In vitro laser ablation of natural marine biofilms.
ABSTRACT We studied the efficiency of pulsed low-power laser irradiation of 532 nm from an Nd:YAG (neodymium-doped yttrium-aluminum-garnet) laser to remove marine biofilm developed on titanium and glass coupons. Natural biofilms with thicknesses of 79.4 +/- 27.8 microm (titanium) and 107.4 +/- 28.5 microm (glass) were completely disrupted by 30 s of laser irradiation (fluence, 0.1 J/cm2). Laser irradiation significantly reduced the number of diatoms and bacteria in the biofilm (paired t test; P < 0.05). The removal was better on titanium than on glass coupons.
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ABSTRACT: Recently Erbium (Er) YAG laser has been developed for dentistry. It may be suitable for periodontal therapy. This study examined the bactericidal effect of the Er: YAG laser on periodontopathic bacteria in vitro. After spreading the bacterial suspension of Porphyromonas gingivalis or Actinobacillus actinomycetemcomitans on agar plates, a single pulse laser was applied to the agar plates at the energy density of 0.04-2.6 J/cm2. The growth of the bacterial colonies on the lased agar plates was examined after anaerobic culture. P. gingivalis colonies were also individually exposed to the single pulse laser at the energy of 1.8-10.6 J/cm2. The colony forming units of the irradiated colonies were counted. Growth inhibitory zones were found at the irradiated sites at the energy of about 0.3 J/cm2 and higher. The survival ratios of the viable bacteria in the lased P. gingivalis colonies decreased significantly at the energy of 7.1 and 10.6 J/cm2, as compared with that of the control. These findings suggest that the Er:YAG laser has a high bactericidal potential at a low energy level.Lasers in Surgery and Medicine 02/1996; 19(2):190-200. · 2.46 Impact Factor
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ABSTRACT: This overview attempts to bring together a widely scattered and disparate literature on marine natural product antifoulants. In particular, studies that have focused on the screening of secondary metabolites are reviewed in the context of the development of new antifouling coatings. Despite considerable progress towards characterising compounds that inhibit settlement of micro- and/or macrofouling, evidence for an ecological role for these compounds is poor. Even if broad spectrum antifoulants of sufficient promise are discovered, major hurdles must be overcome before they can be commercially exploited. Not least are the need to procure sufficient material and the cost of registration. Given these constraints and the relatively small size of the marine coatings market, industrial investment in product research and development appears low. Notwithstanding these problems, some marine organisms are able to maintain a surface that is essentially free of epibionts. Fundamental research on natural antifouling mechanisms is now required. The information gained, in addition to ascertaining the relative importance of physical versus chemical defences to marine organisms, would provide a conceptual framework for the development of novel, and ideally nontoxic, coatings.Biofouling 01/1996; 9(3):211-229. · 3.40 Impact Factor
Article: Biofilms, the customized microniche.Journal of Bacteriology 05/1994; 176(8):2137-42. · 3.19 Impact Factor
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 2004, p. 6905–6908
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 70, No. 11
In Vitro Laser Ablation of Natural Marine Biofilms
Kanavillil Nandakumar,1* Hideki Obika,2Akihiro Utsumi,2
Toshihiko Ooie,2and Tetsuo Yano2
Great Lakes Institute for Environmental Research, University of Windsor, Windsor, Ontario,
Canada,1and Marine Eco-materials Research Group, Marine Resources and Environment
Research Institute, National Institute of Advanced Industrial Science and
Technology (AIST), Hayashi-cho, Takamatsu, Kagawa, Japan2
Received 5 January 2004/Accepted 31 May 2004
We studied the efficiency of pulsed low-power laser irradiation of 532 nm from an Nd:YAG (neodymium-
doped yttrium-aluminum-garnet) laser to remove marine biofilm developed on titanium and glass coupons.
Natural biofilms with thicknesses of 79.4 ? 27.8 ?m (titanium) and 107.4 ? 28.5 ?m (glass) were completely
disrupted by 30 s of laser irradiation (fluence, 0.1 J/cm2). Laser irradiation significantly reduced the number
of diatoms and bacteria in the biofilm (paired t test; P < 0.05). The removal was better on titanium than on
The growth of biofilms on industrially important structures is
harmful. Methods commonly employed to prevent its forma-
tion include chemical treatment of the water column by bio-
cides or coating the surfaces with antifouling paints. As these
methods invariably lead to pollution, environmentally friendly
methods are desirable. Electromagnetic radiations such as UV
and laser are known to cause bacterial mortality (1, 6, 7, 16).
However, the utility of laser irradiation in abating bacterial
attachment and removing biofilm has rarely been studied (10,
11). Earlier studies showed a considerable reduction in the
viable count of the bacterium Pseudoalteromonas carrageeno-
vora at laser fluence of 0.1 J/cm2for short duration (10 min).
The laser-irradiated bacterial biofilms took considerable time
to reach the preirradiated level (15). These observations led us
to study the impacts of laser irradiation on natural biofilms
developed on experimental coupons exposed to seawater. In
this article, we summarize the impacts with emphasis on the
bacterial and diatom components of the natural biofilms.
Experimental coupons. Borosilicate laboratory glass slides
and titanium (Japanese Industrial Standard grade 1) sheets
were cut into sections 2 by 1 by 0.1 cm and used as experimen-
tal coupons. These materials were selected because (i) they
were nontoxic, inert, and resistant to corrosion and (ii) to
compare laser impacts on biofilms developed on metallic and
nonmetallic hydrophilic surfaces. For example, some part of
the irradiation passes through glass, while it gets absorbed
and/or reflected from titanium. The temperature rise in the
medium and on the coupon surface during irradiation was
determined by using a temperature probe (Custom thermom-
eter CT-2310). The titanium coupons were used in the as-
Laser. The laser used (GCR-170; Spectra-Physics) for this
study was an Nd:YAG (neodymium-doped yttrium-aluminum-
garnet) laser in the 2nd harmonic mode, delivering green light
at 532 nm. The pulse width and repetition rate of this laser
were 5 ns and 10 Hz, respectively. The peak power was 20 MW
cm?2, and the fluence (intensity of laser irradiation expressed
as joules per square centimeter) per pulse tested was 0.1 J
cm?2(kept the same as in our previous studies) (10–12, 14).
Laser irradiation in the green light area was used because its
attenuation rate in the water column is low.
Natural biofilm. In May 2003, 20 coupons each of titanium
and glass were fixed onto an assembly and suspended in coastal
seawater at a 1-m depth from a floating raft off Shikoku, Japan
(a facility of Akashio Research Laboratory, Kagawa Prefec-
ture, Japan). While the titanium coupons were suspended with
plastic wires, the glass coupons were suspended after fixing
them in the slits of a plastic frame. The surface seawater
temperature during the study varied between 21 and 22°C. The
coupons were suspended for 5 days, and by this time, biofilms
with thicknesses of 79.4 ? 27.8 ?m (titanium) and 107.4 ?
28.5 ?m (glass) (n ? 20) had developed on their surfaces. The
measurements were made with a micro-gauge fitted in the
focus knob of the Nikon microscope. The microscope was
focused initially on the bare coupon surface followed by the
top of the biofilm: thus, the difference in readings gave the
thickness. The coupons were brought back to the laboratory by
keeping them immersed in the seawater collected from the site
and were used immediately (within 1 h) for the experiment.
The total culturable viable count (TVC) of bacteria and phy-
toplankton composition of the water column on the first and
fifth days of suspension were also determined. The TVC was
determined by the plate count method (ZoBell marine agar
medium; Difco, Detroit, Mich.), while water fixed in Lugol’s
iodine was analyzed for phytoplankton composition after set-
tling in a settling chamber.
Initial TVC and diatom count. Biofilm from the coupons (in
duplicate) was removed with a sterile toothbrush into a known
volume of autoclaved microfiltered (0.2-?m pore) aged seawa-
ter (AMASW). After vortexing for 3 to 5 min to disperse the
clumps (samples were observed under a microscope for the
clump dispersion), subsamples were plated with ZoBell marine
agar plates to estimate TVC. For the remaining sample, a
* Corresponding author. Mailing address: Department of Biology,
Lakehead University, 955 Oliver Rd., Thunderbay, ON, Canada P7B
5E1. Phone: (807) 766-7151. Fax: (807) 346-7796. E-mail: nandakumar
hemocytometer was used to determine diatom density and
species composition (13). Sixteen fields were counted for each
sample. The diatoms were identified with the help of identifi-
cation keys (3, 20).
Laser irradiation. One side of each coupon was wiped off
with a sterile cotton plug before being placed in the irradiation
chambers (sterile glass dishes 4 by 3 cm) containing 10 ml of
AMASW. This volume left 3 to 4 mm of water above the
coupon surface (determined with Vernier calipers) when laid
horizontally. Coupons in quadruplicate were irradiated from
the top for 30 s and 5 and 10 min. Nonirradiated coupons
served as a control.
Observations. The irradiated coupons were immediately
scraped out with a sterile toothbrush into a known volume of
AMASW inside a laminar flow chamber. TVC was estimated
by plating techniques using ZoBell marine agar. One each of
the nonirradiated and 10-min-irradiated coupons was observed
under an environmental scanning electron microscope (Hita-
chi, Tokyo, Japan) as well as a scanning electron microscope
(S-2460N; Hitachi). In addition, the surface damage on the
titanium coupons irradiated for 10 min was observed under an
atomic force microscope (AFM; Nanopics 2100; Seiko Instru-
ments, Chiba, Japan; images not included here).
Biofilm area cover. Biofilm area cover on irradiated (10 min)
and nonirradiated coupons was estimated from their micro-
scopic images (10 each) by a spread dot method (9). For this
purpose, a transparent sheet of the size of the images with dots
at a 1-mm2interval was laid over the images. All data were log
transformed before the statistical analysis.
The diatom density in the water column varied between 2 ?
102and 3 ? 102cells/ml, while TVC varied between 0.8 ? 104
and 1.3 ? 104cells/ml. The major diatom species found were
Nitzshia sp., Amphora sp., and Skeletonema costatum.
Natural biofilm. The major diatom species found were Ba-
cillaria sp., Nitzschia longissima, Navicula sp., Cylindrotheca sp.,
and Amphora sp. Of the total diatom density, Nitzschia sp. and
Navicula sp. accounted for 50 to 90%. Laser irradiation for 10
min dislodged a significant portion of the biofilm from the
titanium coupons, while some parts remained on the glass (Fig.
1). The area the biofilm covered was significantly lower on the
FIG. 1. Environmental scanning electron microscopic images of nonirradiated and irradiated coupons with marine natural biofilm. (A) Tita-
nium control coupon with biofilm, (B) titanium coupon after irradiation (fluence, 0.1 J/cm2for 10 min), (C) glass control coupon with biofilm, and
(D) glass coupon after irradiation (fluence, 0.1 J/cm2for 10 min).
6906 NANDAKUMAR ET AL.APPL. ENVIRON. MICROBIOL.
irradiated coupons (10 min) than the nonirradiated coupons
(t test with paired two samples for mean, P ? 0.001; n ? 10
images analyzed). Irradiation resulted in deformed biofilms
and broken diatom cells (Fig. 2). After 30 s of irradiation, 71.7
and 74.8% of diatoms were dislodged, and by 10 min of irra-
diation, 94.9 and 87.4% of diatoms disappeared from the bio-
films on titanium and glass coupons, respectively (Fig. 3).
Compared to controls, diatom number was significantly re-
duced (t test with paired two samples for mean, P ? 0.05; n ?
4 coupons for each comparison).
TVC. The biofilm TVC was reduced from 15 to 90%, de-
pending on the duration of irradiation. The reduction in TVC
compared to the control after 5 and 10 min of laser irradiation
was found to be significant (t test with paired two samples for
mean, P ? 0.05; n ? 4 coupons). The TVC reduction on glass
coupons was slightly less than that for the titanium coupons.
On glass coupons, the TVC was reduced from 52.5 to 84.3% by
30 s and 10 min of irradiation, respectively.
The attachment and growth of microorganisms on material
surfaces lead to the formation of biofilm, which is described as
a consortium of bacteria, microalgae, and protozoa with their
exudates (2, 5). Although incongruity exists, biofilm is de-
scribed as an intermediary step in the biofouling growth. Also,
it has been shown to influence the initiation of biocorrosion of
material surfaces (17). Although efforts to restrict biofilm for-
mation have focused on chemical means, environmentally be-
nign methods such as the use of antibacterial materials (18),
electrochemical techniques (8), and the use of bioactive com-
pounds (4) are gaining prominence. The present study dem-
onstrates the possibility of using pulsed low-power laser irra-
diation as a technique to dislodge biofilm from hard surfaces.
The initial substratum surface temperature of 24.7°C was
FIG. 2. Scanning electron microscopy image of broken diatom cells
after laser irradiation for 10 min with a fluence of 0.1 J/cm2.
FIG. 3. Variation observed in the total number of diatoms on titanium (A) and glass (B) and TVC of bacteria on titanium (C) and glass (D) on
the experimental coupons before and after laser irradiation. Con, control.
VOL. 70, 2004LASER ABLATION OF BIOFILMS6907
increased to 32.8°C after irradiation for 10 min with a fluence
of 0.1 J/cm2. Thus, although pulsed laser irradiation resulted in
the removal of bacteria from the material surface, it is unlikely
that mortality was due to the rise in the medium temperature
(19, 21). However, of the two types of coupons studied, more
effective biofilm removal was observed on titanium than on
glass coupons. This could be due to the difference in the ma-
terial properties. For example on titanium coupons, laser irra-
diation resulted in aberrations (AFM images as in reference
14) that also lead to the removal of biofilms.
Observation of biofilm area cover, diatom density, and TVC
of bacteria before and after the laser irradiation showed that
the irradiation resulted in a significant reduction in all three
parameters. However, the reduction in bacterial TVC was not
as prominent as in diatoms. In summary, irradiation for a very
short duration removed a significant portion of the biofilm
from the coupon surfaces, while any parts remaining were
composed chiefly of dead cells. The results from both types of
substrata showed that low-power pulsed laser irradiation has
the potential to act as a tool to remove biofilm from solid
K.N. acknowledges the New Energy Development Organization
(NEDO), Tokyo, Japan, for financial assistance in the form of a
NEDO postdoctoral fellowship.
We thank the two anonymous reviewers for useful comments. David
Kelly of GLIER, University of Windsor, Windsor, Ontario, Canada, is
thanked for comments on the manuscript.
1. Ando, Y., A. Aoki, H. Watanabe, and I. Ishikawa. 1996. Bactericidal effect of
erbium YAG laser on periodontopathic bacteria. Lasers Surg. Med. 19:190–
2. Characklis, W. G., G. A. McFeters, and K. C. Marshall. 1990. Physiological
ecology in biofilm systems, p. 341–393. In W. G. Characklis and K. C.
Marshall (ed.) Biofilms. John Wiley & Sons, Inc., New York, N.Y.
3. Chihara, M., and M. Murano. 1997. An illustrated guide to marine phyto-
plankton in Japan. Tokai daigaku syuppankai (Tokai University Press), To-
4. Clare, A. S. 1996. Marine natural products antifoulants: status and potential.
5. Costerton, J. W., Z. Lewandowski, D. DeBeer, D. Caldwell, D. Korber, and
G. James. 1994. Biofilms, the customized microniche. J. Bacteriol. 176:
6. Deckelbaum, L. I. 1994. Cardiovascular applications of laser technology.
Lasers Surg. Med. 15:315–341.
7. Kawamoto, K., N. Senda, K. Shimada, K. Ito, Y. Hirano, and S. Murai. 2000.
Antibacterial effect of yellow He-Ne laser irradiation with crystal violet
solution on Porphyromonas gingivalis: an evaluation using experimental rat
model involving subcutaneous abscess. Lasers Med. Sci. 15:257–262.
8. Matsunaga, T., T. Nakayama, H. Wake, M. Takahashi, M. Okochi, and N.
Nakamura. 1998. Prevention of marine biofouling using a conductive paint
electrode. Biotechnol. Bioeng. 59:374–378.
9. Nandakumar, K. 1996. Importance of timing of panel exposure on the
competitive outcome and succession of sessile organisms. Mar. Ecol. Prog.
10. Nandakumar, K., H. Obika, T. Shinozaki, T. Ooie, A. Utsumi, and T. Yano.
2002. Impact of pulsed Nd:YAG laser irradiation on the growth and mor-
tality of a biofilm forming marine bacteria Pseudoalteromonas carrageeno-
vora. Biofouling 18:123–127.
11. Nandakumar, K., H. Obika, T. Shinozaki, T. Ooie, A. Utsumi, and T. Yano.
2002. Inhibition of bacterial attachment by Nd:YAG laser irradiations: an
in-vitro study using marine biofilm forming bacteria Pseudoalteromonas car-
rageenovora. Biotechnol. Bioeng. 80:552–558.
12. Nandakumar, K., H. Obika, T. Shinozaki, T. Ooie, A. Utsumi, and T. Yano.
2003. Laser impact on the marine planktonic diatoms: an experimental study
using a flow-cytometric system. Biofouling 19:133–138.
13. Nandakumar, K., H. Matsunaga, and M. Takagi. 2003. Microfouling studies
on the steel making slag and concrete test pieces exposed to seawater at the
Chiba coast, Japan. Biofouling 19:257–267.
14. Nandakumar, K., H. Obika, T. Shinozaki, T. Ooie, A. Utsumi, and T. Yano.
2004. In-vitro laser ablation of laboratory developed marine biofilm. Bio-
technol. Bioeng. 86:729–736.
15. Nandakumar, K., H. Obika, A. Utsumi, T. Ooie, and T. Yano. 2004. Recolo-
nization of laser ablated bacterial biofilm. Biotechnol. Bioeng. 85:185–189.
16. Sarkar, S., and M. Wilson. 1993. Lethal photosensitization of bacteria in
subgingival plaque from patients with chronic periodontitis. J. Periodontal
17. Sreekumari, K. R., K. Nandakumar, and Y. Kikuchi. 2001. Bacterial adhe-
sion to weld metals: significance of substratum microstructure. Biofouling 17:
18. Sreekumari, K. R., K. Nandakumar, T. Yokota, and Y. Kikuchi. 2002. Lab-
oratory assay of antibacterial properties and corrosion behaviour of silver
alloyed AISI type 304 stainless steel and its welds. Corrosion 2002, paper no.
02469. NACE International, Houston, Tex.
19. Ward, G. D., I. A. Watson, D. E. S. Stewart-Tull, A. C. Wardlaw, R. C. Wang,
M. A. Nutley, and A. Cooper. 2000. Bactericidal action of high-power
Nd:YAG laser light on Escherichia coli in saline suspension. J. Appl. Micro-
20. Yamaji, I. 1984. Illustrations of the marine plankton of Japan. Hoikusha
Publishing Company Ltd., Osaka, Japan.
21. Yeo, C. B. A., I. A. Watson, D. E. S. Stewart-Tull, A. C. Wardlaw, and G. N.
Armstrong. 1998. Bactericidal effects of high-power Nd:YAG laser radiation
on Staphylococcus aureus. Pure Appl. Optics 7:643–655.
6908NANDAKUMAR ET AL.APPL. ENVIRON. MICROBIOL.