AVS2013 Session BI+AI+BA+IS-MoA: Biofouling

Biofouling of surfaces causes numerous problems in a wide range of industries such as shipping, health care, oil and gas production and food production. Of specific interest to the current work is the accelerated corrosion of metals that can arise as a consequence of bacterial biofilm formation, which is commonly known as microbiologically influenced corrosion (MIC).

The initial attachment of bacteria to a surface is one of the first steps in the process of biofouling. The attachment is dependent upon a large number of factors, which are broadly related to the properties of the bacteria, substrate/surface and environment. Changes in these properties can not only influence the initial attachment step, but also the interrelated production of extracellular polymeric substances (EPS) by the bacteria and the subsequent corrosion.

A large amount of the work performed to date on bacterial attachment in relation to MIC has focused on stainless steels, possibly due to reports of rapid failures of these materials such as through thickness pitting of piping welds. These studies have highlighted how a range of material properties (e.g. chemical composition, surface roughness, grain size and boundaries) can influence attachment and biofilm formation on steel surfaces. This range of influences means that a high level of care must be taken when designing and carrying out bacterial attachment tests in order to avoid the situation where a number of material variables affect the outcome of a single test. For example one of the criticisms of some of the previous work in this area is the lack of control of surface roughness of the substrates used in the studies.

In this work we will report results of studies of the initial attachment and EPS production of E. coli bacteria on highly polished carbon steel samples, with a number of different microstructures, for a number of different test media. We have found that the microstructure and test medium can have a significant effect on the rate of bacterial attachment, the distribution of attached bacteria, the onset of EPS production and the corrosion of samples immersed in E. coli inoculated test media.

Zwitterionic surfaces are a class of coatings that receive increasing attention due to their good antifouling performance.¹ Since early work on protein resistance of mixed, charged self-assembled monolayers (SAMs), charge neutrality seems to be a prerequisite for their inert properties. ^2,3 Similar to established non-fouling ethylene glycol chemistries, zwitterionic systems rely on a strong hydration of the coating. In this study we attempt a systematic analysis to which extend charge neutrality and the chemical nature of the charged groups affect their antifouling performance. Positively charged trimethylammonium terminated thiols were therefore mixed with sulfonate-, carboxylate- and phosphonate-terminated undecanethiols in varying ratios. Optimized preparation conditions and surface analysis will be presented that demonstrates successful assembly of the coatings and characterizes their physicochemical properties. The antifouling properties were tested against a range of laboratory organisms such as diatoms and spores of algae and compared to protein resistance. The obtained trends will be discussed and correlated with field experiments in the real marine environment.

(1) Chen, S.; Jiang, S. 2008 A new avenue to nonfouling materials. Advanced Materials, 20, 335-338.

(2) Holmlin, R. E.; Chen, X. X.; Chapman, R. G.; Takayama, S.; Whitesides, G. M. 2001 Zwitterionic SAMs that resist nonspecific adsorption of protein from aqueous buffer. Langmuir, 17, 2841-2850.

(3) Chen, S. F.; Yu, F. C.; Yu, Q. M.; He, Y.; Jiang, S. Y. 2006 Strong resistance of a thin crystalline layer of balanced charged groups to protein adsorption. Langmuir, 22, 8186-8191.

Biofilms are multicellular communities of bacteria that are often found attached to surfaces and cause significant problems in medical, industrial, and marine settings. Cells within biofilms are enmeshed in an extracellular polymeric matrix comprised of polysaccharides, proteins, lipids, and nucleic acids. Over the past decade, extracellular DNA (eDNA) has been found to be essential for biofilm formation by many species of bacteriawhere it is thought to function as an intercellular “glue” that binds cells together. Interestingly, whilst it has been known for over a decade that eDNA is essential during the early stages of biofilm development by the opportunistic pathogen Pseudomonas aeruginosa, the precise roles of eDNA in this process have yet to be elucidated. We have used advanced techniques in microscopy, computer vision and image informatics to explore the roles of eDNA during early biofilm development and during active expansion of biofilms formed by P.aeruginosa. Many species of bacteria, including P. aeruginosa utilize type IV pili mediated twitching motility to actively translocate across solid and semi-solid surfaces. Twitching motility can manifest as a complex, multicellular behavior that enables the active expansion of bacterial biofilms. Under appropriate conditions, such as those encountered at the interface of a glass coverslip and semi-solid nutrient media, the expanding biofilm can develop dramatic networks of intersecting trails. Our analyses reveal that at the leading edge of the interstitial biofilm, highly coherent groups of bacteria migrate across the surface of the semi-solid media, and in doing so, create furrows along which following cells preferentially migrate. This leads to the emergence of a network of trails that guide mass transit toward the leading edges of the biofilm. We have determined that eDNA facilitates efficient traffic flow throughout the expanding biofilm by maintaining coherent cell alignments, thereby avoiding traffic jams and ensuring an efficient supply of cells to the migrating front. Our analyses reveal that eDNA also co-ordinates the movements of cells in the leading edge rafts and is required for the assembly of cells into aggregates that forge the interconnecting furrows. Our observations have revealed that large-scale self-organization of cells in actively expanding biofilms of P. aeruginosa occurs through construction of an intricate network of furrows that is facilitated by eDNA.

The characterisation and identification of individual bacteria using Raman spectroscopy can aid in rapid, in situ microbiological diagnosis and hence timely, appropriate treatment and control measures [1, 2]. Appropriate sample preparation methods and experimental conditions are crucial to avoid some potential difficulties in analysing the information-rich Raman spectra from bacterial cells. In this study, the Raman spectra of fresh and stored samples of bacterial isolates (Escherichia coli) were analysed to determine any variations caused by sample processing. Analysis based on principal components suggests that different methods of sample preparation and storage affect the spectral components associated with different biochemical compounds in bacterial cells. The effect of long term storage in glycerol stock at freezing temperatures on the Raman spectrum of cells from the early exponential phase was observed in this study and found to modify the bacteria cells. Furthermore, the presence of extracellular polymeric substance (EPS) matrix around bacterial cells at later stages of the growth cycle provide higher resistance to environmental stress compared with other phases. Based on these results, a specific experimental protocol has been developed in order to obtain interpretable, comparable and reliable Raman data from bacterial samples.

Keywords: Raman spectroscopy; Bacterial identification; Sample preparation.

References

[1] W. E. Huang, R. I. Griffiths. Anal. Chem. 2004, 76(15): 4452-4458.

[2] T. J. Moritz, S. T. Douglas. J. Clin. Microbiol. 2010, 48(11): 4287-4290.