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Biofouling: The Journal of Bioadhesion and Biofilm


Research
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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
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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 Legislation on the use of biocides used is becoming more
Biolm is a bacterial community which adheres to restrictive due to European regulations (Directive 1998/
biotic and abiotic surfaces and is embedded in a 8/EC) which control their use, especially by banishing
polymeric matrix composed mainly of polysaccharides, organostannic compounds in antifouling paints. There-
proteins, nucleic acids (Flemming and Wingender fore, it is essential to look for environmentally friendly
2001; Sutherland 2001; Allison 2003; Branda et al. antibiolm agents.
2005) and inorganic compounds such as salts The biolm matrix is mainly composed of water
(Compere et al. 2001). Biolm develops in many elds, (97%) and extracellular polymeric substances (EPS)
for example, health (nosocomial infection), industry made up of polysaccharides, proteins, nucleic acids,
(food, pulp and paper, textiles, wastewater treatment), lipids, mineral ions and various cellular debris
equipment in contact with natural waters (vessels, (Sutherland 2001). It fullls dierent functions such as
pipelines, harbour facilities, aquaculture equipment, providing an adhesive foundation, structural integrity,
marine sensors, cooling water system). In this publica- bacterial protection and intercellular communication
tion, the focus is on preventing bacterial adhesion on (Allison 2003; Branda et al. 2005). When focusing on
solid/liquid interfaces in the marine environment. hydrolysing organic compounds involved in marine
Marine biolms have an inuence on the adhesion of biolms, hydrolases in commercial enzymatic prepara-
marine macroorganisms leading to fouling (Holmstrom tions are good antibiolm agent candidates due to
et al. 1992; Wieczorek and Todd 1998; Qian et al. 2003). (i) their availability (some are already produced at an
To combat fouling, one strategy is to control biolm industrial scale); (ii) their biodegradability (unlike to the
development during the rst step of fouling adhesion by long persistence of organometallic compounds) and
physical or chemical technology. Chemical techniques (iii) their weak toxicity (unlike oxidase enzymes).
use biocide products such as metal compounds (tin, Hydrolases may inhibit adhesion by degrading biolm
copper), oxidant compounds (chloride, bromide, ozone) matrix molecules rather than by killing marine micro-
or synthetic non-oxidant compounds (algicide, bacter- and macroorganisms.
icide, fungicide usually used in agriculture). It has been In this study, the adhesion of a natural pioneer
shown that antifouling paints with organostannic agents 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 before measuring the amount of released reducing
and one lipase in natural sterile sea water. Inhibition sugars. The enzymatic activity of glucanase, xylanase,
activity on microbial adhesion was measured in terms pectinase, amylase and cellulase was determined using b-
of the prevention of adhesion and the detachment of glucan, xylan, pectin, soluble starch and carboxymethyl
adhered bacteria using a new test in polystyrene cellulose as substrates, with middle viscosity, respec-
microplates adapted to screen antifouling agents in tively. One unit of glycosidase activity (UG, UX, UPe,
marine conditions (Leroy et al. 2007). While some UA and UC) was dened as the amount of enzyme
enzymes partly inhibited adhesion of Pseudoaltero- capable of forming one reducing sugar glucose equiva-
monas sp. D41, others increased it. 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
Materials and methods release of p-nitrophenol during the hydrolysis of p-
Except for commercial enzymes, all the chemicals and nitrophenyl palmitate (C16:0) (Kilcawley et al. 2002).
reagents were of analytical grade from Sigma-Aldrich. Absorbance was measured every minute for 10 min at
258C. To initialize the reaction, lipase solution in
natural sea water (previously ltered through a
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Enzymes and enzymatic activities 0.22 mm membrane) was added to 50 mM p-nitrophe-


Commercial enzymatic preparations manufactured for a nyl palmitate solution in acetonitrile. One unit of lipase
range of industrial applications were supplied by dier- activity (UL) was dened as the amount of lipase
ent providers. Four proteases, seven glycosidases and required to produce an absorbance unit change per
one lipase were tested. Table 1 summarizes the charac- minute at 258C at pH 8.15.
teristics of the enzymes selected to test against bacterial
adhesion. Heat denatured controls were tested on
bacterial adhesion only for papain, a-amylase, Lipolase The biolm forming bacterium
and Glucanex. They were obtained by heating enzymatic The D41 strain was isolated from a natural marine
preparations at 1008C for 20 min and recovering the biolm on Teon coupons immersed for 24 h in the
supernatants by centrifugation at 10,000g for 10 min bay of Brest (France) (Rubio 2002). For long-term
before using to test bacterial adhesion. In order to storage, pure culture was maintained on Difco Marine
compare the enzymatic eects between each other and in Broth (MB) 2216E (Fischer Scientic Labosi, Elan-
marine conditions, each enzymatic preparation was court, France) with 15% (v/v) glycerol at 7808C. A
quantied using the appropriate assay and substrate in comparative 16S rRNA gene sequences analysis
sterile natural sea water at 258C and at pH 8.15. indicated that the D41 isolate belonged to the genus
Protease activity was measured with azocasein Pseudoalteromonas sp. (Rubio 2002).
(Tomarelli et al. 1949). Savinase (16 L type EX, The bacterial suspension was prepared from Pseu-
Novozymes) was incubated at concentrations ranging doalteromonas sp. D41 grown overnight on Difco
from 0.5 to 10 g l71 in natural seawater (previously Marine Agar (MA) 2216E (Fischer Scientic Labosi,
ltered through a 0.22 mm membrane), at 258C with Elancourt, France) at 258C, scraped and suspended in
2% (w/v) azocasein in phosphate buer of pH 8.15 at 10 ml of natural sea water sterilized by ltration
dierent incubation times from 3 to 12 min. Enzymatic through a 0.22 mm, to reach an OD600 of 2
activity was stopped by adding 10% trichloroacetic (2 6 109 cfu ml71).
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 Bacterial adhesion tests and enzymatic screening
required to produce an absorbance unit change per Adhesion of Pseudoalteromonas sp. D41 adhesion in
minute at 258C at pH 8.15. black polystyrene microtiter plates (Microwell F96
Glycosidase activities were determined by measuring FluoroNunc, Bioblock, Illkirch, France), in sterile
the release of reducing sugars according to the Somogyi seawater, at 208C was performed as described by Leroy
Nelson method using a glucose standard (Somogyi 1960; et al. (2007).
Spiro 1966). Glycosidases were incubated at dierent The enzymes were tested in two ways. For the
concentrations, from 0.005 to 10 g l71 depending on the inhibition of bacterial adhesion (the prevention test),
enzymatic preparation, in natural seawater (previously 50 ml of the enzymatic preparation at the desired
ltered through a 0.22 mm membrane), at 258C with concentration was placed in the wells 1 h before the
12.5 g l71 of the appropriate substrate at dierent bacterial suspension (200 ml per well) and incubated
incubation times from 3 to 12 min. Enzymatic activity for 3 and 24 h at 208C. For the detachment of adhered
was stopped by heating samples for 5 min at 1008C 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 incubating for 24 h at 208C. The wells were then
for 1 h at 208C. In both tests, three washings in washed three times with 36 g l71 sterile NaCl solution
36 g l71 NaCl were performed before xation for 1.5 h and the adhered bacteria were scraped into 250 ml with
at 48C with 200 ml of 36 g l71 sterile NaCl containing 36 g l71 sterile NaCl solution four times. One hundred
2.5% formaldehyde and DAPI staining (4 mg ml71) microlitres of the scraped adhered bacteria were
for 20 min. Another three washings were performed to diluted with appropriate serial tenfold dilutions and
remove excess DAPI and then, the remaining bound 100 ml of the last three dilutions were spread out on
DAPI was solubilised into 95% ethanol for 15 min marine agar, incubated from 24 to 48 h at 258C and
(200 ml). Fluorescence was measured at 350 nm excita- the colony forming units were then enumerated. Nine
tion and 510 nm emission wavelengths using a Genios hundred microlitres of the scraped adhered bacteria
Plus microplate uorescence reader (TECAN, Lyon, were incubated with formaldehyde solution (2.5% nal
France). For each condition, the enzymatic prepara- concentration) for 1.5 h. Five microlitres of this
tions were tested four times and heat denatured solution were then added to 2 ml 36 g l71 sterile
controls twice. They were all tested at dierent NaCl and incubated with triton X-100 solution (0.05%
concentrations using serial twofold dilutions in micro- nal concentration) for 10 min before sonicating the
plate wells. solution for 10 min at 47 kHz. DAPI was then added
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A blank with sterile seawater as well as a control to a nal concentration of 4 mg ml71 for 20 min. The
with bacterial suspension without enzymes was in- DAPI stained bacterial solution was then ltered onto
cluded in each column of the experimental microplate. a 0.22 mm sterile black polycarbonate lter (45 mm
The change in bacterial adhesion was calculated as diameter, Isopore GTBP Millipore, Fischer Scientic
the percentage reduction (PR) from the uorescence of Labosi, Elancourt, France). The lters were rinsed
the blank (FB; without bacteria), the uorescence of the with sterile water and mounted on glass slides. The
control (FC; bacteria without enzyme) and the uor- slides were observed under 6100 magnication by
escence of the sample (FS; bacteria with enzyme) epiuorescence microscopy using an Olympus BH201
according to: under WU lter (Olympus, Rungis, France). Approxi-
mately 1250 cells were counted per lter from the 25
PR fFC  FB FS  FB =FC  FB g  100 dierent elds. This experiment was carried out in four
wells of one microplate.
The concentrations necessary to reach a reduction
percentage of 50% adhesion (C50) were calculated
from a loglog plot of PR against enzymatic concen- Epiuorescence microscopy counts of total
trations. Standard deviation was calculated from the adhered bacteria
four C50s calculated for each experiment and p values Lab-TekTM wells with a glass surface (Lab-TekTM
were calculated with the t-test (Excel software). NUNC, Fischer Scientic Labosi, Elancourt, France)
For CTC staining with Amano protease, the were used and 360 ml of sterile natural sea water or
experiment was undertaken according to the preven- 360 ml of Amano protease were added per well and
tion test described above. Amano protease was added incubated for 1 h at 208C without shaking. Five
1 h before the D41 suspension, incubated for 3 and hundred and forty microlitres of bacterial suspension
24 h at 208C. The adhered bacteria were then washed were then inoculated. The covered Lab-TekTM glass
three times with 36 g l71 of sterile NaCl solution, slides were incubated for 24 h at 208C with orbital
stained with CTC (5 mM) for 2 h, washed three times shaking (300 rpm). The non adhered bacteria were
with 36 g l71 sterile NaCl solution, xed for 1.5 h at removed by three successive hand washings. The Lab-
48C with 200 ml of 36 g l71 sterile NaCl containing TekTM wells were rinsed and shaken with 900 ml of
2.5% formaldehyde and nally, the CTC reduced to 36 g l71 sterile NaCl solution. The adhered bacteria
formazan (CTF) via the bacterial respiratory electron were xed for 1.5 h at 48C with 900 ml of 36 g l71
transport chain. The CTF was then solubilised into sterile NaCl solution with 2.5% formaldehyde and
95% ethanol for 15 min. Absorbance was measured at then desalted by successive baths in solutions of
450 nm using the Genios Plus microplate uorescence decreasing salinity. The adhered bacteria were incu-
reader (TECAN, Lyon, France). bated with 900 ml of the DAPI solution (4 mg ml71) for
Regarding the enumeration of total and viable 20 min in the dark. The excess stain was removed by
cultivable adhered bacteria in the polystyrene micro- three hand washings, then the Lab-TekTM wells were
plates, the 24 h adhesion prevention test with Amano removed and the glass surface was dried out. The slides
protease was used as described above: Fifty microlitres were observed under 6100 magnication by epiuor-
of Amano protease or sterile natural seawater were escence microscopy using an Olympus BH201 under
added 1 h before adding the bacterial suspension and UV lter (Olympus, Rungis, France).
Biofouling 15

adhesion for 24 h were negative showing that the total


Results
quantity of adhered bacteria was greater with Amano
Eects of commercial enzymes on Pseudoalteromonas protease than without. This was also the case for
sp. D41 adhesion in seawater microplate tests Shearzyme (Figure 2), Celluclast, a-amylase and
The potential capability of several enzymes to inhibit Pectinex (Figure 3). Umamizyme, papain and Amano
the development of adhesion by Pseudoalteromonas sp. protease (Figure 1), Finizym and Glucanex (Figure 2)
D41 on the polystyrene microplate surface was tested. also promoted bacterial adhesion in the 24 h preven-
As stated previously the enzymes were tested for the tion test at low concentrations and inhibited it at
prevention of bacterial adhesion and for the detach- higher concentrations. This eect was also observed in
ment of adhered bacteria. the detachment test with Glucanex and Shearzyme
The percentage change in bacterial adhesion in (Figure 2), Celluclast, a-amylase and Pectinex
relation to enzymatic activities is shown in Figures 1 (Figure 3). Finizym inhibited bacterial adhesion in
to 3 depending on each enzymatic activity. The inu- the prevention test and increased it in the detachment
ence of enzymatic activities depended on the type of protocol of adhered bacteria (Figure 2).Thus in some
protocol tested. Glucanases and proteases were more enzyme concentrations and depending upon the pro-
eective than the xylanases, amylase, cellulase and tocol used (prevention or detachment of bacterial
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lipase. Savinase was the most eective protease, adhesion), some enzymatic activities were capable of
rapidly reaching the highest reduction percentage at increasing bacterial adhesion.
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 The eect of Amano protease on adhesion of
compare each C50 between all enzymatic preparations, Pseudoalteromonas sp. D41
the C50 were calculated in mg ml71. The results The eectiveness of Amano protease in the prevention
conrmed that Savinase was the most eective of adhesion of D41 was studied for periods of 3 and
enzyme since it needed the lowest concentration to 24 h (Figure 4). In the case of a 3 h adhesion, Amano
reach 50% inhibition. Savinase was more eective at protease inhibited an increasing number of adhered
3 h prevention compared with the detachment of bacteria for both total and respiring cells with
adhered bacteria (result statistically signicant at increasing concentration (Figure 4). However, for the
98%, p 0.02). Moreover, less Savinase was needed 24 h adhesion test, there was an opposite eect, ie the
to prevent bacterial adhesion in the 24 h test compared more concentrated the Amano protease caused an
with 3 h, as indicated by the C50. This result was only increase in the total number of adhered bacteria.
observed for Savinase. This showed that the eective- Adhesion of respiring bacteria was still inhibited at an
ness of Savinase in the prevention of bacterial adhesion average rate of about 25%. The enumeration of total
increased with the incubation time. and also viable and cultivable adhered bacteria in the
Only Glucanex, Lipolase, a-amylase and papain well after scraping conrmed this observation
could be tested after heating since not all the enzymatic (Figure 5). Enumerations of DAPI stained and
preparations in Table 1 could be heat denatured, some cultivable viable bacteria showed an increment of
(generally the liquid ones) became solid, thus testing 99.6% in the total bacterial amount, and an inhibition
was impossible. For Glucanex, a-amylase and Lipo- of 75.1% of the cultivable viable adhered bacterial
lase, heat denatured enzymes presented a partial amount. Surprisingly, the total amount of cells
inhibition of bacterial adhesion whatever the protocol counted using epiuorescence microscopy was lower
tested and approximately the same pattern was than the number counted by plating in the control
observed for the non denatured enzymes. Nevertheless, treatment. DAPI stained cells were sonicated before
lower bacterial inhibition was observed for heat enumeration, suggesting that sonication could partly
denatured Glucanex and Lipolase in the 3 h prevention lead to the lysis of cells, as previously observed
test compared with non-heat denatured Glucanex and (Garabetian et al. 1999; Fykse et al. 2003). This
Lipolase when the highest concentrations were used. phenomenon did not interfere with the observations of
Only heat denatured papain showed less or no total DAPI cell increments and cultivable viable cell
bacterial inhibition. No heat-denatured enzyme pre- inhibition in the presence of Amano protease since
parations showed any residual enzymatic activity enumeration of DAPI stained cells in the controls was
(Leroy 2006). compared with that in Amano protease, and in the
Amano protease was partially able to prevent D41 same way for cultivable viable cell enumeration.
adhesion for 3 h but it increased the nal quantity of The pattern of surface coverage by adhering
D41 bacterial cells if they were allowed to settle for Pseudoalteromonas sp. D41 in the presence of Amano
24 h (Figure 1). The reduction percentages after D41 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 molecules or adhesins. Mutanase and dextranase,
observed by Petitt et al. (2004) in regards to spores of eective against mutan and dextran EPS, have been
marine macro-organisms and larvae. Additives or shown to inhibit the formation of dental biolms by
denatured enzymes could be adsorbed onto the surface Streptococcus mutans and Streptococcus sobrinicus
modifying its chemical properties and leading to dier- (Wiater et al. 2004). Dispersin B catalyses the
ent bacterial adhesion. Further studies with enzymes hydrolysis of b-1,6-N-acetyl-D-glucosamine adhesin
after re-purication from commercial preparations (Ramasubbu et al. 2005) and inhibits Escherichia coli
should be undertaken. However, the eectiveness of en- and Staphylococcus epidermis biolms (Kaplan et al.
zymes such as Glucanex and papain in the 3 h preven- 2004; Itoh et al. 2005). More recently, the susceptibility
tion test seems to be due to specic enzymatic eects. of staphylococcal biolms to enzymatic treatments has
The antibiolm eectiveness of hydrolases could be been shown to depend on their chemical composition.
assigned to the hydrolysis of a substrate involved in Staphylococcal biolms producing poly-N-acetylglu-
bacterial adhesion such as EPS, Quorum Sensing (QS) cosamine were sensitive to Dispersin B while staphy-
lococcal biolms producing proteins were sensitive to
proteases (Chaignon et al. 2007). Target substrates
Table 2. Concentration (mg ml71) to achieve a reduction of could also be molecules implicated in QS pathway like
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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 been observed when an insucient amount of biocide
involvement of b-glucans in the rst adhesion step of was applied (Grant and Bott 2005). This increase in
Pseudoalteromonas sp. D41. Epiuorescence micro- adhered bacterial cells could be related to sedimenta-
scopy of 24 h D41 adhesion on glass slides supports tion, the accumulation of planktonic bacteria, or to cell
these observations, showing good uorescence using growth within the biolm whereas Amano protease in
the b-glucan stain calcouor white (Leroy 2006). addition to its enzymatic activity could be a substrate
Some enzymes increased the quantity of adhered supporting bacteria growth. Dead cells in clusters may
bacteria relative to the untreated control. In the also be a nutrient source, allowing other cells to
presence of Amano protease, adhesion of Pseudoalter- develop. Moreover, the enumeration of viable and
omonas sp. D41 was prevented if the cells were allowed cultivable bacteria and the epiuorescence microscopic
to adhere for 3 h, but after 24 h, adhesion was counts of DAPI stained adhered bacteria conrmed
increased. Since Amano protease is not naturally that the number of total adhered bacteria was greater
uorescent and has no anity to DAPI (Leroy when Amano protease was used. The number of
2006), it is concluded that the observed uorescence respiring adhered bacteria (CTC stain) and viable
after DAPI staining in the presence of Amano protease cultivable adhered cells were not shown to increase in
is not an artifact. Biolm accumulation has already relation to the number of total adhered cells (DAPI
Downloaded by [Universita degli Studi di Torino] at 12:24 02 June 2013

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
Figure 5. Eect of Amano protease in prevention of D41 enzymatic preparations can inhibit adhesion of marine
adhesion on a polystyrene surface in a microplate and Pseudoalteromonas sp. D41 in natural sterile seawater.
measured after 24 h. The total number (&) and the number Savinase was the most eective in the test conditions
of viable cultivable () adhered bacteria per cm2 are shown. used and this may be related to proteins directly
Amano protease was pre-incubated in sea water in involved during adhesion (Leroy 2006) and surface-
microplate at 1 UP ml71 for 1 h, then Pseudoalteromonas
sp. D41 was added per well and incubated for 24 h at 208C in enriched proteins of Pseudoalteromonas sp. D41
sterile natural seawater. SD was calculated from four (Pradier et al. 2005). Moreover, Alcalase, an enzymatic
experiments. preparation based on subtilisin like Savinase, inhibits

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).
Biofouling 21

the adhesion of fouling macro-organisms (Pettitt et al. Compere C, Bellon-Fontaine M-N, Bertrand P, Costa D,
2004). However, unlike the results of Pettitt et al. Marcus P, Poleunis C, Pradier C-M, Rondot B,
(2004) the eectiveness of Savinase seems to be stable, Walls MG. 2001. Kinetics of conditioning layer forma-
even better, when bacterial adhesion in the prevention tion on stainless steel immersed in seawater. Biofouling.
17:129145.
test was increased from 3 to 24 h. This result diers
Dong YH, Xu JL, Li XZ, Zhang LH. 2000. AiiA, an enzyme
from those observed by Pettitt et al. (2004) using
that inactivates the acylhomoserine lactone quorum-
Alcalase on the adhesion of cypris larvae and spores of sensing signal and attenuates the virulence of Erwinia
green algae or diatoms, suggesting that biochemical carotovora. Microbiology. 97:35263531.
molecules involved in bacterial adhesion for 24 h did Flemming HC, Wingender J. 2001. Relevance of microbial
not change signicantly. Thus, it is evidently better to extracellular polymeric substances (EPSs)-Part I: Struc-
use subtilisin preparations such as Savinase or Alcalase tural and ecological aspects. Water Sci Technol. 43:18.
during the rst stages of bacterial or macro-organism Fykse EM, Olsen JS, Skogan G. 2003. Application of
adhesion, by conditioning the surface after immobili- sonication to release DNA from Bacillus cereus for
zing this enzyme directly onto the surface or by quantitative detection by real-time PCR. J Microbiol
incorporating it into an antifouling paint. Methods. 55:110.
Garabetian F, Petit M, Lavandier P. 1999. Does storage
To conclude, hydrolases were shown to reduce and
Downloaded by [Universita degli Studi di Torino] at 12:24 02 June 2013

aect epiuorescence microscopic counts of total


enhance bacterial adhesion, depending on the time of bacteria in freshwater samples? C R Acad Sci III. 322:
exposure, and the concentration and the nature of the 779784.
enzymes. The enzyme Savinase appeared to be very Grant DM, Bott TR. 2005. Biocide dosing strategies for
eective in the marine test conditions used. In addition biolm control. Heat Trans Eng. 26:4450.
to previous antifouling studies (Pettitt et al. 2004), this Hahn Berg IC, Kalfas S, Malmsten M, Arnebrant T. 2001.
result also emphasizes the high antifouling potential of Proteolytic degradation of oral biolms in vitro and in vivo:
subtilisin. potential of proteases originating from Euphausia superba
for oral plaque control. Eur J Oral Sci. 109:316324.
Acknowledgements Holmstrom C, Rittschof D, Kjelleberg S. 1992. Inhibition of
settlement of larvae of Balanus amphitrite and Ciona
The authors acknowledge ANRT (Association Nationale de
la Recherche et Technique, France) and Mexel S.A. (France) intestinalis by a surface-colonising marine bacterium.
for nancial support for C. Leroy. Appl Environ Microbiol. 58:21112115.
None of the authors have competing nancial interests Itoh Y, Wang X, Hinnebush BJ, Preston JF III, Romeo T.
with any of the companies whose products were compared in 2005. Depolymerization of beta-1,6-N-acetyl-D-glucosa-
the study. These interests include full or partial project mine disrupts the integrity of diverse bacterial biolms.
funding, direct investments, reimbursed speaking arrange- J Bacteriol. 187:382387.
ments or consulting fees. No conicts of interest exist. Johansen C, Falholt P, Gram L. 1997. Enzymatic removal
and disinfection of bacterial biolms. Appl Environ
Microbiol. 63:37243728.
Kaplan JB, Ragunath C, Velliyagounder K, Fine DH,
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