BioLocus A/S

– Developing sustainable AF coatings for the

future

Martin Hangler, Ib Schneider & Knud Allermann (BioLocus A/S)

The settlement of different macro-and microorganisms on marine surfaces, a process known as

biofouling (Fig. 1), constitutes a major economic problem for the maritime industry. At present

antifouling (AF) coatings containing toxic biocides and heavy metals are used on installations and

ships. However, unwanted adverse effects of the biocides such as toxicity to non-target organisms,

imposex in gastropods and increased multiresistance amongst bacteria have been observed.

Therefore, new alternative agents are needed.

Fouling organisms use biological adhesives in their

initial attachment process and a way to disrupt this is

by employing adhesive degrading enzymes. Profound

effect on the settlement of algal spores and barnacle

larvae has been shown (Pettit et al. 2004; Dobretsov et

al. 2007).The small research based company BioLocus

A/S focus on the concept of employing biodegradable

enzymes to AF paints. When certain enzymes are

applied to a polishing waterbased paint, it maintains

profound antifouling effect for one fouling season (Fig. 2). Therefore, the product Coatzyme© is a

good alternative to traditionel paints in the pleasure boat market but an AF effect of one year does

not comply with the docking intervals of commercial vessels.

BioLocus aims to develop an enzyme-based AF coating

capable of matching the requirements of the maritime

industry. Our initial goal is to develop a coating with good

AF performance of 2-3 years in tropical waters.

In that respect we focus on two aspects of our research in this

presentation: 1. Effect of enzymes on marine biofilms - and

2. Retaining, dispersing and stabilising enzymes in coatings

Figure 2. Testpanels in Elsinore harbour at the end of the fouling

season (October 2006). Upper left is an un-treated control, the grey panel

is a copper based product and the marked panel is our best test-paint

Figure 1: Yacht after one year in seawater

1. Effect of enzymes on marine biofilms.

Any marine surface under static conditions, even when protected by biocides (e.g. copper and TBT)

is rapidly covered by a biofilm. When TBT is present the thickness of the film does not exceed 20

µm after one year, while in the absence of TBT and presence of copper it exceeds 50 µm (Jackson

& Jones 1988).

The thickness of the biofilm can affect AF paint performance, through decreasing the release rate of

the biocides. and the biocides may be degraded within the biofilm (Yebra et al 2006).

For small persistent molecules like different aromatic herbicides and organnometallic compounds,

the diffusion rate through the biofilms may be neglectable. But when the antifouling effects of the

enzymes are evaluated, the degradation and diffusion rates should both be considered. Therefore,

the effectiveness of enzymes on macrofoulers, needs to be interpreted alongside their

corresponding effect on biofilms.

We have tested a model enzyme product on a multispecies biofilm at various concentrations (Fig 3).

Our model enzyme inhibits biofilm formation in a non-toxic manner at low concentrations as the

growth of free-flowing bacteria (planktonic fraction) is not affected. At high concentrations the

effect may be toxic as the industrial enzyme-product contains additives with antibacterial effect. An

enzyme product without these additives shows no toxic effect on the planktonic fractions regardless

of the concentrations (Hangler et al. submitted). Furthermore the stability and effectiveness was

increased when the enzyme was immobilised.

This leads us to a second aspect of our research - The immobilisation.

Inhibition of biofilm development

Protein concentration (mg/mL)

3.75 1.875 0.375 Control

OD

595

0,0

0,5

1,0

1,5

2,0

2,5

Effect on planktonic fractions

Protein concentration (mg/mL)

3.75 1.875 0.375 Control

OD

595

0,0

0,1

0,2

0,3

0,4

0,5

0,6

A B

Figure 3: Effect of enzyme on biofilm development (A) and planktonic growth (B). Briefly, different dissolutions of an

industrial enzyme product (E) were added to the bottom of the wells in a 96 well TC plate. After evaporation, addition of bacteria

and media (B+M) and 16 hours incubation the planktonic bacteria were transferred to clean wells, while the developed biofilms

were stained with crystal violet (CV) (O´Toole and Kolter 1998). The biofilm formation was evaluated with native (black) and

denatured (grey) enzyme

2. Retaining, dispersing and stabilising enzymes in coatings

A high initial release of enzyme to the surrounding water is observed from paints when the enzyme

has not been immobilised. With our new technology the enzymes are retained in the coating and a

close to constant enzyme activity is observed over time at the paint surface.

A linear relationship exists between

enzyme-concentration in the paint and

enzyme-activity at the surface (Fig 4).

Enzymes are thus dispersed evenly in

the coating with our new technology.

To verify the antifouling effect in situ, exposure studies have been conducted both in temperate and

tropical waters and in collaboration with a large shipping company we are conducting tests on a

large commercial vessel, currently operating in tropic waters (Fig 5).

Conclusion

In recent years the development of sustainable AF coatings have centered on two strategies. One is

the development of surfaces with low surface energy by treatment with silicone and Teflon

compounds (Foul-Release), and the other focus on environmentally friendly anti fouling agents, for

instance enzyme based coatings. Silicone and Teflon compounds have limited success on ships

operating over large distances with a constant speed about 15 knots and only a few stops, but self -

polishing paints with high copper content and pesticides for control of algae are still applied for

ships with short regular services and frequent stops.

Figure 4. Enzyme activity at the surface of a coating. Three

different concentrations of encapsulated enzyme was added to paints

corresponding to 25%, 50% and 100% respectively. The correlation

between the theoretical activity (X-axis) and the measured activity (Y-

axis) shows an even distribution of the enzymes at the surface of the

coating. (data are means ± Standard error, n=6)

Figure 5. Test of coatings

containing immobilised

enzymes. A 6 month

exposure study in Helsingør

harbour with (A) a

commercial red copper based

and a white enzyme based AF

coating. (B) a 25 m2 test area

on the vessel “TORM

GERD” with blue enzyme

based and red copper based

AF coating (B)

A B

Replacing the toxic chemicals in self-polishing paints with environmentally friendly enzymes are

therefore an attractive alternative to some of todays products. BioLocus is currently optimising our

coatings and the results are promising.

Even though an enzyme based antifouling product complying to EU regulations (i.e the Biocidal

Products Directive (BPD) has yet to hit the market, BioLocus is confident that we are not far from

reaching our goal of a sustainable AF coating matching the toxic products of today.

References and information:

1. Dobretsov, S., Xiong, H., Xu, Y., Levin, LA.,Qian, P. (2007) Marin. Biotechnol. 9; 388-297

2. Jackson, SM., Jones,.EBG., (1988). Int. Biodeterio. 24; 277-287

3. Hangler, M., Burmølle, M., Schneider, I., Allermann, K., Jensen, B., (2008), submitted for

Biofouling

4. O´Toole, GA., Kolter, R., (1998). Mol. Microbiol. 28; 449-461

5. Pettitt, ME., Henry, SL., Callow, ME., Callow, JA., Clare, AS. (2004). Biofouling. 20; 299-

311

6. Yebra, DM., Kiil, S., Dam-Johansen, K., Weinell, CE., (2006). AlChE. 52; 1926-1940

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