[biofilms & biodiversity]

The Sequence of Fouling of Engineering
Materials In the Sea

R.E. Baier, Ph.D.
Professor and Executive Director
Industry/University Cooperative Research Center for Biosurfaces

How Do Biofilms Form? | Bioadhesion and the Surface of Materials
PMMA Chemistry | Frequently Asked Questions | Reference

How Do Biofilms Form?

[Step 1]

Step 1

All fouling events in natural seawater begin with a spontaneous deposition of a "primer" coat of natural, high-polymer film. This process takes about one to three days and no further fouling by particulates, living or dead-occurs until the primer coat is in place. The film components are most often derived from the trace background amounts of decaying animal and vegetable matter-in essence, the "humic acid of the sea."

How well the primer coat adheres to the original material surface determines how well the thickening fouling layers adhere. This process can be controlled by adjusting the original surface properties of the engineering material of choice.

Figure 1. This photograph (2600x) shows a film on a test panel that has been deliberately scratched to illustrate its thinness.

[Step 2]

Step 2

The second step in the fouling of materials in the sea is selective binding to the primer film by short rod shaped bacteria. These are not usually the most abundant organisms present, or the fastest swimmers. Occasionally, some long-tailed organisms- known as prominent members of very nutrient poor communities- join in the first wave of bacterial colonization. In deep ocean waters, this colonization is noted after about three days of material exposure. In more concentrated, coastal waters, it can begin sooner.

Figure 2. A scanning electron microscope "shadowgraph" of the earliest attached organisms.

[Step 3]

Step 3

After about three days of exposure, the materials' surfaces are increasingly dominated by the long-tailed "prosthecate" microorganisms, reproducing, growing, and secreting cellulosic slimes.

Figure 3. This scanning electron "shadowgraph" illustrates the slime producing process. Note the gray areas around the bacteria.

[Step 4]

Step 4

As the extrusion of the slime patches continues around the microbes, there is further adsorption and spreading of the humic-like substances and sometimes the selective enrichment of metal-containing compounds over the entire surface zone.

Figure 4. The extrusion of the slime producing process around the microbes.

[Step 5]

Step 5

Apparently taking advantage of the secure anchorage and nutrient-rich organic film on the solid surface, the attached organisms respond to this "escape from starvation" by extending their tails and continuing to reproduce. Meanwhile, attachment of new organisms of divers character-rod-shaped, spherical, and filamentous-continues on top of the original conditioning coat. When anti-fouling methods are practiced-such as chlorination or the use of copper or tin-laden paints-the extension of the long membranous tails is usually suppressed. The organisms respond to poisons, however, by making much greater amounts of protective slime, which in turn rapidly thickens the fouling layer.

Figure 5. This "shadowgraph" illustrates the long membranous tails extended by the microbes in an effort to increase their nutrient uptake.

[Step 6]

Step 6

As the filamentous appendages of the early colonizers continue to grow, one also observes the attachment of algae spores, some diatoms, and inorganic (salt-like or mineral scale) particles. It is at this stage, usually a week or so after the first exposure to seawater, that attachment of larval forms of sessile organisms-barnacles, for example-is often noted. The engineering material surface has now become a slimy, fibrous mat that is quite effective in simply entrapping passing particulate debris. Optical, flow, and heat transfer properties of the original solid materials are seriously degraded after this time.

Figure 6. An increase in the filamentous appendages of the microbes is illustrated by this "shadowgraph". At this point, other organisms such as algae, diatoms and larval forms of invertebrates begin to attach.

[Step 7]

Step 7

In locations away from sunlight, where alga growth is limited, the film often becomes completely overlain with silt and other mineral deposits. When the original engineering materials are treated to display appropriately low energy (non-stick) surface properties, it is at this stage that sufficient mechanical "coupling" to the flowing water is present to peel away the fouling layers. Such re-entrainment can lead to meaningful lengthened operating times for the numerous-periscope windows, heat exchangers, and hull surfaces, for example.

Figure 7. Scanning electron micrograph illustrating the complex fouling layer after more than week of exposure to seawater. This layer is composed of entrapped and attached organisms in the process of of mineralization and binding.

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Relationship of Bioadhesion
and the Surface of Materials

The degree of biofouling on surfaces of engineered materials will vary depending upon the critical surface tension of the coating, the chemical make-up of the surface, and the biological material that is causing the fouling. Low critical surface tension coatings tend to discourage the initial onset of the biofouling process in comparison to higher critical surface tension coatings. Acrylic (plexiglass) discs, as utilized in the biofilms and biodiversity experiment, has a high critical surface tension which initiates biofouling rapidly. In fact, acrylic tends to be one of the highest energy surfaces while Teflon is a low energy surface. Conditioning films that form on surfaces tend to lower the energy-state of the surface and the rate of biofouling decreases proportionally. However, biofouling will continue to go through a succession pattern consisting of larger micro and macro invertebrate species until the space available has been completely "fouled."

The discs used in the biofilms and biodiversity lab are made from a polymer represented by the initials PMMA or polymethylmethacrylate. The common trade names for this material are Perspex, Lucite, and Plexiglas. Some other uses of PMMA are covers on automobile headlight, airplane canopies, and sneeze-guards over salad bars.

When dissolved, it is used for "bone cement" around artificial hips. In the solid state, it can be used to make dentures. All in all, it is a good surface for biofilm attachment, which are the ocean's equivalent of dental plaque!

The relationship between biofilm attachment strength and surface properties of man-made materials, as measured by surface energy or "critical surface tension", is not a straight line (it is a straight line for all synthetic materials sticking to each other, like fiberglass and resin in making ski poles and circuit boards).

A part of the reason comes from the fact that protein has become the "universal" glue substance in nature. Proteins (as in the operations of the body's immune system) are able to recognize many modern materials (Teflon, for example, was only accidentally invented in the late 1940's) as typical "foreign bodies" to be walled off with scar-like capsules as an attempted defense mechanism.

The surface that proteins, after some millions of years of co-evolution, have the least-aggressive interaction with is that made up from closely-packed methyl (CH3) groups like those at the ends of lipids in cell membranes.

A CST (critical surface tension) value, measured from the shapes of simple liquids in contact with the surfaces, between 20 and 30 milliNewtons per meter characterizes these essentially nonstick surfaces in biology terms, not including the 18 mN/m value for Teflon.

That is why real gourmet chefs do not use Teflon cookware (which really does allow some sticking). Instead they prefer well-cured iron skillets and woks, which become coated with a millionth of an inch of fatty molecule residues (butter, olive oil, etc), with their methyl groups pointing upward, giving the true easy-release or fouling-free surface for cooked foods.

Proteins are natural adhesives, developed over millions of years. Proteins will recognize and stick to all types of surfaces natural and man-made. This sticking is similar to the proteins of the immune system that attach to and deactivate foreign bodies within an individual. The molecular surface that proteins have the least attraction to are those made from closely packed methyl groups, CH3, which are similar to those found in cell membranes.

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PMMA Chemistry

[PMMA diagram]

CH3-O-C=O is the pendant methylacrylate group that sticks out from every alternate carbon in the polymer PMMA backbone, connected from the central carbon (shown at left). Although the exterior CH3 group can cause rapid association with similar hydrophobic side- chains of proteins, it is the -O-C=O (carboxyl) segment which interacts (albeit more slowly, because of the time it takes to displace the bound water from this vinegar-like segment) with similar (in Nature, "like likes like"..."birds of a feather flock together) side chains from nearby regions of the protein. These dipole-dipole (like magnets interacting) relationships get stronger as they slowly align to perfect matching----causing the protein to flatten and dehydrate in the process.

What is left is a thin, tight, glue line to which pioneering microbes and larvae and spores will attach substantially more strongly than to the juicy, hydrated, fluffy proteins attached only tentatively to the adjacent methyl groups. If you closely pack the methyl groups at the ends of long carbon chains from alternative carbons--masking access to the -O-C=O group-- you have the coating on the back side of Scotch tape. This nonstick coating is what turned Scotch tape into a real product, since they had the "stick-um" around for years but couldn't make a roll from it.

Try this for biofouling resistance: the backside of Scotch tape will be more resistant to retention (attachment is out of our control) of arriving biodeposits than almost anything you stick the tape to!

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Frequently Asked Questions

Surface Structure of the PMMA (acrylic) Discs

If methyl groups are the least "attractive" molecules to proteins, why is the surface structure of the polymethylmethacrylate (acrylic) discs attractive to proteins?

There are 2 methyl groups as the name suggests- methyl methacrylate- in PMMA. The methyl groups keep the material supple and non-brittle and allow the polymer chains to slide over one another on the surface. However, the acrylate pieces (vinegar-like structures) are still abundant and increase the surface reactivity, which significantly binds down proteins on the surface.

The Chemistry of Biofilms and Cooking

Would it be appropriate to say that chefs use a butter and oils (natural sources of methyl groups) as that millionth of an inch layer to keep proteins in food from sticking to the pan?

No. The chefs use the butter and oil to do the job with a quick approach that can be done "dry" with a properly "cured" skillet.

How does the curing of the iron or stainless steel skillet help the non-stick process?

The process of "curing" is the slow simmering in (with the help of a little water) of the breakdown products of the butter, oil, lard, tallow and other natural greases that are used in the cooking process. The fatty acids from the greases actually form covalent compounds, or soap-like chemicals, with the iron called iron sterate and iron palmitate as the millionth of an inch thick release layer. This release layer has closely packed methyl groups on the surface with no show through to the original metal. If never cleaned with anything more than a damp rag, the "cured" skillet will retain its non-stick properties almost forever.

How can methyl groups become closely packed to form this virtually non-stick layer?

The end group CH3's can become closely packed by actually attaching the 18-carbon long side chain (called octadecyl, or stearyl) to the oxygen atom of the acrylate, rather than just a short stubby little single methyl group. The long carbon chains, like sheafs of wheat in the field, are able to stand tall and support one another, even in the wind, so that the "heads" are all uppermost and aligned, blocking access to the sticky "soil" beneath. So, the Scotch tape backing is actually the octadecylester of polymethylacrylate. Stearates are the main side chains, by the way, of biological fats, and the word steatapygous means "fat butt". Look up callipygous for a more flattering definition!

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Baier, Robert E. 1984. Initial Events in Microbial Film Formation in Marine Biodeterioration: An Interdisciplinary Study, Proceedings of the Symposium on Marine Biodeterioration, pp. 57-62. Uniformed Services University of Health Sciences, 20-23 April 1981 (pdf).

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