Biofouling, 2003 Vol 19 (Supplement), pp 197–205

The Development of a Marine Natural Product-based

Antifouling Paint
J GRANT BURGESSa,*, KENNETH G BOYDa, EVELYN ARMSTRONGa, ZHONG JIANGa, LIMING YANa,
MATZ BERGGRENb, ULRIKA MAYb, TONY PISACANEc, ÅKE GRANMOb and DAVID R ADAMSd
a
School of Life Sciences, Heriot-Watt University, Riccarton, Edinburgh EH14 4AS, UK; bKristinebergs Marine Biological Station, P1 2130, S-450, 34
Fiskebäckskil, Sweden; cX-Gnat Laboratories Ltd, Cumbernauld, Scotland, UK; dDepartment of Chemistry, Heriot-Watt University, Riccarton,
Edinburgh EH14 4AS, UK

(Received 29 August 2002; in final form 28 October 2002)

Problems with tin and copper antifouling compounds have
highlighted the need to develop new environmentally
friendly antifouling coatings. Bacteria isolated from living
surfaces in the marine environment are a promising source
of natural antifouling compounds. Four isolates were used
to produce extracts that were formulated into ten water-
based paints. All but one of the paints showed activity
against a test panel of fouling bacteria. Five of the paints
were further tested for their ability to inhibit the settlement
of barnacle larvae, Balanus amphitrite, and algal spores of
Ulva lactuca, and for their ability to inhibit the growth of
U. lactuca. Two paints caused a significant decrease in the
number of settled barnacles. One paint containing extract
of Pseudomonas sp. strain NUDMB50-11, showed excellent
activity in all assays. The antifouling chemicals responsible
for the activity of the extract were isolated, using bioassay
guided fractionation, and their chemical structures
determined.
Keywords: antifouling paint; marine bacteria; natural products
INTRODUCTION
The effects of metal-based antifouling paints on the
ecology of the marine environment have been the
subject of intense debate (Foster, 1994). The most
widely used chemical antifoulant, tri-n-butyl tin
(TBT), accumulates in marine sediments and a
number of studies have demonstrated its
negative effect on several marine species (Stewart
& Thompson, 1997; Hashimoto et al., 1998; Grinwis
et al., 1998; Matthiessen & Gibbs, 1998; Fisher et al.,
1999). As a result, the use of TBT has been restricted
in Europe, Canada, and Japan. Copper-based paints
are currently used as an alternative to TBT-contain-
ing paints, however, the toxicity of copper to some
species of marine algae and the ability of shellfish to
accumulate the metal has increased calls for
legislation against the use of such paints (Claisse &
Alzieu, 1993; Batley et al., 1994). Another widely used
co-biocide is the herbicide Irgarolw (2-methylthio-4-
tert-butyl-amino-6-cyclopropylamino-s-triazine)
(Thomas, 2001), however there is growing concern
over its possible impact on aquatic and saltmarsh
plants (Gibbon, 1995).
Fouling may be controlled by foul-release coat-
ings, which have low surface energies and do not
contain biocides. Attached organisms are removed
from vessels as a result of frictional forces caused by
their motion through the water (Bultman & Griffith,
1994; Kavanagh et al., 2001 and references therein).
While effective on fast-moving vessels, these coat-
ings are not suitable for use on the majority of
vessels. Electrochemical technologies have also been
examined but are expensive to apply to large
structures (Nakasono et al., 1993).
Natural products from marine organisms can be
used as replacements for the chemicals commonly
used in antifouling coatings (Clare, 1996a). Many
sessile marine animals are free from biofouling and
produce metabolites that demonstrate antifouling
properties, presumably as a means of protection
from colonisation by fouling organisms or to reduce
competition for space in highly competitive environ-
ments (Wahl et al., 1994). Some seaweed species can
also interfere with bacterial colonization on their

*Corresponding author; fax: þ44-131-451-3009; e-mail: j.g.burgess@hw.ac.uk

ISSN 0892-7014 print/ISSN 1029-2454 online q 2003 Taylor & Francis Ltd
DOI: 10.1080/0892701031000061778
198 J G BURGESS et al.

TABLE I Marine organisms used a source of epiphytic bacteria been determined. The development of large-scale
fermentation conditions for production of these
Sample Species antifouling extracts is also reported.
Alga Fucus serratus
Fucus spiralis
Laminaria sp.
Palmaria palmata MATERIALS AND METHODS
Corallina officinalis
Chondrus crispus
Poryphera umbilicas
Isolation of Epiphytic Bacteria
Ulva lactuca
Codium fragile sp. atlanticum
Seaweed surfaces were thoroughly washed with
Mastocarpus stellatus sterile seawater, so that only bacteria with a strong
Leathesia difformis affinity for the host were sampled (Gil-Turnes et al.,
Himanthalia elongata
Delesseria sanguinea
1989; Cheng et al., 1999; Jiang et al., 1999). Seaweed
Dilesia carnosa fronds were swabbed with a sterile cotton tip to
Halidrys siliquosa obtain epiphytic bacteria (Boyd et al., 1998; 1999a).
Nudibranch Archidoris pseudoargus
Sponge Haliclona viscose
Marine agar (MA, DIFCO), seawater agar and
Halichondria bowerbanki seaweed agar were used for cultivation. In order to
Myxilla incrustans better reflect the nutrients available to algal-
Halichondria panicea
Pachymatisma johnstonia
associated bacteria in their natural environment, a
Featherstar Antedon bifida range of media were also made to mimic the
Starfish Asterias rubens chemical composition of particular seaweeds from
Bryozoan Membranipora membracea
Flustra folacia
which bacteria were isolated. These media were
Sea anemone Actinia equina incorporated with commercially available sugars,
Tunicate Ascidia mentula such as fucoidan, mannitol, galactose and glucose
Botyllus schlosseri
Ascidiella aspersa
which were added after autoclaving to a final
Sea hare Aplysia punctata concentration of 0.1%. Media incorporating alginate
and carboxymethyl cellulose were also prepared by
incorporating these materials prior to autoclaving. In
surfaces by the release of antifouling compounds addition two other gel-forming polysaccharides,
(de Nys et al., 1995; Dobretsov & Qian, 2002). carrageenan type I and carrageenan type II, extracted
In addition, recent studies have highlighted import- from red algae, were used to make agar plates. Plates
ant roles for bacteria colonising the surfaces of were incubated at either 13 or 208C. Colonies were
marine organisms. Bacteria growing on the surfaces selected to make pure cultures by re-streaking. Six
of larvae of some crustaceans produce antimicrobial hundred and fifty isolates were obtained from the
compounds which protect the developing larvae surfaces of a range of marine algae and invertebrates
from fungal infection (Gil-Turnes et al., 1989; (Table I).
Gil-Turnes & Fenical, 1992). Biofilms of the
bacterium, Pseudoalteromonas tunicata, isolated from Antibacterial Assays
the surface of a tunicate, showed antifouling activity
against Balanus amphrite and Ciona intestinalis larvae Antibiotic assays against nine fouling bacteria were
( James et al., 1996; Holmstrom & Kjelleberg, 1999). carried out using either a standard disk diffusion
Bacteria isolated from the surface of seaweeds have assay (Mearns-Spragg et al., 1998; Boyd et al., 1999a)
also been shown to release compounds that repel or a well assay. For the well assay, 10 mm diameter
other fouling bacteria, suggesting that they may wells were cut in MA plates that had been freshly
protect the seaweed from fouling by other organisms swabbed with a lawn of each fouling strain. To these
(Boyd et al., 1998; 1999a; 1999b; Burgess et al., 1999). wells, 100 ml of the cell free supernatant were added.
Incorporation of natural products into antifouling The plates were incubated overnight at 288C. Strains
paints is an approach that has been tried by a number giving inhibition zones greater than 11 mm were
of groups (Willemsen & Ferrari, 1993; Armstrong considered to be antibiotic producers. Forty two
et al., 2000b; Peppiat et al., 2000). However, there have (6.5%) of the 650 isolates exhibited antibacterial
been few attempts to define the chemistry of the activity against at least one of the fouling strains.
extracts used. This paper reports the development of
an antifouling coating incorporating extracts from a
Preparation of Antifouling Extracts from Marine
marine bacterium as the active principal. The coating
Bacteria
exhibited good activity against marine bacteria,
barnacle larvae and algal spores, which suggests Of the forty two antibacterial compound-producing
potential use as an antifouling paint. Moreover, the isolates, strains FS55, NUDMB50-11, MAI-11-01-CP
chemical structure of the active compounds has and II-111-5, exhibited antibacterial activity against
 MARINE NATURAL PRODUCT-BASED AF PAINTS 199

more than two marine fouling bacteria. They were

Overall antibacterial activity of paint
compared with resin controls

therefore selected to provide active extracts, from

supernatants and cell extracts, for addition to paints
(Table II). These four isolates were grown in either
marine broth or SM9 media (Stead et al., 1996).
Supernatant extracts were prepared by chromato-
2
2

2
2
þ
þ
þ
þ
þ

graphy on Amberlite XAD16 resin and eluted with a

gradient of increasing acetonitrile in water. Active
fractions were combined and the acetonitrile evapo-
rated. The aqueous solutions were then either
extracted with dichloromethane or freeze dried and
resuspended in methanol. Cell extracts were pre-
pared by extracting cell pellets with methanol. In the
bacteria killed by the paint

case of a Pseudomonas sp. NUDMB50-11 (Peppiatt

The number of strains of
a panel of 9 fouling

et al., 2000), a portion of the cell extract did not

solubilise in methanol and this was dissolved in
TABLE II Summary of antibacterial activity of paints formulated from bacterial extracts

water.
4
2
7
4
1
4
1
2
0

Extraction and Characterisation of Antifouling

Metabolites from Pseudomonas sp. NUDMB50-11
þ ¼ significantly different compared with the resin negative controls; 2 ¼ not significantly different compared with the resin negative controls

A Pseudomonas sp., NUDMB50-11, was grown in 5 l

of SM9 medium at 288C with shaking for 5 d. The
culture was centrifuged (2500 rev min21, 1 h) and the
Type of extract used in the paint

H2O soluble residue of cells

supernatant extracted with 5 l of chloroform.

The chloroform extract (2.0 g) was subjected to
silica gel column chromatography and eluted
with a chloroform methanol gradient (0 , 100%
methanol). Fractions with similar patterns on TLC
supernatant

supernatant

supernatant

supernatant

were combined and 15 fractions were obtained.

Further silica gel column chromatography and
cell

cell

cell

cell

crystallisation, combined with comparison of their

2D NMR spectral data with literature values, were
used for identification of the chemical structures of
these metabolites.
Archidoris pseudoargaus (Nudibranch)

Determination of Minimum Inhibitory

Palmaria palmata (Red alga)
Fucus serratus (Brown alga)

Laminaria sp. (Brown alga)

Concentration (MIC)
Isolated from

The five metabolites isolated from the NUDMB50-11

culture were dissolved in methanol. The methanol
solutions were applied to antibiotic assay discs to
give a series of discs containing 50, 10, 5, 1, 0.5 and
0.1 mg of metabolite. Marine agar plates were
inoculated with the target bacteria and the disks
applied. MIC values were determined from the
minimum concentration giving a zone of clearing
MAI-11-01-CP (Bacillus licheniformis )

around the disk, as described previously (Brown &

Wallace, 1992).
NUDMB50-11 (Pseudomonas sp.)

II-111-5 (Bacillus subtilis )

Incorporation of Bacterial Extracts into Paints

FS55 (Bacillus pumilus )

Supernatant and cell extracts (Table II) from FS55,

NUDMB50-11, MAI-11-01-CP and II-111-5 cultures
were dissolved in minimal volumes of either
methanol or dichloromethane. The solutions were
Strain

mixed with a water based paint resin, Revacryl 380

(Harlaw Chemical Company Ltd). The concentration
200 J G BURGESS et al.

of the cell extracts was 28 mg ml21, while that of the cups (diameter 40 mm, height 35 mm) containing
supernatant was 20 mg ml21. All paints were applied painted test panels and 6 ml of filtered seawater. The
in the same manner and thoroughly dried prior to use. spores were allowed to settle for 2 h in darkness after
which the filtered seawater was exchanged for 10 ml
of fresh culture medium. The spores were left to
Antibacterial Assays on Paint Formulations
germinate at 198C under fluorescent lamps at an
The assay was carried out as described by irradiance of 50 mmol m22 s21 (PAR) and a photo-
Armstrong et al. (2000b). Fouling bacteria, 5.1, 4.2, period of 16 h light per day (Nordby, 1977; Hattori &
5.4, 132-10, 132-11, 134-15, 137-5, 137-8 and 137-9 Shizuri, 1996). After 6– 7 d, the test panels were
were used as target strains. Significant differences observed microscopically (320 £ ) using UTHSCSA
ðp , 0:05Þ between the effects of each paint were (Image Tool for Windows version 2.00) for result
determined using Kruskal – Wallis One Way ANOVA analysis. Three images were randomly selected from
ranks followed by Student –Newmann –Keuls all each replicate image covering about 4.5 mm2. Images
pairewise multiple comparison procedure. were analysed for the number of algal germlings per
mm 2, the relative size of the germlings and
percentage cover.
Barnacle Settling Assay
Larvae were obtained from adult barnacles collected
from the Swedish west coast (April –September). RESULTS
They were continuously fed with the microalga
Isochrysis galbana and the fairy shrimp Artemia salina. Purification of Antifouling Metabolites from
Newly settled barnacles were kept in aquaria with Marine Epibiotic Bacteria
flowing, filtered seawater. Larvae were collected by
Over 650 isolates of marine epibiotic bacteria from
a filter system consisting of four filters of decreasing
different sources were screened (Table I). Four of
mesh size (450 –490 mm), connected to the outlet of
the aquarium. The method of rearing larvae was
modified from Clare et al. (1995) and Clare (1996b).
Larvae were transferred to a 1.5 l flask with aerated
seawater (278C) and fed with a mixture of I. galbana
and Thalassiosira weissflogi. After 10 –12 d, the larvae
had reached the cyprid stage and were ready to
settle. Settling tests were performed using plastic
Petri dishes (Bibby Sterilin, diameter 50 mm). The
inside was painted with three layers of the test paint.
A drying time of 24 h was allowed for each layer.
After drying, the painted Petri dishes were filled
with filtered seawater to remove any possible toxic
residues from the paint itself. After 3 d, the Petri
dishes were replenished with 5 ml of fresh filtered
seawater and 25 –35 cyprids transferred to each and
settlement assayed after 12 d.

Macroalgal Settling Assay

Mature spore-producing fronds of the green macro-
alga Ulva lactuca were collected and incubated in
aerated seawater (178C) at an irradiance of approxi-
mately 100 mmol m22 s21 (PAR) for 16 h per day.
Roughened PVC test panels ð30 £ 30 mm2 Þ were
painted with three layers of test paints containing the
different extracts.
Zoospores of U. lactuca were released and
prepared as described by Callow et al. (1997). The
spores were counted by means of a Coulter
Counterw (MODEL ZF) and diluted with fresh
medium to 30,000 cells ml21. The test procedure
FIGURE 1 Metabolites with antifouling activity isolated from
modified from Fletcher (1989) was used. Spore a culture of the nudibranch-associated Pseudomonas sp.,
suspension (300 ml) was added to plastic settling NUDMB50-11.
 MARINE NATURAL PRODUCT-BASED AF PAINTS
TABLE III Minimum inhibitory concentrations of individual compounds isolated from NUDMB50-11 against a panel of fouling bacteria
Fouling bacteria (strain designations)
Compound
4.2 5.1 5.4 132-10
1 , ¼5 , ¼5 , ¼5 , ¼5
2 , ¼ 10 , ¼ 10 , ¼ 10 .50
3 , ¼5 , ¼5 , ¼5 , ¼5
4 , ¼ 0.5 , ¼1 , ¼ 10 , ¼5
5 , ¼5 , ¼ 50 , ¼ 50 , ¼ 50
Unit ¼ ug
the most active were used to produce extracts that
were incorporated into antifouling coatings (Table
II). One of the most active isolates was a Pseudomonas
sp., strain NUDMB50-11. Five metabolites were
isolated from a 5 l fermentation of NUDMB50-11 in
MSM9 medium (Figure 1). Of 15 fractions, fraction 3
gave phenazine-1-carboxylic acid (185 mg) (Stead
et al., 1996). Sep-Pak silica gel cartridge column
chromatography (chloroform-methanol gradient)
and reversed phase preparative TLC (methanol/-
water: 60/40) of fraction 4 gave 2-n-hyptyl quinol-4-
one (8 mg) and 2-n-nonylquinol-4-one (5 mg) (Deb-
itus et al., 1998). Silica gel preparative TLC (chloro-
form/methanol: 95/5) of fractions 5 and 6 gave
1-hydroxyphenazine (20 mg) and phenazine-1-car-
boxylic acid (Fernandez & Pizarro, 1997). Sep Pak C18
cartridge column chromatography (water-methanol
gradient: 0 , 100% methanol) and crystallisation
(acetone and methanol) of fractions 10 and 11 gave
pyolipic acid (25 mg) (Itoh et al., 1971; Syldatk et al.,
1985). The spectral data of pyolipic acid were
assigned in detail using 2D NMR.
Determination of MICs
Phenazine-1-carboxylic acid showed the most
marked activity with an MIC of 5 mg against seven
of the nine strains, and values of 1 and 10 mg against
strains 134-15 and 132-11 respectively by the
standard disc diffusion assay. In contrast, 1-hydro-
xyphenazine proved to be less active, only showing
activity against five of the nine strains and MIC
values were more than 10 mg. Both the quinol-4-ones
showed similar MICs. Pyolipic acid had an MIC
approximately of 50 mg against all of the strains
(Table III).
Antibacterial Activity of Paint Formulations
All the resin paint-extract preparations were active
against fouling strain 4.2. The cell and supernatant
extracts of FS 55 were not significantly more active
than the controls of resin alone or resin with
methanol when tested against strain 4.2. All
NUDMB50-11 extracts were significantly more active
than controls against strain 4.2 and extracts had
201
132-11 134-15 137-5 137-8 137-9
, ¼ 10 , ¼1 , ¼5 , ¼5 , ¼5
, ¼ 50 , ¼ 10 .50 .50 .50
, ¼ 0.5 , ¼ 10 , ¼ 50 , ¼ 50 .50
, ¼ 0.5 , ¼ 10 .50 .50 .50
, ¼ 50 , ¼ 50 , ¼ 50 , ¼ 50 .50
a pronounced effect in inhibiting the growth of the
other strains. Almost all of the coatings were more
active than the commercial antifouling paint. None of
the paints were active against fouling strain I-137-9.
However the NUDMB50-11 extract was active
against the strain before incorporation into the
paint base. Additionally, FS55 extracts were no
longer active against strain 5.1 once incorporated
into the paint (Table II).
In order to compare the bacterial extracts used in
each paint, the results were calculated to give a
measure of the zone of clearing obtained per mg of
pure extract. The NUDMB50-11 and methanol
soluble cell extract and supernatant extract were
the most active, being significantly better than all of
the other extracts against strains 5.4 and I-137-9. The
NUDMB50-11 water-soluble extract was more effec-
tive than that of FS55 when tested against 4.2, but the
reverse was true against strain I-137-8.
In the second set of assays another five paint
formulations were tested. FS55 supernatant and cell
extracts were again tested as well as cell and
supernatant extracts of MA 11-01-CP and the cell
extract of II-111-5. The supernatant extract of FS55
was active against 4.2 and I-137-5 and the cell extract
from FS55 was active against I-137-8 (compared with
the previous batch of paint made from FS55 which
was active against 4.1 and I-137-8). The paint made
from the supernatant extract of MAI-11-01-CP was
active against I-137-8, I-137-9 and 1-132-11 and the
cell extract paint of the same species active was only
against I-137-9. The paint made from II-111-5 was not
significantly more active than controls against any of
the fouling strains. From overall results pooled
across all nine fouling strains, the FS55 supernatant
and both supernatant and cell extract paints from
MAI-11-01-CP were significantly more effective than
all the negative controls and the commercial Marinex
marine antifouling paint. There was no significant
difference among these paints (Table II).
Barnacle Settlement Assay
Five of the paints exhibiting antifouling activity in
the antibacterial assays described above were used in
barnacle settlement assays. Two of the paints showed
202 J G BURGESS et al.

FIGURE 2 Settling frequency of barnacle larvae in Petri dishes treated with antifouling paints. Settling frequency is expressed as a
percentage of the negative controls. Vertical lines ¼ 95% confidence limits. NUD S ¼ NUDMB50-11 supernatant extract paint; FS55
S ¼ FS55 supernatant extract paint; MA S ¼ MAI-11-01-CP supernatant extract paint; FS55 C ¼ FS55 cell extract paint; 111-5 ¼ II-111-5 cell
extract paint.

a significant decrease in the number of settled inhibition of the settling frequency compared to the
barnacles in comparison with the control (Figure 2). negative control.
The paints of interest were those formulated from the
NUDMB50-11 supernatant ðp , 0:05Þ: All other
paints showed no significant antisettlement activity. DISCUSSION

The first stage of biofilm development is the

Algal Settlement Assay
Of the five paints described above, the paint
containing NUDMB50-11 supernatant showed a
significant inhibition in the settling frequency and
also the growth rate of U. lactuca compared to the
control (Figures 3 and 4). Paint containing super-
natant of MAI-11-01-CP showed a significant
adsorption of organic macromolecules to form a
conditioning film (Wahl, 1989). This is closely
followed by the attachment of bacteria and other
unicellular organisms. It has been suggested that
effective inhibition of biofilm formation at this early
stage would lead to a surface that lacks the necessary
characteristics to permit the settlement of larvae of

FIGURE 3 Settling frequency of algal spores in Petri dishes treated with antifouling paints. Settling frequency is expressed as a
percentage of the negative controls. Vertical lines ¼ 95% confidence limits. NUD S ¼ NUDMB50-11 supernatant extract paint; FS55
S ¼ FS55 supernatant extract paint; MA S ¼ MAI-11-01-CP supernatant extract paint; FS55 C ¼ FS55 cell extract paint; 111-5 ¼ II-111-5 cell
extract paint.
 MARINE NATURAL PRODUCT-BASED AF PAINTS
FIGURE 4 Area covered by algal spores in Petri dishes treated with tested antifouling paints. The areas are expressed as a percentage of
the negative control. Vertical lines ¼ 95% confidence limits. NUD S ¼ NUDMB50-11 supernatant extract paint; FS55 S ¼ FS55 supernatant
extract paint; MA S ¼ MAI-11-01-CP supernatant extract paint; FS55 C ¼ FS55 cell extract paint; 111-5 ¼ II-111-5 cell extract paint.
macrofoulers (Satuito et al., 1997; Wieczorek & Todd,
1997). Therefore, the development of a paint with
antibacterial activity may disrupt the early stages of
biofilm development, and provide an effective
antifouling coating for the protection marine struc-
tures. In addition, the use of a water-based paint,
Revacryl 380, would overcome the adverse environ-
mental and health impacts of solvent based paints.
The study of natural product antifoulants has
included the assessment of the activity of crude
extracts or isolated metabolites in antifouling assays
(Armstrong et al., 2000a; 2000b). Although there are
some reports of paint formulations incorporating
characterised natural products (Shin & Smith, 2001),
studies using microbial extracts are rare. Extracts of
the sea pansy, Renilla reniformis, added to commer-
cially available paint and encapsulated in metallic
microtubules (Price et al., 1994) were effective in
controlling biofouling over short periods in the
marine environment. Paint formulations incorpora-
ting extracts of sponges were also active in barnacle
settling assays (Willemsen & Ferrari, 1993) and in a
separate study, extracts of sponges and a gorgonian
were shown to be active against tube worms when
mixed with abietic acid and coated onto panels
(Bakus et al., 1994). However, no attempts have been
made to identify the active compounds in these
coatings.
Most of the bacteria isolated in the present study
which showed good antifouling activity were species
of Bacillus and Pseudomonas. This may reflect the
culture methods used rather than be an indication of
species diversity in the natural surface films. Five
compounds exhibiting antifouling activity were
isolated and characterised from a culture of the
most active strain, NUDMB50-11. All five com-
pounds have been reported to be secondary
metabolites of Pseudomonas and their antimicrobial
203
activities have been documented (Leisinger &
Margraff, 1979; Turner & Messenger, 1986). Pyolipic
acid has also been reported to exhibit mycoplasma-
cidal and antiviral activities in vitro (Leisinger &
Margraff, 1979), and was reported as a biosurfactant
with potential for application in combating marine
oil pollution (Vandyke et al., 1993; Lang &
Wullbrandt, 1999). However, this is the first report
describing the antifouling activity of these com-
pounds. The compounds represent three diverse
classes of molecule with different physical properties
and antimicrobial activities. This range of physical
properties and activities may, when the extract is
incorporated into a paint, give active components
that are released at different rates from the painted
surface and have differential activities against a
range of target organisms.
Clare (1996a) reviewed antifouling natural pro-
ducts from marine sources identifying a number of
compounds with antifouling activities. However, the
vast majority of these were classified as antifoulants
on the basis of a single assay. The antifouling activity
of one bacterium has however been studied in some
detail. Pseudoalteromonas tunicata, isolated from the
surface of the tunicate Ciona intestinalis, produced at
least five extracellular compounds which inhibited
the settlement or development of a range of surface
colonising species (Holmstrom & Kjelleberg, 1999).
The compounds, which have not been identified,
inhibit the settlement of invertebrates and algal
spores, growth of bacteria and fungi and surface
colonisation by diatoms.
In addition, sea trials were also conducted in two
different locations, one in Scotland and one in
Sweden (data not shown). In contrast with the
laboratory assays, the field trials showed the paints
had little effect on either the onset or degree of
macrofouling. This appears to be a common theme in
204 J G BURGESS et al.
attempts to develop antifouling coatings whereby
the promise of laboratory assays are rarely if ever
confirmed by field trials (Costerton & Lappin-Scott,
1995; Matsumura et al., 2000). It could be the case that
the active metabolites are leached from the paint too
quickly and therefore any antifouling activity is lost
over a fairly short time. In a previous study, the paint
formulation system described here was shown to
rapidly leach antimicrobial metabolites into seawater
(Peppiatt et al., 2000). Thus, future improvements are
aimed at developing coatings which release these
compounds more slowly.
In conclusion, the strategy adopted in this study
has identified a number of surface associated
bacteria capable of producing metabolites that,
when incorporated into paint, retain their antifouling
activity. Five metabolites exhibiting antifouling
activity were purified and identified. Broad spec-
trum antifouling activity was achieved by combi-
nations of these simple antimicrobial compounds.
This work demonstrates the potential of marine
bacteria in the production of antifouling coatings
based on biodegradable natural products rather than
the toxic compounds in current use.
Acknowledgements
The authors thank Tony Clare (Department of
Marine Science and Coastal Management, University
of Newcastle) for contributing his expertise and
advice regarding the development of the barnacle
settling assays. The authors thank the European
Commission (MAS3-CT98-0162) for financial
support.
References
Armstrong E, Boyd K G, Burgess J G (2000a) Prevention of marine
biofouling using natural compounds from marine organisms.
Biotechnol Annu Rev 6: 221–241
Armstrong E, Boyd K G, Pisacane A, Peppiatt C J, Burgess J G
(2000b) Marine microbial natural products in antifouling
coatings. Biofouling 16: 215–224
Bakus G J, Wright M, Khan A K, Ormsby B, Gulko D A, Licuanan
W, Carriazo E, Ortiz A, Chan D B, Lorenzana D, Huxley M P
(1994) Experiments seeking marine natural antifouling com-
pounds. In: Thompson M F, Nagabhushanam R, Sarojini R and
Fingerman M (eds) Recent Developments in Biofouling Control. A
A Balkema, Rotterdam, pp 373–381
Batley G E, Chapman J C, Wilson S P (1994) Environmental
impacts of antifouling technology. In: Kjelleberg S, Steinberg P
(eds) Biofouling: Problems and Solutions. UNSW, Sydney, pp 49 –53
Boyd K G, Adams A R, Burgess J G (1999a) Antibacterial and
repellent activity of marine bacteria associated with algal
surfaces. Biofouling 14: 227 –236
Boyd K G, Mearns-Spragg A, Burgess J G (1999b) Screening of
marine bacteria for the production of microbial repellents using
a spectrophotometric chemotaxis assay. Mar Biotechnol 1:
359–363
Boyd K G, Mearns-Spragg A, Brindley G, Hatzidimitrou K, Rennie
R, Bregu M, Hubble M O, Burgess J G (1998) Antifouling
potential of epiphytic marine bacteria from the surfaces of algae.
In: Le Gal Y, Muller-Feuga A (eds) Marine Microorganisms for
Industry. Editions IFREMER, Plouzané, France, pp 128 –136
Brown B A, Wallace R J Jr (1992) Broth microdilution MIC test for
Nocardia spp. In: Clinical Microbiology Procedures Handbook,
Section 5: Antimicrobial Susceptibility Testing. American
Society for Microbiology Book Division, Washington, DC,
p 5.12.1
Bultman J D, Griffith J R (1994) Fluoropolymer and silicone
fouling-release coatings. In: Thompson M F, Nagabhushanam R,
Sarojini R, Fingerman M (eds) Recent Developments in Biofouling
Control. A A Balkema, Rotterdam, pp 383–389
Burgess J G, Jordan E M, Bregu M, Mearns-Spragg A, Boyd K G
(1999) Microbial antagonism: a neglected avenue of natural
products research. J Biotechnol 70: 27–32
Callow M E, Callow J A, Pickett-Heaps J, Wetherbee R (1997)
Primary adhesion of Enteromorpha (Chlorophyta, Ulvales)
propagules: quantitative settlement studies and video
microscopy. J Phycol 33: 938–947
Cheng X C, Jensen P R, Fenical W (1999) Luisols A and B, new
aromatic tetraols produced by an estuarine marine bacterium of
the genus Streptomyces (Actinomycetales). J Nat Prod 62: 608 –610
Claisse D, Alzieu C (1993) Copper contamination as a result of
antifouling paint regulations? Mar Pollut Bull 26: 395– 397
Clare A S (1996a) Marine natural product antifoulants: status and
potential. Biofouling 9: 211–229
Clare A S (1996b) Signal transduction in barnacle settlement:
calcium revisited. Biofouling 10: 141–159
Clare A S, Thomas R F, Rittschof D, (1995) Evidence for the
involvement of cyclic AMP in the pheromonal modulation of
barnacle settlement. J Exp Biol 198: 655–664
Costerton J W, Lappin-Scott H (1995) Introduction to microbial
biofilms. In: Costerton J W, Lappin-Scott H (eds) Microbial
Biofilms. Cambridge University Press, Cambridge, pp 1–11
Debitus C, Guella G, Mancini I, Waikedre J, Guemas J-P, Nicolas J
N, Pietra F (1998) Quinolones from a bacterium and tyrosine
metabolites from its host sponge, Suberea creba from the Coral
Sea. J Mar Biotechnol 6: 136 –141
Dobretsov S V, Qian P-Y (2002) Effect of bacteria associated with
the green alga Ulva reticulata on marine micro- and macrofoul-
ing. Biofouling 18: 217–228
Fernandez R O, Pizarro R A, (1997) High-performance liquid
chromatographic analysis of Pseudomonas aeruginosa phena-
zines. J Chromatog A 77: 99–104
Fisher W S, Oliver L M, Walker W W, Manning C S, Lytle T F (1999)
Decreased resistance of eastern oysters (Crassosterea virginica )
to a protozoal pathogen (Perkinsus marinus ) after
sublethal exposure to tributyltin oxide. Mar Environ Res 47:
185–201
Fletcher R L (1989) A bioassay technique using the marine fouling
green alga Enteromorpha. Int Biodeterior 25: 407 –422
Foster A C (1994) Current antifouling technologies. In: Kjelleberg
S, Steinberg P (eds) Biofouling: Problems and Solutions. UNSW,
Sydney, pp 44–48
Gibbon P (1995) Molluscan shellfisheries in estuaries. In: Earl R C
(ed) Marine Environmental Management: Review of Events in 1994
and Future Trends. Candle Cottage, Kempley, Gloucestershire,
UK, p 83
Gil-Turnes M S, Fenical W, (1992) Embryos of Homarus
americanus are protected by epibiotic bacteria. Biol Bull 182:
105–108
Gil-Turnes M S, Hay M E, Fenical W (1989) Symbiotic marine
bacteria chemically defend crustacean embryos from a
pathogenic fungus. Science 246: 116–118
Grinwis G C M, Boonstra A, van den Brandhof E J, Dormans J A M
A, Englesma M, Kuiper R V, van Loveren H, Wester P W, Vaal M
A, Vethaak A D, Vos J G (1998) Short-term toxicity of bis(tri-n-
butylyin) oxide in flounder (Platichthys flesus ): pathology and
immune function. Aquat Toxicol 42: 15–36
Hashimoto S, Watanabe M, Noda Y, Hayashi T, Kurita Y, Takasu Y,
Otsuki A (1998) Concentration and distribution of butyltin
compounds in a heavy tanker route in the Strait of Malacca and
in Tokyo Bay. Mar Environ Res 45: 169– 177
Hattori T, Shizuri Y (1996) A screening method for antifouling
substances using spores of the fouling macroalga Ulva
conglobata. Fish Sci (Tokyo) 62: 955– 958
Holmstrom C, Kjelleberg S (1999) Marine Pseudoalteromonas
species are associated with higher organisms and produce
biologically active extracellular agents. FEMS Microbiol Ecol 30:
285–293
 MARINE NATURAL PRODUCT-BASED AF PAINTS
Itoh S, Honda H, Tomita F, Suzuki T (1971) Rhamolipids produced
by Pseudomonas aeruginosa grown on n-paraffin. J Antibiotics 14:
855–859
James S G, Holmstrom C, Kjelleberg S (1996) Purification and
characterisation of a novel antibacterial protein from the marine
bacterium D2. Appl Environ Microbiol 62: 2783–2788
Jiang Z D, Jensen P R, Fenical W (1999) Lobophorins A and B, new
antiinflammatory macrolides produced by a tropical marine
bacterium. Bioorg Med Chem Lett 9: 2003–2006
Kavanagh C J, Schultz M P, Swain G W, Stein J, Truby K,
Darkangelo Wood C (2001) Variation in adhesion strength of
Balanus eburneus, Crassosterea virginica and Hydroides dianthus to
fouling release coatings. Biofouling 17: 155–167
Lang S, Wullbrandt D (1999) Rhamnose lipids-biosynthesis,
microbial production and application potential. Appl Microbiol
Biotechnol 51: 22–32
Leisinger T, Margraff R (1979) Secondary metabolites of the
fluorescent pseudomonads. Microbiol Rev 43: 422–442
Matsumura K, Hills J M, Thomason P O, Thomason J C, Clare A S
(2000) Discrimination at settlement in barnacles: laboratory and
field experiments on settlement behaviour in response
to settlement-inducing protein complexes. Biofouling 16:
181–190
Matthiessen P, Gibbs P E (1998) Critical appraisal of the evidence
for tributyltin-mediated endocrine disruption in mollusks.
Environ Toxicol Chem 17: 37 –43
Mearns-Spragg A, Bregu M, Boyd K G, Burgess J G (1998) Cross-
species induction and enhancement of antimicrobial activity
produced by epibiotic bacteria from marine algae and
invertebrates, after exposure to terrestrial bacteria. Lett Appl
Microbiol 27: 142–146
Nakasono S, Burgess J G, Takahashi K, Murayama C, Nakamura S,
Matsunaga T (1993) Electrochemical prevention of biofouling
with a carbon-chloroprene sheet. Appl Environ Microbiol 59:
2757–2762
Nordby O (1977) Optimal conditions for meiotic spore formation
in Ulva mutabilis. Bot Mar 20: 19–28
de Nys R, Steinberg P, Willemsen P, Dworjanyn S A, Gabelish C L,
King R J (1995) Broad spectrum effects of secondary metabolites
from the red alga Delisea pulchra in antifouling assays. Biofouling
8: 259–271
Peppiatt C J, Armstrong A, Pisacane A, Burgess J G (2000)
Antibacterial activity of resin based coatings containing marine
microbial extracts. Biofouling 16: 225–234
205
Price R R, Patchan M, Clare A S, Rittschof D, Bonaventura J (1994)
Performance enhancement of natural antifouling compounds
and their analogs through microencapsulation and controlled
release. In: Thompson M-F, Nagabhushanam R, Sarojini R,
Fingerman M (eds) Recent Developments in Biofouling Control.
A A Balkema, Rotterdam, pp 321 –334
Satuito C G, Shimizu K, Fusetani N (1997) Studies on the factors
influencing larval settlement in Balanus amphitrite and Mytilus
galloprovinvialis. Hydrobiologia 358: 275–280
Shin H W, Smith C M (2001) Antifouling activity of six nontoxic
chemicals on spore attachment of Ulva fasciata. J Environ Biol 22:
145–151
Stead P, Rudd B A M, Bradshaw H, Noble D, Dawson M J (1996)
Induction of phenazine biosynthesis in cultures of Pseudomonas
aeruginosa by L-N-(3-oxohexanoyl)homoserine lactone. FEMS
Microbiol Lett 140: 15–22
Stewart C, Thompson J A J (1997) Vertical distribution of butyltin
residues in sediments of British Columbia harbours. Environ
Technol 18: 175–202
Syldatk C, Lang S, Wagner F, Wray V, Witte L (1985) Chemical and
physical characterization of four interfacial-active rhamnolipids
from Pseudomonas spec. DSM 2874 grown on n-alkanes.
Z Naturforsch [C] 40: 51–60
Thomas K V (2001) The environmental fate and behaviour
of antifouling paint booster biocides: a review. Biofouling
17: 73– 86
Turner J M, Messenger A J (1986) Occurrence, biochemistry and
physiology of phenazine pigment production. Adv Microbial
Physiol 27: 211–274
Vandyke M I, Couture P, Brauer M, Lee H, Trevors J T (1993)
Pseudomonas aeruginosa Ug2 rhamnolipid biosurfactants-struc-
tural characterization and their use in removing hydrophobic
compounds from soil. Can J Microbiol 39: 1071–1078
Wahl M (1989) Marine epibiosis. I. Fouling and antifouling: some
basic aspects. Mar Ecol Prog Ser 58: 175– 189
Wahl M, Jensen P R, Fenical W (1994) Chemical control of bacterial
epibiosis on ascidians. Mar Ecol Prog Ser 110: 45–57
Wieczorek S K, Todd C D (1997) Inhibition of bryozoan and
ascidian settlement by natural multispecies biofilms: effects of
film age and the roles of active and passive algal attachment.
Mar Biol 128: 463– 473
Willemsen P R, Ferrari G M (1993) The use of anti-fouling
compounds from sponges in anti-fouling paints. Surface Coat Int
10: 423– 427