialysis of the PE showed that substances inhibitory to the settlement of C. intestinalis and P. praeputialis larvae produced by strains Ni1-LEM and HA had molecular weights of less than 3,500 Da.
Figure 6a shows the results with the EP fractions obtained by separation with an HPLC column on larvae settlement of the two ascidian species used in this study. Only the four- and five-minute column eluates of the PE from bacterial strain Ni1-LEM inhibited larval settlement of the two ascidian species (Figure 6a). The remaining fractions produced no significant differences from the controls (ANOVA, p < 0.05).
The present results are in general agreement with results obtained by other authors working with various species as listed in Table 2.
The results of these studies and those of the present study showed that bacteria present in biofilms may have an important role in the development of trophic successions in benthic micro systems based on inhibitory ability of microfouling components of the intertidal zone. The presence of the inhibitory bacteria can influence the normal development and survival of some invertebrates and algae (Egan et al. 2001). The inhibitory ability of these biofilms is lost after killing with chloroform (Figure 2), suggesting that the production of inhibitory substances is an activity inherent to the metabolism of living cells. In preliminary test chloroform fumes, no affects the activity inhibitory (data unpublished).
Incorporation of bacterial inhibitory substances in a gel matrix is a model system representing a method for applying antifouling substances onto surfaces in need of antifouling protection in the sea. The antifouling compounds can be incorporated into the matrix at concentrations higher than those in the natural environment without altering the physical characteristics of the settlement surface (Henrikson and Pawlik, 1995). This technique is valuable for assaying the antifouling effects of secondary metabolites produced by marine organisms (Henrikson and Pawlik, 1998). PhytagelTM was chosen for the present study due to its stability when compared with other potentially useful polymers. Henrikson and Pawlik (1995) have reviewed these properties related to their in situ studies with extracts from marine invertebrates. As noted above, the inhibitory ability of bacterial extracellular products was not inhibited by their inclusion in Phytagel, with one bacterial strain (HA) as an exception (Figure 3). The observed increases of effectiveness of the bacterial extracellular products with higher concentration (Figure 4 and Figure 5) were similar to the results of Thiyagarajan et al. (1999), where low concentrations of extracts did not affect settlement of B. meticulatus, but high concentrations did.
Our results suggest that the bacterial inhibitory substances are thermostable, proteic or peptidic substance of less than 3500 Da in MW (strain Ni1-LEM); this agrees with Holmström and Kjelleberg (1999) and Egan et al. (2001) that low molecular weight peptides or proteins inhibited the settlement of bacteria, fungi, microalgae, algal spores, and invertebrates. In contrast, Dobretsov and Qian (2004) found that inhibitory substances isolated from bacteria from the soft coral Dendronephthya sp had a molecular weight of greater than 100 KDa.
The results obtained from the HPLC analyses suggested that the protein or peptide present in the active fraction had polar characteristics (in preliminary experiments it was test, data no show) (Figure 6), similarly to that reported by Egan et al. (2001), polar proteins and low molecular weight, which inhibited the germination of Ulva lactuca spores. Previously, Kawamata et al. (1994) reported that substances from the soft coral Dendronephthya sp which inhibited the settlement of Balanus amphitrite larvae were water soluble and of low molecular weight. Although, we can not dismissed the presence of other compounds of other chemical nature present in the bacterial supernatants.
Indirect evidence, as well as newly emerging experimental evidence, suggests that many marine species have variable abilities for protecting themselves from overgrowth by natural competitors and neighbours within their communities. Protective substances may be produced directly by the organisms, or may be products from microbial symbionts or incidental microbial film-formers in the habitat. In any case, extracellular metabolites with antifouling activity are metabolic products representing an energy cost to the producers, which may be why in the few reports we have seen (Table 2) bacterial antifouling activity has been relatively low (only 20% of the strains were active in the present study). It is of pressing interest to find unique substances of bacterial or other origin which would be useful for incorporation into antifouling coatings of practical use. As is well known for existing chemical antifouling coatings such as those containing copper or TBT, they are designed to release considerable quantities of highly toxic substances to be effective over time. It is therefore essential to find naturally repellent products with a broad spectrum activity and capable of being made available in commercial quantities.
The present study is part of the beginning of an effort to find natural products which in the future will fulfil emerging needs for antifouling products which repel classically damaging invertebrates, while at the same time allowing the growth of desirable species in aquaculture.
authors thank Drs. Suelen Egan and Staffan Kjelleberg of the Center
for Marine Biofouling and Bioinvasion,
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