Impact of silver nanoparticles on natural marine biofilm bacteria

The use of nanoparticles has increased dramatically in recent years, with silver being the most widely used in consumer and medical products. Silver is a known bactericide and is found in goods such as cosmetics, surfaces, plastics and clothing. This increases their potential release and accumulation in the environment. Bacteria in nature are largely found as biofilms and perform or contribute to a number on essential environmental processes, and are at risk from the accumulation of silver nanoparticles.

The authors of this paper investigated the impact of well characterised silver nanoparticles to natural marine biofilms.

Biofilms were grown on acid-washed and sterilised glass slides at the western marina of Singapore during September and October 2007. The slides were placed parallel to each other using plastic frame holders to separate them by approximately 1cm. Holders were then submerged for 3 days at a depth of around 1m. After the three days, the biofilms were transported to laboratories in seawater.

Silver nanoparticles in the seawater were characterised (using a number of methods, which are discussed in the paper, but wouldn’t fit into this blog).

Extracellular polymeric substances were lectin stained to quantify the effects of silver nanoparticles on marine biofilms and examined, analysing silver content and the genetics of the bacterial community present.

Even though the biofilms were grown in an industrious harbour, the background levels of silver in sea-water were always below the limits of detection. The amount of silver uptake by the biofilms per unit of volume was between 1.3- 2.1 and 17.1- 19.5 for 20µg L^-1 and 200µg L^-1, and almost 10 times greater for 2000µg L^-1 which was 172.6- 235.2. These results show a linear dose dependence.

The investigators then checked for the effects of the silver nanoparticles on the biofilms. This was done by fluorescent labelling with site-specific lectins and examined using ISA-2. The 4 day old biofilms (exposed for 24h to 200µg L^-1 and 2000µg L^-1 of silver nanoparticles) had a significantly lower volume and biomass than controls of the same age; although there was no significant difference between the controls and biofilms exposed to 20µg L^-1.The results obtained in this study strongly show effects on marine biofilms of waterbourne exposure to silver nanoparticles. Exposure and uptake of silver nanoparticles at concentrations over 200µg L^-1 creates an overall thinning effect on the biofilm and molecular analysis displayed thatb the highest concentration of silver nanoparticles impeded biofilm colonisation and development.

Fabrega, J., Zhang, R., Renshaw, J. C., Liu, W-T., Lead, J. R., 2011. Impact of silver nanoparticles on natural marine biofilm bacteria. Chemosphere. 85 (6): 961-966.

Building bacterial biofilms

Biofilms are complex structures which are created by the attachment and growth of microorganisms on available substrates. Biofilms are formed in succession, starting with pioneers and later colonisers; but very little is known about a biofilms early formation; despite it being relevant to many sectors of marine ecology, such as larval recruitment, settlement and dynamics of microbial communities. It is thought that a biofilm commences with the adsorption of a film of polysaccharides, proteins, lipids, nucleic acids and aromatic amino acids, and is modified to create a stable climax community by secondary microorganisms/ colonisers, following the reproduction, growth and death of the pioneers. The coral surface mucus layer (SML) provides the perfect surface for the formation of the biofilm. Microbial biofilms can be established and maintained in three ways:

1) Microbes continually settle or are trapped by the SML, but not form a stable community due to the constant sloughing off of the layer.

2) A semi-established community may form in the SML of coral species which periodically shed their mucus as a tunic (e.g. Porites spp.)

3) Microbes may settle in the SML and/or coral tissues and become established, forming a distinct community from that of the water column.

Specific physical and chemical properties of the various mucus’ created by different corals may effect, and therefore explain, differences in microbial communities found in biofilms of different species; there is also an idea that differences may occur between communities due to the settlement surface offered.

While many studies have employed the use of flat settlement surfaces, this project used artificial corals coated in agar to test for different effects of surface shape and chemical composition on the development of a microbial biofilm community over 96 hours. The results were compared to the surrounding water column and a major reef building coral A. muricata; in both summer and winter.

The initial experiment used 4 types of agar coating for the artificial coral: plain agar, agar plus mucus, agar plus exudates from healthy coral and agar plus exudates from stressed coral; however there was no significant difference between the 16s rRNA gene bacterial assemblages settling, therefore, only plain agar was used for further temporal analysis.

Microscope slides were used to compare a smooth surface to the artificial coral nubbins, and gave a significant difference in microbial assemblage, with the nubbins giving a much more diverse community; tested for using DGGE; however, they found that 22% of variance could be put down to season alone, but after finding no ribotypes exclusive to one season meant significant differences were due to shifts in dominance of particular ribotypes. Large fluctuations in diversity between replicates in the first 12h, indicated that the initial settlement period is highly dynamic, but after this time becomes a lot more stable.

Vibrio species seemed to be opportunistic bacteria, as they appeared in early periods around 2h but were absent by 6h, apparently outcompeted. The idea of the Vibrio species being opportunistic ties in well with their role in coral bleaching; and as they are out competed it would be interesting to see if Flavobacteria sp., Glaciecola sp., and Klebsiella sp. etc. would prevent coral bleaching?

This study showed that there is a strong correlation between early colonisers of the SML and the surface type for settlement; and as shown before, a greater bacterial diversity was found on a more textured surface when compared to a smooth one. One of the reasons suggested for this is because a textured surface provides shelter from hydrodynamic processes such as wave action, and to further this investigation, it would be fascinating to see what effect would arise using the same principles, in a different marine environment, where hydrodynamic processes will not play much of a role, such as a sheltered lagoon.

A review of:

Sweet, M.J., Croquer, A., and Bythell, J.C. (2011). Development of Bacterial Biofilms on Artificial Corals in Comparison to Surface-Associated Microbes of Hard Corals. PLoS ONE.

Vol. 6. (6): e21195.doi:10.1371/journal.pone.0021195