BIOFILMS:

SUMMARY: Adhered aggregates of bacterial cells can be far more resistant to chemical attack than is commonly appreciated, exceeding the resistance of any other known natural material.

Bacteria often exist as adhered cellular aggregates (biofilms), which helps them resist antibiotics, extreme environmental conditions, and other life-threatening situations. Biofilms also have commercial value, a potential upcoming example being the production of hydrogen, an environmentally-friendly fuel.

The protective benefits of biofilms is likely derived from the extracellular matrix, a filter typically comprised of proteins and carbohydrates. Although biofilms are known to be very robust (e.g. providing some protection against bleach for one hour), why they are so robust remains a mystery.

Joanna Aizenberg (Harvard University, United States) and coworkers are helping to unravel this mystery. They have found that some bacterial biofilms repel a wide range of solvents, and even resist gas penetration.

Resistance to penetration.

The scientists studied biofilms of Bacillus subtilis, a common bacterium that readily forms large (centimeter-scale) biofilms on both liquid and solid surfaces. Liquid-repelling properties were measured via the contact area of a drop of liquid on the biofilm; a larger angle (e.g. greater than 90°) between the drop and the biofilm means more repulsion.

Ethanol, up to a concentration of 80% in water, gives a contact angle between roughly 135° and 145°, dropping significantly at higher concentrations. This is very different from typical wetting behavior of synthetic materials, which give a roughly linear decrease in contact angle with increasing liquid concentration, and which commonly wet at a much lower ethanol concentration (e.g. 20%).

Especially noteworthy is that ethanol concentrations of 60% are generally considered optimal for defeating bacteria. Clearly, when bacteria are in the biofilm state, larger concentrations are needed.

Isopropanol, methanol, and acetone, each at 50% concentration, are all defeated by the biofilms. These solvents are commonly thought to kill bacteria, but clearly not in the biofilm state.

Even commercial biocides such as Lysol (benzalkonium chloride), Hibiclens (chlorhexidine), and drain opener (the latter after 10 seconds of exposure) exhibit a large contact angle with the biofilms. Clorox bleach and drain opener (the latter after 5 minutes of exposure), however, do likely penetrate into the biofilms.

No effect of either biofilm age (between 3 days and 2 weeks) was observed. The liquid repelling property of the biofilms is clearly persistent.

Surprisingly, the biofilms were even resistant to metal oxide gases (the penetration of which is visible via X-ray imaging). Alumina (Al₂O₃) and hafnia (HfO₂) gas each failed to penetrate the biofilms much more than 10 micrometers in depth.

Mechanism of resistance.

What is it about the biofilms that imparts such a remarkable resistance to chemical attack, in either the vapor or liquid state? The scientists asked this question from both a biochemical and geometric viewpoint.

They genetically engineered a number of bacterial strains. One underexpressed carbohydrates in the extracellular matrix, one underexpressed proteins, and the other overexpressed both.

The strain with reduced carbohydrates in the extracellular matrix was largely wetted by 50% solutions of either ethanol, isopropanol, methanol, or acetone. The strain with reduced proteins, and the strain with extra carbohydrates and proteins, featured somewhat reduced repellancy.

Given that these results are in line with gas penetration experiments, this says that the carbohydrates (and to a lesser extent the proteins) in the extracellular matrix are largely responsible for the chemical resistance of the biofilms. Since epoxy molds of the biofilms failed to mimic the resistance seen of the biofilms themselves, the altered geometry of the carbohydrate-deficient biofilms was not the sole cause of failed resistance.

Implications.

It's clear that antimicrobials targeted to biofilms need to be designed with extreme resistance in mind. However, these biofilm results aren't entirely bad news; on the contrary, they imply that bacteria may be very useful as cheap, renewable, broadly-useful nonwetting surfaces, the synthetic design of which has (until now) been very challenging.

NOTE: The scientists' research was directly funded by the BASF Advanced Research Initiative at Harvard University, and indirectly by the United States Department of Energy.

Epstein, A. K., Pokroya, B., Seminara, A., & Aizenberg, J. (2010). Bacterial biofilm shows persistent resistance to liquid wetting and gas penetration Proceedings of the National Academy of Sciences : 10.1073/pnas.1011033108