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Biofouling: The Journal of Bioadhesion and Biofilm

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Effects of commercial enzymes on the adhesion of a

marine biofilm-forming bacterium
a b a b c d
C. Leroy , C. Delbarre , F. Ghillebaert , C. Compere & D. Combes
a
Laboratoire de Biotechnologie et Molcules Marines IFREMER Nantes, France
b
Mexel S.A. Verberie, France
c
Service Interfaces et Capteurs IFREMER Brest, France
d
Laboratoire Biotechnologie-Bioprocds, UMR CNRS 5504, UMR INRA 792, INSA Toulouse,
France
Published online: 04 Dec 2007.

To cite this article: C. Leroy , C. Delbarre , F. Ghillebaert , C. Compere & D. Combes (2008): Effects of commercial enzymes
on the adhesion of a marine biofilm-forming bacterium, Biofouling: The Journal of Bioadhesion and Biofilm Research, 24:1,
11-22

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 Biofouling
Vol. 24, No. 1, January 2008, 1122

Eects of commercial enzymes on the adhesion of a marine biolm-forming bacterium

C. Leroya,b**, C. Delbarrea, F. Ghillebaertb, C. Comperec* and D. Combesd
a
Laboratoire de Biotechnologie et Molecules Marines IFREMER Nantes, France; bMexel S.A. Verberie, France;
c
Service Interfaces et Capteurs IFREMER Brest, France; dLaboratoire Biotechnologie-Bioprocedes, UMR CNRS 5504,
UMR INRA 792, INSA Toulouse, France
(Received 25 May 2007; nal version received 31 October 2007)

The antifouling potential of commercial hydrolases, four proteases, seven glycosidases and one lipase was evaluated
on the adhesion of marine Pseudoalteromonas sp. D41. The experimental method, adapted to screen antifouling
agents, was based on bacterial adhesion in natural sterile sea water in a microtiter plate and on total biomass
quantication by the uorescent dye DAPI (4[prime]6-diamidino-2-phenylindole). Savinase (subtilisin) was the most
Downloaded by [Universita degli Studi di Torino] at 12:24 02 June 2013

eective hydrolase in both the prevention of bacterial adhesion and the removal of adhered bacteria. However, some
enzymatic preparations tested such as Amano protease were not only ineective but also increased the number of
adhered bacterial cells. Enumeration using epiuorescence microscopy of CTC (5-cyano-2,3-ditolyl tetrazolium
chloride) and DAPI stained adhered D41 cells conrmed these observations. Overall, these results demonstrated that
hydrolases could either prevent adhesion and remove adhered bacterial cells eectively, or conversely increase
bacterial adhesion, depending on enzymatic concentrations and the type of enzymes tested.
Keywords: adhesion; enzymes; Pseudoalteromonas; antifouling; marine biolm

are toxic to marine ora and fauna (Alzieu 1996, 1998).

Introduction
Biolm is a bacterial community which adheres to
biotic and abiotic surfaces and is embedded in a
polymeric matrix composed mainly of polysaccharides,
proteins, nucleic acids (Flemming and Wingender
2001; Sutherland 2001; Allison 2003; Branda et al.
2005) and inorganic compounds such as salts
(Compere et al. 2001). Biolm develops in many elds,
for example, health (nosocomial infection), industry
(food, pulp and paper, textiles, wastewater treatment),
equipment in contact with natural waters (vessels,
pipelines, harbour facilities, aquaculture equipment,
marine sensors, cooling water system). In this publica-
tion, the focus is on preventing bacterial adhesion on
solid/liquid interfaces in the marine environment.
Marine biolms have an inuence on the adhesion of
marine macroorganisms leading to fouling (Holmstrom
et al. 1992; Wieczorek and Todd 1998; Qian et al. 2003).
To combat fouling, one strategy is to control biolm
development during the rst step of fouling adhesion by
physical or chemical technology. Chemical techniques
use biocide products such as metal compounds (tin,
copper), oxidant compounds (chloride, bromide, ozone)
or synthetic non-oxidant compounds (algicide, bacter-
icide, fungicide usually used in agriculture). It has been
shown that antifouling paints with organostannic agents
Legislation on the use of biocides used is becoming more
restrictive due to European regulations (Directive 1998/
8/EC) which control their use, especially by banishing
organostannic compounds in antifouling paints. There-
fore, it is essential to look for environmentally friendly
antibiolm agents.
The biolm matrix is mainly composed of water
(97%) and extracellular polymeric substances (EPS)
made up of polysaccharides, proteins, nucleic acids,
lipids, mineral ions and various cellular debris
(Sutherland 2001). It fullls dierent functions such as
providing an adhesive foundation, structural integrity,
bacterial protection and intercellular communication
(Allison 2003; Branda et al. 2005). When focusing on
hydrolysing organic compounds involved in marine
biolms, hydrolases in commercial enzymatic prepara-
tions are good antibiolm agent candidates due to
(i) their availability (some are already produced at an
industrial scale); (ii) their biodegradability (unlike to the
long persistence of organometallic compounds) and
(iii) their weak toxicity (unlike oxidase enzymes).
Hydrolases may inhibit adhesion by degrading biolm
matrix molecules rather than by killing marine micro-
and macroorganisms.
In this study, the adhesion of a natural pioneer
marine biolm-forming bacterium was tested in the

*Corresponding author. Email: chantal.compere@ifremer.fr

**Present address: Groupe dEtude des Interactions Hote-Parasite, UPRES-A 3142, Laboratoire de Parasitologie-Mycologie, 4,
rue Larrey, 49933 Angers Cedex 9, France

ISSN 0892-7014 print/ISSN 1029-2454 online

2008 Taylor & Francis
DOI: 10.1080/08927010701784912
http://www.informaworld.com
 12 C. Leroy et al.
presence of hydrolases such as proteases, glycosidases
and one lipase in natural sterile sea water. Inhibition
activity on microbial adhesion was measured in terms
of the prevention of adhesion and the detachment of
adhered bacteria using a new test in polystyrene
microplates adapted to screen antifouling agents in
marine conditions (Leroy et al. 2007). While some
enzymes partly inhibited adhesion of Pseudoaltero-
monas sp. D41, others increased it.
Materials and methods
Except for commercial enzymes, all the chemicals and
reagents were of analytical grade from Sigma-Aldrich.
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Enzymes and enzymatic activities
Commercial enzymatic preparations manufactured for a
range of industrial applications were supplied by dier-
ent providers. Four proteases, seven glycosidases and
one lipase were tested. Table 1 summarizes the charac-
teristics of the enzymes selected to test against bacterial
adhesion. Heat denatured controls were tested on
bacterial adhesion only for papain, a-amylase, Lipolase
and Glucanex. They were obtained by heating enzymatic
preparations at 1008C for 20 min and recovering the
supernatants by centrifugation at 10,000g for 10 min
before using to test bacterial adhesion. In order to
compare the enzymatic eects between each other and in
marine conditions, each enzymatic preparation was
quantied using the appropriate assay and substrate in
sterile natural sea water at 258C and at pH 8.15.
Protease activity was measured with azocasein
(Tomarelli et al. 1949). Savinase (16 L type EX,
Novozymes) was incubated at concentrations ranging
from 0.5 to 10 g l71 in natural seawater (previously
ltered through a 0.22 mm membrane), at 258C with
2% (w/v) azocasein in phosphate buer of pH 8.15 at
dierent incubation times from 3 to 12 min. Enzymatic
activity was stopped by adding 10% trichloroacetic
acid. The absorbance of enzymatically hydrolyzed
casein was determined at 450 nm. One unit of protease
activity (UP) was dened as the amount of enzyme
required to produce an absorbance unit change per
minute at 258C at pH 8.15.
Glycosidase activities were determined by measuring
the release of reducing sugars according to the Somogyi
Nelson method using a glucose standard (Somogyi 1960;
Spiro 1966). Glycosidases were incubated at dierent
concentrations, from 0.005 to 10 g l71 depending on the
enzymatic preparation, in natural seawater (previously
ltered through a 0.22 mm membrane), at 258C with
12.5 g l71 of the appropriate substrate at dierent
incubation times from 3 to 12 min. Enzymatic activity
was stopped by heating samples for 5 min at 1008C
before measuring the amount of released reducing
sugars. The enzymatic activity of glucanase, xylanase,
pectinase, amylase and cellulase was determined using b-
glucan, xylan, pectin, soluble starch and carboxymethyl
cellulose as substrates, with middle viscosity, respec-
tively. One unit of glycosidase activity (UG, UX, UPe,
UA and UC) was dened as the amount of enzyme
capable of forming one reducing sugar glucose equiva-
lent per minute at 258C at pH 8.15.
Lipase activity was determined by measuring the
increase in absorbance at 400 nm produced by the
release of p-nitrophenol during the hydrolysis of p-
nitrophenyl palmitate (C16:0) (Kilcawley et al. 2002).
Absorbance was measured every minute for 10 min at
258C. To initialize the reaction, lipase solution in
natural sea water (previously ltered through a
0.22 mm membrane) was added to 50 mM p-nitrophe-
nyl palmitate solution in acetonitrile. One unit of lipase
activity (UL) was dened as the amount of lipase
required to produce an absorbance unit change per
minute at 258C at pH 8.15.
The biolm forming bacterium
The D41 strain was isolated from a natural marine
biolm on Teon coupons immersed for 24 h in the
bay of Brest (France) (Rubio 2002). For long-term
storage, pure culture was maintained on Difco Marine
Broth (MB) 2216E (Fischer Scientic Labosi, Elan-
court, France) with 15% (v/v) glycerol at 7808C. A
comparative 16S rRNA gene sequences analysis
indicated that the D41 isolate belonged to the genus
Pseudoalteromonas sp. (Rubio 2002).
The bacterial suspension was prepared from Pseu-
doalteromonas sp. D41 grown overnight on Difco
Marine Agar (MA) 2216E (Fischer Scientic Labosi,
Elancourt, France) at 258C, scraped and suspended in
10 ml of natural sea water sterilized by ltration
through a 0.22 mm, to reach an OD600 of 2
(2 6 109 cfu ml71).
Bacterial adhesion tests and enzymatic screening
Adhesion of Pseudoalteromonas sp. D41 adhesion in
black polystyrene microtiter plates (Microwell F96
FluoroNunc, Bioblock, Illkirch, France), in sterile
seawater, at 208C was performed as described by Leroy
et al. (2007).
The enzymes were tested in two ways. For the
inhibition of bacterial adhesion (the prevention test),
50 ml of the enzymatic preparation at the desired
concentration was placed in the wells 1 h before the
bacterial suspension (200 ml per well) and incubated
for 3 and 24 h at 208C. For the detachment of adhered
bacteria (the detachment test), the enzymes were added
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Table 1. Characteristics of commercial enzymes tested.

Optima
Commercial Microorganism
name Manufacturer source EC number Name (Class) Form pH Tp (8C) Application

Amano Protease A Unipex Aspergillus oryzae EC3.4.24.39 Deuterolysine, (metalloendopeptidase) Powder 7 50 Protein process
Papain Sigma Aldrich Papaya latex EC3.4.22.2 Papan (cysteine endopeptidase) Powder 67 25 Varied (foods,
pharmaceutical,
textile)
Umamizyme Unipex Aspergillus oryzae EC3.4.11.1 Leucyl aminopeptidase Powder 8 45 Brewing
(aminopeptidase)
Savinase 16 L Novozymes Bacillus sp. EC3.4.21.62 Subtilisin (serine endopeptidase) Liquid 48.5 50 Textile
type EX
Celluclast 1.5 L Novozymes Trichoderma reesei EC3.2.1.4 Cellulase Liquid 4.56 5065 Varied cellulose
process
Pulpzyme HC Novozymes Bacillus sp. EC3.2.1.8 Endo-1,4-b-xylanase Liquid 7 50 Pulp and paper
Glucanex 200G Novozymes EC3.2.1.58 Glucan 1,3-b-glucosidase, b-glucanase Powder Oenology
b-1,6-glucanase, protease, chitinase,
cellulase
Finizym 200 L Novozymes Aspergillus niger EC3.2.1.6 Endo-1,3(4)-b-glucanase cellulase, Liquid 5 60 Brewing
xylanase, pentosanase and arabanase
Shearzyme 500 L Novozymes Aspergillus oryzae EC3.2.1.8 Endo-1,4-b-xylanase, endo-1,3(4)- Liquid 5 70 Wheat process
EC3.2.1.6 b-glucanase, cellulase
EC3.2.1.4
a-Amylase Novozymes Bacillus sp. EC3.2.1.1 a-amylase Liquid
Pectinex Ultra SPL Novozymes Aspergillus aculeatus EC3.2.1.15 Polygalacturonase hemicellulase, Liquid 3.5 35 Fruit juice
arabanase, b-glucanase et xylanase process
Lipolase 100 L Novozymes Aspergillus oryzae EC3.1.1.3 Lipase (phosphoric monoester hydrolases) Liquid Detergent
composition
Biofouling
13
 14 C. Leroy et al.
3 h after the D41 bacterial suspension and incubated
for 1 h at 208C. In both tests, three washings in
36 g l71 NaCl were performed before xation for 1.5 h
at 48C with 200 ml of 36 g l71 sterile NaCl containing
2.5% formaldehyde and DAPI staining (4 mg ml71)
for 20 min. Another three washings were performed to
remove excess DAPI and then, the remaining bound
DAPI was solubilised into 95% ethanol for 15 min
(200 ml). Fluorescence was measured at 350 nm excita-
tion and 510 nm emission wavelengths using a Genios
Plus microplate uorescence reader (TECAN, Lyon,
France). For each condition, the enzymatic prepara-
tions were tested four times and heat denatured
controls twice. They were all tested at dierent
concentrations using serial twofold dilutions in micro-
plate wells.
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A blank with sterile seawater as well as a control
with bacterial suspension without enzymes was in-
cluded in each column of the experimental microplate.
The change in bacterial adhesion was calculated as
the percentage reduction (PR) from the uorescence of
the blank (FB; without bacteria), the uorescence of the
control (FC; bacteria without enzyme) and the uor-
escence of the sample (FS; bacteria with enzyme)
according to:
PR fFC  FB FS  FB =FC  FB g  100 dierent elds. This experiment was carried out in four
The concentrations necessary to reach a reduction
percentage of 50% adhesion (C50) were calculated
from a loglog plot of PR against enzymatic concen-
trations. Standard deviation was calculated from the
four C50s calculated for each experiment and p values
were calculated with the t-test (Excel software).
For CTC staining with Amano protease, the
experiment was undertaken according to the preven-
tion test described above. Amano protease was added
1 h before the D41 suspension, incubated for 3 and
24 h at 208C. The adhered bacteria were then washed
three times with 36 g l71 of sterile NaCl solution,
stained with CTC (5 mM) for 2 h, washed three times
with 36 g l71 sterile NaCl solution, xed for 1.5 h at
48C with 200 ml of 36 g l71 sterile NaCl containing
2.5% formaldehyde and nally, the CTC reduced to
formazan (CTF) via the bacterial respiratory electron
transport chain. The CTF was then solubilised into
95% ethanol for 15 min. Absorbance was measured at
450 nm using the Genios Plus microplate uorescence
reader (TECAN, Lyon, France).
Regarding the enumeration of total and viable
cultivable adhered bacteria in the polystyrene micro-
plates, the 24 h adhesion prevention test with Amano
protease was used as described above: Fifty microlitres
of Amano protease or sterile natural seawater were
added 1 h before adding the bacterial suspension and
incubating for 24 h at 208C. The wells were then
washed three times with 36 g l71 sterile NaCl solution
and the adhered bacteria were scraped into 250 ml with
36 g l71 sterile NaCl solution four times. One hundred
microlitres of the scraped adhered bacteria were
diluted with appropriate serial tenfold dilutions and
100 ml of the last three dilutions were spread out on
marine agar, incubated from 24 to 48 h at 258C and
the colony forming units were then enumerated. Nine
hundred microlitres of the scraped adhered bacteria
were incubated with formaldehyde solution (2.5% nal
concentration) for 1.5 h. Five microlitres of this
solution were then added to 2 ml 36 g l71 sterile
NaCl and incubated with triton X-100 solution (0.05%
nal concentration) for 10 min before sonicating the
solution for 10 min at 47 kHz. DAPI was then added
to a nal concentration of 4 mg ml71 for 20 min. The
DAPI stained bacterial solution was then ltered onto
a 0.22 mm sterile black polycarbonate lter (45 mm
diameter, Isopore GTBP Millipore, Fischer Scientic
Labosi, Elancourt, France). The lters were rinsed
with sterile water and mounted on glass slides. The
slides were observed under 6100 magnication by
epiuorescence microscopy using an Olympus BH201
under WU lter (Olympus, Rungis, France). Approxi-
mately 1250 cells were counted per lter from the 25
wells of one microplate.
Epiuorescence microscopy counts of total
adhered bacteria
Lab-TekTM wells with a glass surface (Lab-TekTM
NUNC, Fischer Scientic Labosi, Elancourt, France)
were used and 360 ml of sterile natural sea water or
360 ml of Amano protease were added per well and
incubated for 1 h at 208C without shaking. Five
hundred and forty microlitres of bacterial suspension
were then inoculated. The covered Lab-TekTM glass
slides were incubated for 24 h at 208C with orbital
shaking (300 rpm). The non adhered bacteria were
removed by three successive hand washings. The Lab-
TekTM wells were rinsed and shaken with 900 ml of
36 g l71 sterile NaCl solution. The adhered bacteria
were xed for 1.5 h at 48C with 900 ml of 36 g l71
sterile NaCl solution with 2.5% formaldehyde and
then desalted by successive baths in solutions of
decreasing salinity. The adhered bacteria were incu-
bated with 900 ml of the DAPI solution (4 mg ml71) for
20 min in the dark. The excess stain was removed by
three hand washings, then the Lab-TekTM wells were
removed and the glass surface was dried out. The slides
were observed under 6100 magnication by epiuor-
escence microscopy using an Olympus BH201 under
UV lter (Olympus, Rungis, France).

Results
Eects of commercial enzymes on Pseudoalteromonas
sp. D41 adhesion in seawater microplate tests
The potential capability of several enzymes to inhibit
the development of adhesion by Pseudoalteromonas sp.
D41 on the polystyrene microplate surface was tested.
As stated previously the enzymes were tested for the
prevention of bacterial adhesion and for the detach-
ment of adhered bacteria.
The percentage change in bacterial adhesion in
relation to enzymatic activities is shown in Figures 1
to 3 depending on each enzymatic activity. The inu-
ence of enzymatic activities depended on the type of
protocol tested. Glucanases and proteases were more
eective than the xylanases, amylase, cellulase and
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lipase. Savinase was the most eective protease,
rapidly reaching the highest reduction percentage at
the lowest proteolytic activity used in the well,
whatever the preventive or curative protocol used
(see Figure 1). The C50 values are given in Table 2. To
compare each C50 between all enzymatic preparations,
the C50 were calculated in mg ml71. The results
conrmed that Savinase was the most eective
enzyme since it needed the lowest concentration to
reach 50% inhibition. Savinase was more eective at
3 h prevention compared with the detachment of
adhered bacteria (result statistically signicant at
98%, p 0.02). Moreover, less Savinase was needed
to prevent bacterial adhesion in the 24 h test compared
with 3 h, as indicated by the C50. This result was only
observed for Savinase. This showed that the eective-
ness of Savinase in the prevention of bacterial adhesion
increased with the incubation time.
Only Glucanex, Lipolase, a-amylase and papain
could be tested after heating since not all the enzymatic
preparations in Table 1 could be heat denatured, some
(generally the liquid ones) became solid, thus testing
was impossible. For Glucanex, a-amylase and Lipo-
lase, heat denatured enzymes presented a partial
inhibition of bacterial adhesion whatever the protocol
tested and approximately the same pattern was
observed for the non denatured enzymes. Nevertheless,
lower bacterial inhibition was observed for heat
denatured Glucanex and Lipolase in the 3 h prevention
test compared with non-heat denatured Glucanex and
Lipolase when the highest concentrations were used.
Only heat denatured papain showed less or no
bacterial inhibition. No heat-denatured enzyme pre-
parations showed any residual enzymatic activity
(Leroy 2006).
Amano protease was partially able to prevent D41
adhesion for 3 h but it increased the nal quantity of
D41 bacterial cells if they were allowed to settle for
24 h (Figure 1). The reduction percentages after D41
Biofouling 15
adhesion for 24 h were negative showing that the total
quantity of adhered bacteria was greater with Amano
protease than without. This was also the case for
Shearzyme (Figure 2), Celluclast, a-amylase and
Pectinex (Figure 3). Umamizyme, papain and Amano
protease (Figure 1), Finizym and Glucanex (Figure 2)
also promoted bacterial adhesion in the 24 h preven-
tion test at low concentrations and inhibited it at
higher concentrations. This eect was also observed in
the detachment test with Glucanex and Shearzyme
(Figure 2), Celluclast, a-amylase and Pectinex
(Figure 3). Finizym inhibited bacterial adhesion in
the prevention test and increased it in the detachment
protocol of adhered bacteria (Figure 2).Thus in some
enzyme concentrations and depending upon the pro-
tocol used (prevention or detachment of bacterial
adhesion), some enzymatic activities were capable of
increasing bacterial adhesion.
The eect of Amano protease on adhesion of
Pseudoalteromonas sp. D41
The eectiveness of Amano protease in the prevention
of adhesion of D41 was studied for periods of 3 and
24 h (Figure 4). In the case of a 3 h adhesion, Amano
protease inhibited an increasing number of adhered
bacteria for both total and respiring cells with
increasing concentration (Figure 4). However, for the
24 h adhesion test, there was an opposite eect, ie the
more concentrated the Amano protease caused an
increase in the total number of adhered bacteria.
Adhesion of respiring bacteria was still inhibited at an
average rate of about 25%. The enumeration of total
and also viable and cultivable adhered bacteria in the
well after scraping conrmed this observation
(Figure 5). Enumerations of DAPI stained and
cultivable viable bacteria showed an increment of
99.6% in the total bacterial amount, and an inhibition
of 75.1% of the cultivable viable adhered bacterial
amount. Surprisingly, the total amount of cells
counted using epiuorescence microscopy was lower
than the number counted by plating in the control
treatment. DAPI stained cells were sonicated before
enumeration, suggesting that sonication could partly
lead to the lysis of cells, as previously observed
(Garabetian et al. 1999; Fykse et al. 2003). This
phenomenon did not interfere with the observations of
total DAPI cell increments and cultivable viable cell
inhibition in the presence of Amano protease since
enumeration of DAPI stained cells in the controls was
compared with that in Amano protease, and in the
same way for cultivable viable cell enumeration.
The pattern of surface coverage by adhering
Pseudoalteromonas sp. D41 in the presence of Amano
protease was studied on glass slides after DAPI
 16 C. Leroy et al.
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Figure 1. Percentage reduction of bacterial adhesion in a microtiter plate as a function of enzyme concentration in the well of
Savinase, Umamizyme, papain and Amano protease with the three protocols tested: prevention of bacterial adhesion measured
after 3 h (A), after 24 h (B) and detachment of adhered bacteria after 3 h (C). Heat denatured papain was represented as } and
the dashed line. All experimental values are shown; four experiments were carried out per enzyme concentration tested and two
for heat denatured enzyme. UP is used for the protease activity unit.

staining of total bacteria (Figure 6). After 24 h, the Discussion

coverage was relatively uniform and homogeneous Hydrolases had an eect on the adhesion of Pseudo-
without Amano protease (Figure 6A) but when alteromonas sp. D41 in polystyrene microplates when
Amano protease was added clusters of adhered cells tested at 208C in natural seawater. Some signicantly
were formed (Figure 6B). increased adhesion whereas others inhibited it.
 Biofouling 17
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Figure 2. Percentage reduction of bacterial adhesion in a microtiter plate as a function of enzyme concentration in the well of
Finizym, Glucanex, Pulpzyme and Shearzyme with the three protocols tested: prevention of bacterial adhesion measured after
3 h (A), after 24 h (B) and detachment of adhered bacteria after 3 h (C). Heat denatured Glucanex is represented as } and the
dashed line. All experimental values are shown; four experiments carried out per enzyme concentration tested and two for heat
denatured enzyme. UG and UX are used respectively for glucanase and xylanase activities units.

Proteases especially Savinase seemed to be the most either the prevention or detachment of Pseudoalter-
eective enzyme in preventing adhesion onto a omonas sp. D41 whereas b-glucanases like Finizym and
polystyrene surface in marine conditions. Amylase, Glucanex showed limited inhibition of bacterial adhe-
pectinase, cellulase and xylanase were not eective in sion, especially in the prevention test.
 18 C. Leroy et al.
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Figure 3. Percentage reduction of bacterial adhesion in a microtiter plate as a function of enzyme concentration in the well of
Celluclast, a-amylase, Pectinex and Lipolase with the three protocols tested: prevention of bacterial adhesion measured after 3 h (A),
after 24 h (B) and detachment of adhered bacteria after 3 h (C). Heat denatured a-amylase and Lipolase are represented as } and the
dashed line. All experimental values are shown; four experiments were carried out per enzyme concentration tested and two for heat
denatured enzyme. UA, UPe, UL and UC are used, respectively, for amylase, pectinase, lipase and cellulase activities units.

Some bacterial inhibition observed for a-amylase the same pattern of inhibition. This observation
or Lipolase could not be attributed to a specic suggests that additives or denatured enzymes in
enzymatic eect as heat denatured enzymes displayed enzymatic preparations could be involved in the
 Biofouling 19

bacterial inhibition eect. This phenomenon has been
observed by Petitt et al. (2004) in regards to spores of
marine macro-organisms and larvae. Additives or
denatured enzymes could be adsorbed onto the surface
modifying its chemical properties and leading to dier-
ent bacterial adhesion. Further studies with enzymes
after re-purication from commercial preparations
should be undertaken. However, the eectiveness of en-
zymes such as Glucanex and papain in the 3 h preven-
tion test seems to be due to specic enzymatic eects.
The antibiolm eectiveness of hydrolases could be
assigned to the hydrolysis of a substrate involved in
bacterial adhesion such as EPS, Quorum Sensing (QS)
Table 2. Concentration (mg ml71) to achieve a reduction of
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molecules or adhesins. Mutanase and dextranase,
eective against mutan and dextran EPS, have been
shown to inhibit the formation of dental biolms by
Streptococcus mutans and Streptococcus sobrinicus
(Wiater et al. 2004). Dispersin B catalyses the
hydrolysis of b-1,6-N-acetyl-D-glucosamine adhesin
(Ramasubbu et al. 2005) and inhibits Escherichia coli
and Staphylococcus epidermis biolms (Kaplan et al.
2004; Itoh et al. 2005). More recently, the susceptibility
of staphylococcal biolms to enzymatic treatments has
been shown to depend on their chemical composition.
Staphylococcal biolms producing poly-N-acetylglu-
cosamine were sensitive to Dispersin B while staphy-
lococcal biolms producing proteins were sensitive to
proteases (Chaignon et al. 2007). Target substrates
could also be molecules implicated in QS pathway like

adhered bacteria of 50% calculated from a loglog plot of N-acyl homoserine lactone (AHL) involved in a
RP against enzymatic concentration*. biolm composed of Gram negative bacteria. Degra-
Prevention test 3 h- dation by AHL lactonase (Wang et al. 2004) inacti-
Detachment vates QS (Roche et al. 2004) and attenuates the
3h 24 h test virulence of the pathogenic bacterium Erwinia caroto-
vora (Dong et al. 2000). In the present study, the
Savinase 4.6 + 1.4 1.7 + 0.5 6.2 + 0.9
Umamizyme 10.5 + 3.2 30.6 + 1.3 60.3 + 21.0 eectiveness of Savinase could be related to the
Papain{ 12.8 + 3.1 41.4 + 10.7 184.8 + 32.4 physicochemical feature of the external layer of
Amano 38.8 + 10.0 ND{ 79.2 + 29.2 the cells of Pseudoalteromonas sp. D41 which has
protease been shown to be rich in proteins by Fourier transform
Finizym 40.8 + 9.4 44.6 + 0.2 ND{
Glucanex{ 69.9 + 4.9 85.5 + 15.8 39.8 + 4.9 infrared spectroscopy (FTIR), X-ray photoelectron
Pulpzyme 175.1 + 46.3 241.9 + 16.6 178.0 + 29.4 spectroscopy (XPS) and time-of-ight secondary-ion
Shearzyme 183.7 + 16.5 ND{ 201.5 + 19.2 mass spectrometry (ToF-SIMS) (Pradier et al. 2005).
Celluclast 221.9 + 24.5 ND{ 195.4 + 21.3 Moreover, the EPS of Pseudoalteromonas sp. D41 after
a-amylase{ ND{ ND{ 193.8 + 9.0
Pectinex 698.8 + 10.7 926.5 + 118.3 372.0 + 16.3 fermentation and during adhesion on glass slides
Lipolase{ 81.9 + 12.8 183.9 + 42.8 ND{ showed high protein concentrations (Leroy 2006),
thus, it was hypothesised that the Savinase target
*Data are means + SD (n 4). {Enzymes that were also tested heat
denatured. {Some C50s were not determined (ND) because a 50% could be proteins involved in D41 adhesion. On the
reduction of adhesion was not reached. other hand, Finizym and Glucanex have also shown

Figure 4. Percentage reduction of total adhered bacteria (DAPI stained) () and respiring adhered bacteria (CTC stained) (~)
as a function of Amano protease concentration (UP ml71) in prevention of an adhesion after 3 h (A) and after 24 h (B). Solid
and dashed lines represent average data for DAPI and CTC stained adhered bacteria respectively. All experimental values are
shown; four experiments were carried out for DAPI staining () and two for CTC staining (~).
 20 C. Leroy et al.
eectiveness in preventing adhesion suggesting the
involvement of b-glucans in the rst adhesion step of
Pseudoalteromonas sp. D41. Epiuorescence micro-
scopy of 24 h D41 adhesion on glass slides supports
these observations, showing good uorescence using
the b-glucan stain calcouor white (Leroy 2006).
Some enzymes increased the quantity of adhered
bacteria relative to the untreated control. In the
presence of Amano protease, adhesion of Pseudoalter-
omonas sp. D41 was prevented if the cells were allowed
to adhere for 3 h, but after 24 h, adhesion was
increased. Since Amano protease is not naturally
uorescent and has no anity to DAPI (Leroy
2006), it is concluded that the observed uorescence
after DAPI staining in the presence of Amano protease
is not an artifact. Biolm accumulation has already
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Figure 5. Eect of Amano protease in prevention of D41
adhesion on a polystyrene surface in a microplate and
measured after 24 h. The total number (&) and the number
of viable cultivable () adhered bacteria per cm2 are shown.
Amano protease was pre-incubated in sea water in
microplate at 1 UP ml71 for 1 h, then Pseudoalteromonas
sp. D41 was added per well and incubated for 24 h at 208C in
sterile natural seawater. SD was calculated from four
experiments.
Figure 6. Epiuorescence microscope analysis of DAPI stained Pseudoalteromonas sp. D41 adhered to a glass slide after 24 h in
seawater at 208C (A). Amano protease was pre-incubated in seawater at 1 UP ml71 for 1 h, then Pseudoalteromonas sp. D41 was
added to each well and incubated 24 h at 208C in sterile natural seawater (B).
been observed when an insucient amount of biocide
was applied (Grant and Bott 2005). This increase in
adhered bacterial cells could be related to sedimenta-
tion, the accumulation of planktonic bacteria, or to cell
growth within the biolm whereas Amano protease in
addition to its enzymatic activity could be a substrate
supporting bacteria growth. Dead cells in clusters may
also be a nutrient source, allowing other cells to
develop. Moreover, the enumeration of viable and
cultivable bacteria and the epiuorescence microscopic
counts of DAPI stained adhered bacteria conrmed
that the number of total adhered bacteria was greater
when Amano protease was used. The number of
respiring adhered bacteria (CTC stain) and viable
cultivable adhered cells were not shown to increase in
relation to the number of total adhered cells (DAPI
stain). The cells may have entered a dormant state,
non-respiring and non-growing, known as the viable
but non culturable (VBNC) state (Oliver 2005).
The potential of enzymatic preparations against
biolm formation associated with pathogenic bacteria
has already been shown (Boyd and Chakrabarty 1994;
Johansen et al. 1997; Kaplan et al. 2004; Itoh et al.
2005), as well as against dental biolms (Hahn Berg
et al. 2001; Walker et al. 2003; Wiater et al. 2004), and
industrial biolms (Aldridge et al. 1998), but not
against marine biolms under marine conditions. In
the present study, it has been shown that commercial
enzymatic preparations can inhibit adhesion of marine
Pseudoalteromonas sp. D41 in natural sterile seawater.
Savinase was the most eective in the test conditions
used and this may be related to proteins directly
involved during adhesion (Leroy 2006) and surface-
enriched proteins of Pseudoalteromonas sp. D41
(Pradier et al. 2005). Moreover, Alcalase, an enzymatic
preparation based on subtilisin like Savinase, inhibits

the adhesion of fouling macro-organisms (Pettitt et al.
2004). However, unlike the results of Pettitt et al.
(2004) the eectiveness of Savinase seems to be stable,
even better, when bacterial adhesion in the prevention
test was increased from 3 to 24 h. This result diers
from those observed by Pettitt et al. (2004) using
Alcalase on the adhesion of cypris larvae and spores of
green algae or diatoms, suggesting that biochemical
molecules involved in bacterial adhesion for 24 h did
not change signicantly. Thus, it is evidently better to
use subtilisin preparations such as Savinase or Alcalase
during the rst stages of bacterial or macro-organism
adhesion, by conditioning the surface after immobili-
zing this enzyme directly onto the surface or by
incorporating it into an antifouling paint.
To conclude, hydrolases were shown to reduce and
Downloaded by [Universita degli Studi di Torino] at 12:24 02 June 2013
enhance bacterial adhesion, depending on the time of
exposure, and the concentration and the nature of the
enzymes. The enzyme Savinase appeared to be very
eective in the marine test conditions used. In addition
to previous antifouling studies (Pettitt et al. 2004), this
result also emphasizes the high antifouling potential of
subtilisin.
Acknowledgements
The authors acknowledge ANRT (Association Nationale de
la Recherche et Technique, France) and Mexel S.A. (France)
for nancial support for C. Leroy.
None of the authors have competing nancial interests
with any of the companies whose products were compared in
the study. These interests include full or partial project
funding, direct investments, reimbursed speaking arrange-
ments or consulting fees. No conicts of interest exist.
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