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Combinatorial materials research applied to thedevelopment of new surface coatings V. Application of aspinning water-jet for the semi-high throughputassessment of the attachment strength of marine foulingalgae

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throughput assessment of the attachment strength of marine fouling algae', Biofouling, 23:2, 121 - 130To link to this article: DOI: 10.1080/08927010701189583URL: http://dx.doi.org/10.1080/08927010701189583

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Combinatorial materials research applied to the developmentof new surface coatings V. Application of a spinning water-jetfor the semi-high throughput assessment of the attachment strengthof marine fouling algae

FRANCK CASSE1, SHANE J. STAFSLIEN2, JAMES A. BAHR2, JUSTIN DANIELS2,

JOHN A. FINLAY1, JAMES A. CALLOW1 & MAUREEN E. CALLOW1

1The University of Birmingham, School of Biosciences, Birmingham, UK, and 2Center for Nanoscale Science and Engineering,

North Dakota Sate University, Fargo, North Dakota, USA

(Received 4 October 2006; accepted 21 December 2006)

AbstractIn order to facilitate a semi-high throughput approach to the evaluation of novel fouling-release coatings, a ‘spinjet’apparatus has been constructed. The apparatus delivers a jet of water of controlled, variable pressure into the wells of 24-wellplates in order to facilitate measurement of the strength of adhesion of algae growing on the base of the wells. Two algae,namely, sporelings (young plants) of the green macroalga Ulva and a diatom (Navicula), were selected as test organismsbecause of their opposing responses to silicone fouling-release coatings. The percentage removal of algal biofilm waspositively correlated with the impact pressure for both organisms growing on all the coating types. Ulva sporelings wereremoved from silicone elastomers at low impact pressures in contrast to Navicula cells which were strongly attached to thistype of coating. The data obtained for the 24-well plates correlated with those obtained for the same coatings applied tomicroscope slides. The data show that the 24-well plate format is suitable for semi-high throughput screening of the adhesionstrength of algae.

Keywords: Alga, diatom, fouling release, high-throughput screen, silicone elastomer, Ulva, Navicula

Introduction

All surfaces placed in the sea are rapidly colonised by

a consortium of marine organisms specialised for

benthic life. Ships and other marine structures are

traditionally protected from biofouling by biocide-

containing antifouling paints (Turley et al. 2005;

Finnie, 2006, Jelic-Mrcelic et al. 2006) but new

coatings are now required that do not have a negative

impact on the marine environment. The only major

type of non-biocidal coatings currently commercially

available are based on elastomeric polydimethylsi-

loxane (PDMS), the so-called fouling release coat-

ings (e.g. Kavanagh et al. 2001, Stein et al. 2003,

Sun et al. 2004, Wendt et al. 2006). Fouling release

coatings facilitate the weak adhesion of macro-

fouling organisms such as barnacles, tubeworms

and macroalgae (Holm et al. 2006), which are

released under suitable hydrodynamic conditions

(Kavanagh et al. 2005). Finding alternative tech-

nologies is expensive and time consuming as the

number of combinations that need to be synthesised,

characterised and evaluated is vast.

Evaluation of coatings applied to microscope

slides has been used successfully to reveal the

strength of attachment of algal biofilms when a

limited number of coatings are being studied (e.g.

Chaudhury et al. 2005; Gudipati et al. 2005; Tang

et al. 2005; Krishnan et al. 2006a; 2006b; Statz

et al. 2006; Yarbrough et al. 2006). However, the

combinatorial approach adopted by North Dakota

State University (NDSU) has the potential to

generate hundreds of combinations of polymers of

the same generic type (Webster et al. 2004; Webster

2005; 2007). Hence, new screening methods

are required to down-select samples with fouling-

release potential to a number that is manageable for

more extensive biological evaluation, e.g. through

more rigorous assays of coatings applied to slides or

raft panels.

Correspondence: M. E. Callow, The University of Birmingham, School of Biosciences, Birmingham B15 2TT, UK. Fax: þ44(0) 121 414-5447.

E-mail: m.e.callow@bham.ac.uk

Biofouling, 2007; 23(2): 121 – 130

ISSN 0892-7014 print/ISSN 1029-2454 online � 2007 Taylor & Francis

DOI: 10.1080/08927010701189583

Dow

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07 Large scale screening of bioactive compounds

using high-throughput methods are now routinely

used in the pharmaceutical industry. Such methods

using multi-well plates and plate readers have also

been adapted successfully for screening bioactive

compounds against fouling organisms (Bers et al.

2006; Stafslien et al. 2006; 2007a). The present

paper describes a semi-high throughput method

based on 24-well plates to determine the strength

of attachment of algae. The data are compared to

those obtained for the same coatings applied to glass

microscope slides. Two types of fouling algae with

different adhesion characteristics were selected,

namely, the green macroalga Ulva linza and a

diatom, Navicula perminuta.

Ulva (syn. Enteromorpha) is the most common

macroalga that fouls ships and other submerged

structures. Dispersal of Ulva is mainly through

motile, quadriflagellate zoospores (approximately

7 – 8 mm in length), which are released in large

numbers and form the starting point of the assay

(Callow et al. 1997). The swimming spores settle

and adhere to suitable surfaces, adhesion being

mediated through a glycoprotein adhesive (Callow &

Callow, 2006). The settled spores rapidly germinate

into sporelings (young plants), which adhere weakly

to fouling release coatings (Schultz et al. 2003;

Chaudhury et al. 2005).

Slimes dominated by diatoms are the predominant

form of microfouling on all illuminated surfaces

immersed in the sea (Patil & Anil 2005a; 2005b),

including biocidal antifouling paints (Casse & Swain,

2006; Jelic-Mrcelic et al. 2006) and non-biocidal

coatings (Terlizzi et al. 2000; Casse & Swain,

2006). Adhesion is especially tenacious to silicone

fouling-release coatings and diatom slimes are not

released from vessels including those that operate at

high speeds (Terlizzi et al. 2000; Holland et al.

2004).

The ease of removal of biomass from surfaces was

quantified by application of hydrodynamic forces

using a miniaturised water-jet apparatus (‘spinjet’),

specially designed for use on 24-well plates.

Methods

Sample preparation

A number of calibration experiments were per-

formed using untreated 24-well plates (3524, Corn-

ing Incorporated, Costar1). The details of individual

experiments are provided in the Results section. The

coatings used in the assays comprised two fouling-

release siloxanes, namely, Dow Corning’s Silastic1

T-2 and Intersleek1 (International Paint Ltd) and

polyurethane. The coatings were either applied to

glass or aluminium discs that were fixed in the wells

or were directly deposited in the wells as described

by Stafslien et al. (2006).

Standard coatings were applied to glass slides and

24-well plates at NDSU. Prior to coating pre-

paration, glass slides were immersed for 24 h in a

1:3 solution of hydrogen peroxide and sulphuric

acid, respectively (piranha solution). Slides were

removed from the piranha solution and immediately

rinsed with copious amounts of deionised water and

dried at ambient laboratory conditions. Coatings

solutions were then dispensed on piranha treated

slides until complete coverage was achieved. Coat-

ings were applied to 15 mm aluminium discs already

fixed onto the bottom of the wells with epoxy for the

24-well plates as described in Stafslien et al. (2006).

In some experiments, coatings were applied to

15 mm diameter glass coverslips that were subse-

quently adhered to the bottom of the wells using

colourless, white or black epoxy. Preliminary experi-

ments showed no significant difference between

results obtained for both formats.

Leaching of test samples

All 24-well plates were vented for 1 week in a flow

oven at 308C and then pre-leached in deionised

water for 4 weeks with daily exchange of water in the

wells at NDSU prior to shipping to Birmingham

(Stafslien et al. 2006). The slides with experimental

coatings were shipped to Birmingham where they

were leached for 4 weeks in a 30-l tank of re-

circulating deionised water fitted with a carbon filter.

The 24-well plates and slides were equilibrated in

artificial seawater for 2 h before the start of each

experiment.

Ulva sporeling assay in 24-well plates

Fertile Ulva linza was collected from Wembury

Beach, England (508180N; 48020W) and zoospores

were released as described in Callow et al. (1997).

The concentration of spores was adjusted to

56105 spores ml71.

Twelve replicates wells (6 per row) were used for

each treatment. Each well of the 24-well plates was

inoculated with 1 ml of zoospore suspension. The

24-well plates were immediately placed for 2 h in the

dark at 208C to allow the spores to settle. The wells

were then emptied to remove unsettled spores and

1 ml of enriched seawater medium (Starr & Zeikus,

1987) was added per well. The plates were placed in

an illuminated incubator at 188C with a 16:8 light:

dark cycle (photon flux density 46 mmol m72 s71)

for 5 d and the medium changed every 48 h.

The strength of attachment of the biomass was

determined using the spinjet apparatus described

below. The 24-well plates were jetted at a range of

122 F. Casse et al.

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07 impact pressure. One row of 6 replicates was jetted;

the adjacent row was not jetted. Non-jetted samples

provide information on the amount of biomass in the

wells and also serve as controls for calculation of

percentage biomass removal. Biomass was quan-

tified by the fluorescence of chlorophyll, which was

extracted from biomass in the well with 1 ml of

dimethyl sulphoxide (DMSO). The plates were

incubated in darkness for 30 min. After ensuring

adequate mixing, 200 ml of DMSO were pipetted

from each well into wells of a 96-well plate and the

fluorescence read in a Tecan plate reader (GENios

Plus) with chlorophyll filter (excitation wavelength:

360 nm; emission wavelength: 670 nm) connected

to a computer with Magellan v.4.00 software. Each

well reading was based on four spot readings, taken

in a 262 square. All plates were read from

the top. Fluorescence was recorded as Relative

Fluorescence Units (RFU). The mean of six

replicate wells+ 95% confidence limits was calcu-

lated. The strength of attachment data are presented

as percentage removal compared with the controls,

+95% confidence limits derived from arcsine

transformed data.

The distribution of spores deposited in the un-

coated polystyrene wells after 2 h settlement in

darkness was quantified on untreated plates. After

washing, the attached spores were fixed in 2.5%

glutaraldehyde in seawater (Callow et al. 1997).

Settled spores, viewed through the bottom of the

wells, were counted at 1 mm intervals across the

diameter of the well as described in Callow et al.

(2002).

Ulva sporeling assay on coatings applied

to microscope slides

Coated slides (6 replicates per treatment) were

placed in individual compartments of Quadriperm

dishes (Greiner) and 10 ml of a spore suspension

containing 56105 spores ml71 added. After 3 h in

darkness, the slides were washed in artificial seawater

(ASW) to remove any unattached spores. The settled

spores were cultured (Chaudhury et al. 2005) for

5 days under the same conditions as the 24-well

plates. Growth was estimated by direct measure-

ment of fluorescence from the chlorophyll of the

sporelings using a Tecan plate reader (GENios Plus).

Fluorescence was recorded as RFU from direct

readings. The slides (6 replicates) were read from

the top, 300 readings per slide, taken in blocks of

30610. One blank slide of the same coating was

used to obtain a mean background reading and this

value was subtracted from the respective test

surfaces.

The strength of attachment of the sporelings was

determined by jet washing using the water jet

described by Finlay et al. (2002), which was adapted

for use with microscope slides from the original

apparatus (Swain & Schultz, 1996). RFU readings

(80 per slide) were taken from the central part of

the slide that was exposed to the water jet. The

percentage removal was calculated as described

above.

Diatom assays in 24-well plates

The diatom Navicula perminuta was cultured in

natural seawater supplemented with nutrients from

Guillard’s F/2 medium as described in Holland et al.

(2004). Cultures were grown under static conditions

in 250 ml Pyrex conical flasks containing 100 ml

medium in a growth cabinet at 188C with a

16:8 light: dark cycle (photon flux density 21 mmol

m72 s71).

The cell suspension was poured away leaving a

biofilm of cells adhered to the bottom of the flasks.

The biofilm was gently resuspended in artificial

seawater (ASW) before filtering through 20 mm

nylon mesh. The concentration of cells was adjusted

to 46105 cells ml71. Each 24-well plate was

inoculated with 1 ml of cell culture, which was left

for 2 h on the laboratory bench in the light at room

temperature. Quantification of biomass and the

adhesion assay were the same as described above

for Ulva.

The distribution of Navicula cells in uncoated

polystyrene wells after spinjetting was determined

after fixing in 2.5% glutaraldehyde in seawater as

described for Ulva.

Diatom assays on coatings applied to microscope slides

Six replicate slides of each surface placed into in-

dividual compartments of Quadriperm dishes

(Greiner) were inoculated with 10 ml of culture

containing 46105 cells ml71. The dishes were

allowed to stand for 2 h on the bench in the light

at room temperature.

Strength of attachment of cells was determined by

jet washing 3 replicate slides with artificial seawater

using the water jet described by Finlay et al. (2002).

The other 3 slides served as controls. Cells were

fixed in 2.5% glutaraldehyde in seawater, rinsed in

deionised water and air dried. The number of cells

attached was counted using a Zeiss Kontron 3000

image capture analysis system attached to a Zeiss

epifluorescence microscope (Callow et al. 2002).

Counts were made for 30 fields of view within the

area of the slide that was exposed to the water jet.

The number of cells was compared to counts made

on the three unexposed samples. The percentage

removal was calculated from the mean of cell density

before and after jet washing.

Assessment of attachment strength using a spinning water-jet 123

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Spinjet apparatus

The apparatus was designed to evaluate the strength

of attachment of microorganisms in 24-well plates

using a perpendicular water jet that impacts the

bottom of the coated well. The Spinjet represents

further development and miniaturisation of pre-

viously described water jets designed for use with

microscope slides (Finlay et al. 2002) and raft panels

(Swain & Schultz, 1996). The jet of water was

produced by a straight nozzle placed eccentrically on

a rotating shaft, thus the nozzle rotation describes a

circle of 7 mm in diameter inside of a 15 mm

diameter well (Figure 1). Well plates were loaded

manually into the indexing plate of the Spinjet and

clamped in place. The gas supply pressure was then

adjusted to the desired jetting pressure with the

precision pressure regulator. An integral precision

test gauge allowed the pressure to be set to within

+3.5 kPa out of a total range of 0 – 1034 kPa. The

jet duration was then entered in seconds into the

digital timing relay to an accuracy of 100 ms. Each

well jetting was then triggered with a pneumatic foot

switch tethered to the timing relay. The relay also

controlled the starting and stopping of the nozzle

rotation (Figure 2).

Calibration of the Spinjet nozzle was performed

against an analytical balance to generate the relation-

ship between the set dispense pressure and flow rate

over time. Single volumes of jetted water were

collected at specific pressures and jet durations and

weighed with the analytical balance. The flow rates

calculated from these measured volumes were then

divided by the flow area of the nozzle to calculate the

average velocity of the water jet exiting the nozzle.

The average water jet velocities were then used to

calculate the impact pressures impinging on the well

bottoms (Finlay et al. 2002) where r is the fluid

density.

Impact pressure ¼ 1=2 r ðAverage jet velocityÞ2

For the given geometries, a supply pressure of

100 kPa produced an impact pressure of 67 kPa

(Figure 3).

The plates were placed into the water jet holder

and treated at a range of impact pressures with

artificial seawater (Instant Ocean) for 10 s per well.

Time course experiments had established that

increasing the duration of jetting beyond 10 s did

not influence the percentage removal, maximum cell

removal being obtained after 10 s at any single

pressure. For each treatment or experimental coat-

ing, 12 replicates were used; one line of six replicate

wells was sprayed per impact pressure and the

adjacent six served as unsprayed controls.

Figure 1. Spinjet description. (a) Spinjet overview; (b) uncoated 24-well plate loaded into the Spinjet with the water jet on; (c) representation

of the Spinjet off-set nozzle and well geometries. The plates are inverted on the platform and the Spinjet sprays the water into the well from

below via an offset nozzle.

124 F. Casse et al.

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Results

Distribution of settled Ulva spores

The mean spore count taken from transects across

the diameter of wells is presented in Figure 4. The

plot shows that spores were evenly distributed across

the well and had not settled preferentially at the sides

of the well.

Effect of background colour on growth and attachment

strength of Ulva sporelings

Ulva sporelings were cultured on Silastic1 T2 and

polyurethane applied to glass coverslips which were

secured to the bottom of wells by either white or

black epoxy. Biomass data after 5 days of growth on

white vs. black backgrounds is shown in Table I.

Although the total amount of biomass was less on the

Silastic1 T-2 than the polyurethane, there was no

significant difference between the amount of biomass

developed in relation to background colour.

The percentage removal of biomass increased with

increasing impact pressure on both Silastic1 T2 and

polyurethane (Figure 5). Biomass was removed from

the fouling release coating (Silastic1T-2) at a lower

impact pressure than from the polyurethane coating

(Figure 6). At the highest impact pressure tested

(152 kPa) only 40% of the biomass was removed

from the polyurethane compared to over 80% from

Silastic1 T-2. The strength of attachment of the

biomass was not significantly different on black vs.

white surfaces (Table I).

Figure 2. Spinjet process and instrument diagram. Dispensing pressure is supplied from a compressed gas connection to the precision

pressure regulator. Water jetting pressure is then manually set with the precision pressure regulator. Jet duration and rotation are controlled

by the digital timing relay, triggered by a foot switch, while the nozzle rotates at 120 rpm.

Figure 3. Typical calibration data for the Spinjet. Resultant impact

pressures generated from various supply pressures (dispense tank

pressure). Test repeated 3 times on same nozzle with a maximum

SD of 1.02.

Figure 4. Mean number of attached spores per mm2 after 2 h

settlement in the dark. Counts were taken at 1 mm intervals from

the midpoint of the well (zero). Each point is the mean count from

6 replicate wells. Bars¼+95% confidence limits.

Assessment of attachment strength using a spinning water-jet 125

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Attachment strength of Ulva sporelings to coatings

on 24-well plates and slides

The percentage removal of Ulva sporelings growing

on glass and two silicone elastomers, Silastic1 T2

and Intersleek1 deposited in 24-well plates and on

coated microscope slides is shown in Figure 7. The

impact pressure used for the 24-well plates generated

by the spinjet was 89 kPa and slides were subjected

to 73 kPa impact pressure from a water jet. These

data are representative of those obtained in three

separate experiments. Figure 7 shows that the

coatings rank in the same order in terms of biomass

removal and a similar percentage removal of biomass

was obtained by both methods of coating application.

However, since the impact pressures used are slightly

different for the spinjet and the water jet, it is not

appropriate to directly compare the two data sets.

Adhesion strength of Navicula and distribution of cells

after spinjetting

Adhesion strength, expressed as percentage removal,

is presented in Figure 8. These data are representa-

tive of those obtained in three separate experiments.

Approximately 80% of the cells were removed from

polyurethane by an impact pressure of 18 kPa com-

pared to 30% from Silastic1 T-2.

Cell counts across the diameter of uncoated poly-

styrene wells sprayed at three pressures are presented

in Figure 9. The density of cells remaining is fairly

uniform; at the two lowest pressures, namely, 18

and 43 kPa, 27% and 16%, respectively, of the

original biomass remained. At the highest pressure

(152 kPa), the cell density was lower around

Table I. Biomass of Ulva after 5 d growth (RFU) and percentage

removal at 75 kPa of impact pressure on white vs black back-

grounds. RFU means are from 6 replicate wells+95% confidence

limits. Percentage removal data are based on 6 unjetted replicates

and 6 spinjetted replicates. Paired t-tests show no significant

difference between growth or percentage removal on black vs.

white surfaces for either polyurethane or Silastic1 T-2.

Polyurethane Silastic1 T2

Biomass White+ 95%

conf. limits

34805+ 1117 28193+2978

Biomass Black+95%

conf. limits

35345+ 885 29192+2287

% removal White+ 95%

conf. limits

21.7+ 4.2 65.7+4.1

% removal Black+95%

conf. limits

19.7+ 5.0 64.1+5.8

Figure 5. Percentage removal of Ulva sporeling after 5 d growth on

coatings deposited in 24-well plates and hosed at 18, 43, 75, 111

and 152 kPa impact pressure with the Spinjet. Each point is the

mean of 6 replicate wells. Bars¼+95% confidence limits derived

from arcsine transformed data.

Figure 6. Photograph of a 24-well plate after 5 d growth of Ulva

sporeling (row 1 and 3) and after jetting at 42 kPa impact pressure

with the Spinjet (row 2 and 4). Wells in the top two rows contained

glass coverslips coated with polyurethane and the bottom two rows

contained coverslips coated with Silastic1 T2. The glass coverslips

were secured in the wells with clear epoxy glue; 20% and 60% of

the biomass were removed by jetting from the polyurethane and

Silastic-T2, respectively.

Figure 7. Percentage removal of Ulva sporeling after 5 d growth on

coatings deposited in 24-well plates or on glass microscope slides.

The two experimental coatings were deposited directly onto

aluminium discs that were secured in the 24-well plates with

epoxy glue. The wells were subjected to an impact pressure of

89 kPa with the Spinjet and the slides to 73 kPa with the water jet.

Each point is the mean of 6 replicates. Bars¼+95% confidence

limits derived from arcsine transformed data.

126 F. Casse et al.

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the zone of impact of the water jet; 94% of the cells

were removed at this impact pressure.

Attachment strength of Navicula to coatings

on 24-well plates and slides

The percentage removal of Navicula from glass and

two silicone elastomers, Silastic1 T-2 and Inter-

sleek1 deposited in 24-well plates and applied to

microscope slides is shown in Figure 10. The impact

pressure used for the 24-well plates generated by the

spinjet was 31 kPa and slides were subjected to

34 kPa impact pressure from a water jet. The rank

order in terms of percentage removal from the

coatings was the same for both methods of coating

application although the percentage of biomass

removed was slightly higher for the two silicone

elastomers in 24-well plates compared to the slides.

There was a significant difference between glass,

Silastic1 T-2 and Intersleek1 for both the 24-well

plates (F (2, 15)¼ 160, p5 0.05) and slides (F (2,

267)¼ 641, p5 0.05).

Discussion

Algal biofilms develop on all artificial surfaces

immersed in the sea provided there is some

illumination (Callow, 2000). Adhesion of diatoms

is moderated by the production of extracellular

polymeric substances (EPS) comprising various

polysaccharide and glycoprotein components

(Chiovitti et al. 2006). Macroalgal fouling commu-

nities are frequently dominated by Ulva, which has a

different type of adhesion biology to that of diatoms

(see Callow & Callow, 2006). The difference in the

adhesion biology of these two types of fouling algae

necessitates the development of bioassays suitable for

both organisms, since Ulva sporelings adhere only

weakly to silicone elastomers (Chaudhury et al.

2005) while diatoms adhere relatively strongly to

these coatings (Holland et al. 2004). The develop-

ment of coatings to which the adhesion of both

macro- and microfouling organisms is weak is a

target for coatings development (see Krishnan et al.

2006a; 2006b).

A fouling release assay employing Ulva sporelings

and diatoms in 24-well plates provided a convenient

and reproducible semi-high throughput method for

screening coatings in terms of their strength of

attachment. The 24-well plate format allowed good

replication and the data have been shown to be

reproducible. Moreover the large number of indivi-

dual test samples that can be assayed simultaneously,

under the same conditions, increases the usefulness

of the method.

Figure 8. Percentage removal of Navicula after 2 h settlement on

coatings deposited in 24-well plates and jetted at 18, 43, 67, 89

and 111 kPa impact pressure with the Spinjet. Each point is the

mean of 6 replicate wells. Bars¼+95% confidence limits derived

from arcsine transformed data.

Figure 9. Navicula cell density obtained from cell counts across the

middle of polystyrene wells subjected to different impact

pressures; 18 kPa, 43 kPa and 152 kPa impact pressures show

respectively 73%, 84% and 94% removal of cell biomass based on

chlorophyll extraction. Counts were taken at 1 mm intervals. Each

point is the mean of 6 replicate wells. Bars¼+95% confidence

limits.

Figure 10. Percentage removal of Navicula after 2 h settlement on

coatings deposited in 24-well plates or on glass microscope slides.

Aluminium discs were secured in the 24-well plates with epoxy

glue. The wells were subjected to an impact pressure of 31 kPa

with the Spinjet and the slides to 34 kPa with the water jet. Each

point is the mean of 6 replicate wells for the plates and 90 counts

on 3 replicates for the slides. Bars¼+95% confidence limits

derived from arcsine transformed data.

Assessment of attachment strength using a spinning water-jet 127

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used reliably, venting and leaching of the 24-well

plates must be considered carefully. One week of

venting in an air flow cabinet followed by leaching for

4 weeks with daily water exchange appeared to be

adequate for most of the surfaces, including silicone

elastomers cured with dibutyltin compounds.

Ulva spores settled evenly on the surface of the

wells provided a low spore inoculum was employed.

Spores are known to respond to topographic features

(Hoipenkeimer-Wilson et al. 2004; Carman et al.

2006) and at high concentrations (416106 ml71),

they settle preferentially around the edges of the

wells. Germination and growth were not significantly

affected by background colour in the 24-well plates,

in contrast to slides, where a significantly slower rate

of spore germination and growth on black surfaces

compared to white surfaces are seen (unpublished

data). Since the strength of adhesion of Ulva

sporelings to fouling release silicone elastomers is

also related to the stage of growth (Schultz et al.

2003), the 24-well plate format would be preferred to

a slide format if dark-coloured coatings were being

investigated. The different performance of the two

methods in relation to background colour may be

due to the different reflective properties of the multi-

well plates compared with glass slides, the former

having many reflective surfaces that may serve to

minimise differences in light fields experienced by

the organisms. The influence of background colour

on the development of algal fouling on panels

immersed in the ocean has recently been shown by

Swain et al. (2006).

Adhesion strength, measured as percentage re-

moval of biomass, was positively correlated with

impact pressure provided by the spinjet for both

algae. Moreover, the percentage removal data for the

24-well plates strongly correlated with those obtained

for the same coatings applied to glass microscope

slides, which were hosed using a standard water jet

(Finlay et al. 2002). Ulva sporelings were weakly

attached to the silicone elastomers (Silastic1 T-2

and Intersleek) and strongly attached to glass, which

concurs with previous observations (Chaudhury

et al. 2005; Krishnan et al. 2006a; 2006b). Con-

versely, diatoms adhered relatively strongly to the

silicone elastomers compared to glass, in agreement

with published data (Holland et al. 2004). All of the

adhesion strength data from 24-well plates concurs

with those obtained previously for Ulva (Chaudhury

et al. 2005; Krishnan et al. 2006a) and diatoms

(Holland et al. 2004).

Cell counts across the bottom of wells containing

adhered diatoms following exposure to different

spinjet pressures showed that cell removal was

broadly even across the diameter of the well. The

differential removal seen at the highest impact

pressure is not considered to be important since

down-selection of formulations would never be

based on percentage removal values 490% in view

of the asymptotic nature of the removal curves.

Visual observation of Ulva biofilms also indicated an

even removal of biofilm from the well. Shear forces

produced by the impinging jet on the biofilm samples

are concentrated in a circular region of high shear

that reaches a maximum value at:

tmax ¼ 0:32ðimpact pressure of jetÞ=ðH=dÞ2

where H¼distance from nozzle to surface, d¼nozzle diameter and impact pressure is 1/2 rV2

(Beltaos & Rajaratnam, 1974). Although this equa-

tion is for a static non-rotating jet, it can be applied

to the Spinjet due to the relatively slow rotational

speed of the nozzle. For example, the rotational

speed of the impact region of the water jet is 4.4 cm

s71 as it traces a 7 mm diameter circle in the well

bottom and the velocity of the water jet is almost

three orders of magnitude higher than the rotational

velocity at 1000 – 3000 cm s71 over the pressure

range used for testing. Therefore, it is assumed that

the rotation speed of the jet has a negligible addition

to the speed of the impacting water jet at the leading

edge of rotation and the radial shear region is

approximately symmetrical. With the given nozzle

geometries an impact pressure of 50 kPa will

produce a tmax of *15 Pa. The circular high shear

region is *3 – 4 mm in diameter and sweeps out an

area equal to *55% of the total well bottom area.

The remainder of the well bottom is cleaned less

vigorously by shear forces that are less than tmax. The

nozzle rotates within the well twice a second or 20

times during a typical 10 sec jetting to ensure that

the wells are exposed to a repeatable shearing action

regardless of nozzle starting and end position. The

complete mechanism of biofilm removal from an

impinging jet involves the shear force as well as the

normal force of the jet as it impinges on the coating

sample. The resultant removal force on the biofilm is

quite complex and not fully understood. Modelling

of the water jet shear forces and how they correlate to

those experienced on the side of a ship was outside

the scope of this work. However, the goal of the

Spinjet design was to deliver a consistent water jet

shearing action (sum of all forces) at a specified

supply pressure so that coatings could be ranked by

their relative cleanability. This was accomplished

through the close control of water jet pressure, water

jet duration, and water jet impingement region

reproducibility.

In summary, the data have shown that the spinjet

delivers a suitable range of impact pressures that

facilitate measurement of the adhesion strength of

algae to non-biocidal coatings deposited in 24-well

128 F. Casse et al.

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07 plates. The plate format is also suitable to monitor

the efficacy of fouling release coatings that incorpo-

rate a tethered biocide (Thomas et al. 2004). The

combination of 24-well plate assays employing

bacteria (Stafslien et al. 2007a; 2007b) and algae

will contribute data on which the down-selection of

coatings for further development can be based.

Acknowledgements

This study was carried out with support from the US

Office of Naval Research in the form of grants

N00014-03-1-0509 to JAC & MEC and N00014-02-

1-0794 and N00014-03-1-0702 to NDSU.

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