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Applied and Environmental Microbiology, November 2004, p. 6905-6908, Vol. 70, No. 11
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.11.6905-6908.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

In Vitro Laser Ablation of Natural Marine Biofilms

Kanavillil Nandakumar,1* Hideki Obika,2 Akihiro Utsumi,2 Toshihiko Ooie,2 and Tetsuo Yano2

Great Lakes Institute for Environmental Research, University of Windsor, Windsor, Ontario, Canada,1 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

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 µm (titanium) and 107.4 ± 28.5 µm (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.

The growth of biofilms on industrially important structures is harmful. Methods commonly employed to prevent its formation include chemical treatment of the water column by biocides 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 carrageenovora at laser fluence of 0.1 J/cm2 for 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 experimental 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 thermometer CT-2310). The titanium coupons were used in the as-received condition.

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 Prefecture, 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 µm (titanium) and 107.4 ± 28.5 µm (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 phytoplankton 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 settling 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-µm pore) aged seawater (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 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 identification 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 (Hitachi, 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 Instruments, Chiba, Japan; images not included here).

Biofilm area cover.

Biofilm area cover on irradiated (10 min) and nonirradiated coupons was estimated from their microscopic 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-mm2 interval was laid over the images. All data were log transformed before the statistical analysis.

The diatom density in the water column varied between 2 x 102and 3 x 102 cells/ml, while TVC varied between 0.8 x 104 and 1.3 x 104 cells/ml. The major diatom species found were Nitzshia sp., Amphora sp., and Skeletonema costatum.

Natural biofilm.

The major diatom species found were Bacillaria 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 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 irradiation, 94.9 and 87.4% of diatoms disappeared from the biofilms on titanium and glass coupons, respectively (Fig. 3). Compared to controls, diatom number was significantly reduced (t test with paired two samples for mean, P < 0.05; n = 4 coupons for each comparison).



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  		FIG. 1. Environmental scanning electron microscopic images of nonirradiated and irradiated coupons with marine natural biofilm. (A) Titanium control coupon with biofilm, (B) titanium coupon after irradiation (fluence, 0.1 J/cm2 for 10 min), (C) glass control coupon with biofilm, and (D) glass coupon after irradiation (fluence, 0.1 J/cm2 for 10 min).



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  		FIG. 2. Scanning electron microscopy image of broken diatom cells after laser irradiation for 10 min with a fluence of 0.1 J/cm2.



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  		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.

TVC.

The biofilm TVC was reduced from 15 to 90%, depending 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 described 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 formation have focused on chemical means, environmentally benign methods such as the use of antibacterial materials (18), electrochemical techniques (8), and the use of bioactive compounds (4) are gaining prominence. The present study demonstrates the possibility of using pulsed low-power laser irradiation as a technique to dislodge biofilm from hard surfaces.

The initial substratum surface temperature of 24.7°C was 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 material properties. For example on titanium coupons, laser irradiation 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 surfaces.

ACKNOWLEDGMENTS

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.


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FOOTNOTES
 
* 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.kanavillil{at}lakeheadu.ca. Back

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Applied and Environmental Microbiology, November 2004, p. 6905-6908, Vol. 70, No. 11
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.11.6905-6908.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.




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