Rhamnolipid mediated disruption of marine Bacillus pumilus biofilms

https://doi.org/10.1016/j.colsurfb.2010.07.013Get rights and content

Abstract

Removal of detrimental biofilms from surfaces exposed in the marine environment remains a challenge. A strain of Bacillus pumilus was isolated from the surface of titanium coupons immersed in seawater in the vicinity of Madras Atomic Power Station (MAPS) on the East coast of India. The bacterium formed extensive biofilms when compared to species such as Bacillus licheniformis, Pseudomonas aeruginosa PAO1 and Pseudomonas aureofaciens. A commercially available rhamnolipid was assessed for its ability to inhibit adhesion and disrupt pre-formed B. pumilus biofilms. The planktonic growth of B. pumilus cells was inhibited by concentrations >1.6 mM. We studied the effect of various concentrations (0.05–100 mM) of the rhamnolipid on adhesion of B. pumilus cells to polystyrene microtitre plates, wherein the effectiveness varied from 46 to 99%. Biofilms of B. pumilus were dislodged efficiently at sub-MIC concentrations, suggesting the role of surfactant activity in removing pre-formed biofilms. Scanning electron microscopy (SEM) confirmed the removal of biofilm–matrix components and disruption of biofilms by treatment with the rhamnolipid. The results suggest the possible use of rhamnolipids as efficient anti-adhesive and biofilm-disrupting agents with potential applications in controlling biofilms on surfaces.

Introduction

Several species of Bacillus inhabit coastal and marine environments, where they form important members of the marine bacterial communities [1]. Members of this genus have been observed on titanium surfaces that are generally used for the manufacture of condensers and other heat exchangers in power plants [2]. The bacteria that belong to Bacillus are resistant to environmental conditions, such as low nutrient availability, irradiation and chemical disinfectants [3]. They are known to produce exopolysaccharides and organic acids that accelerate corrosion of steel [4]. Microbial adhesion is the first step in the establishment of microbial communities on surfaces, commonly termed as biofilms. Such biofilms can potentially cause enormous damage by leading to the formation of more complex biofouling [5]. In industrial applications that use seawater, problems such as reduced heat transfer across heat exchanger surfaces, mechanical blockages in cooling water pipes and enhanced corrosion of structural materials occur due to the growth of biofilms [6]. Biofilms and biofouling can be controlled by mechanical, chemical and thermal treatments. Conventional antifouling biocides have limited effectiveness against biofilm bacteria in industrial settings [7]. Bacteria in biofilms are generally protected against the onslaught of biocides [8]. Biofilm formation is initiated when a group of primary colonizing bacteria attach to a surface freshly exposed to the aqueous environment. The control of primary colonizing bacteria in particular is thus important in finding an effective solution. Along with the killing of biofilm bacteria, their removal from the surface is desirable [8]. Biosurfactants are quite effective for such combined applications.

Biosurfactants of microbial origin are reported to have anti-adhesive and biofilm disruption abilities [9], [10]. Fungal and bacterial biofilms have shown to be disrupted by enzymatically synthesized surfactants such as lauroyl glucose [11]. In recent years, rhamnolipids derived from Pseudomonas aeruginosa have emerged as an important group of biosurfactants with several applications; they have also been produced on a commercial scale [12]. Moreover, they display diversity in structure, are environment-friendly and are effective at low concentrations and under extreme conditions. They are safe and hence are being considered as an alternative to conventional antimicrobial agents in a wide variety of industrial areas [13]. Despite their potential, there are only a few studies on the interactions of biosurfactants with bacterial cells and their role in anti-adhesion [14], [12], [15] and biofilm dispersion [10], [16]. To date, the applications of rhamnolipids have been studied mainly with respect to disruption of clinically significant biofilms, and to the best of our knowledge, information on rhamnolipid mediated growth inhibition and removal of biofilms associated with marine environment either alone or in combination with biocides is lacking.

The objective of the present study was to assess the effect of rhamnolipid on the adhesion and biofilm formation by a potent biofilm forming bacterium isolated from condenser material exposed to seawater. The biofilm forming bacterium used in the present study was isolated from titanium surfaces exposed near the seawater intake point of Madras Atomic Power Station (MAPS) and characterized using 16S rRNA gene sequencing.

Section snippets

Titanium coupon preparation and isolation of marine bacteria

Titanium coupons (2.5 cm × 2.5 cm) were polished upto 400 grit and immersed in seawater at Kalpakkam (12°33″ N and 80°11″ E) near the cooling water intake point of Madras Atomic Power Station (MAPS) on the Bay of Bengal coast of India. After 2 days, the coupons were removed, rinsed twice with phosphate buffer (0.1 M, pH 7.0) to remove loosely bound cells. The biofilm was scraped into known volume of phosphate buffer with a sterile nylon brush. The biofilm was disrupted by vortexing, serially diluted

Identification of the dominant marine biofilm bacterium

A surface, when exposed to seawater, is approached and colonized by microbial cells. They establish microcolonies with the help of sticky exopolymeric substances (EPS), which further facilitate association of other organisms. It is believed that the initial settlers known as primary colonizing bacteria have a significant role in the subsequent development of complex multi-species biofilm communities, which ultimately leads to biofouling on surfaces [23]. Different species of Bacillus are

Conclusions

A marine isolate from titanium surfaces exposed to seawater was identified as B. pumilus on the basis of phenotypic, biochemical and 16S rRNA sequencing. The bacterium formed biofilms more efficiently than Ps. aureofaciens and B. licheniformis in polystyrene microtitre plates and on glass surfaces. Rhamnolipid at sub-MIC concentrations was effective as an anti-adhesive and biofilm-disrupting agent against the B. pumilus isolate. Scanning electron microscopic observations confirmed the role of

Acknowledgements

DD would like to thank BARC-University of Pune collaborative research programme for financial support. The authors would like to thank Dr. Pattanathu KSM Rahman for providing the rhamnolipids.

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