One of the old adages of cleaning is that equipment should look clean, smell clean and feel clean. What this means is that plant sanitarians should use their senses as part of monitoring the efficacy of a cleanup operation. It is fairly easy to see if a piece of equipment has been cleaned. If one walks into a plant and sees residual product all over lines or there is polymer buildup on a fryer, it should be fairly obvious that the operation is not paying sufficient attention to their cleanup operations.

Your nose can also be a tool for evaluating efficacy of cleaning. If a during a walkthrough, you pick up butyric odors in a tomato plant, the smell of fermentation in a fruit operation or a distinct putrid note emanating from a drain, it is again obvious that the cleaning program needs attention. The sense of touch may also be used. If you run your hand over a piece of equipment and it feels “slimy,” you have a problem. Specifically, the slime may indicate that you have a biofilm problem.

So what are biofilms? They are layers of bacteria that attach to surfaces and to one another with the help of polymeric materials, which trap other bacteria, debris and nutrients (Figure 1). As these build up, a microbial film, or biofilm, becomes established. Biofilms were first identified in the mid-1970s.[1] Biofilms may be beneficial or detrimental, depending upon where they are found. In the production of some fermented foods, biofilms are an essential element for optimum production. During the production of vinegar, acetic acid bacteria are allowed to grow on wood chips. The biofilm that is formed helps make the conversion of substrate to acid more efficient. The greatest concern is that biofilms may contribute to the production of contaminated products—products that are subject to economic spoilage or may contain microorganisms of public health significance.

Other problems that biofilms may create are lowered heat transfer due to their buildup, promotion of corrosion, fouling of probes and plugging of filters and strainers. Thus, they not only pose a potential health problem, they can adversely affect operating efficiencies, which can cost the food processing operation money.

The bottom line is that biofilms provide bacteria with a competitive advantage. They provide protection to the organisms, they provide the bacteria with a source of food and nutrients, they allow the organisms within the films to act synergistically and they provide an area for reproduction. By understanding how biofilms are formed, food companies will be able to develop better sanitation procedures and control measures to more effectively prevent their formation in the first place.

Formation of Biofilms
Biofilm formation is not something that occurs “suddenly.” When one applies a paint or varnish to a surface, the material coats the surface leaving behind a film of coating. If a piece of metal is dipped in oil, a film of oil remains on the surface. Biofilms need to form. As they form and develop, it becomes harder and harder to remove them. It is for this reason that sanitation or hygiene programs need to be carried out on a regular basis.

The first step in biofilm formation involves the attachment of the organisms to the surface. This initial attachment is very weak, involving what is known as van der Waals forces. At this point in the life of a biofilm, removal is fairly easy. The infant “film” consists of organic film and the weakly bound bacteria. There are many different bacteria that can form biofilms. These include non-pathogenic bacteria, such as Psuedomonas fragi, Enterococcus spp. and Pseudomonas flourescens, and microorganisms of public health significance, such as Salmonella and Listeria monocytogenes. The fact that pathogens can form biofilms makes them a real concern to the food processing industry. During the next phase, organisms attach themselves to the surface by means of tendrils or filaments, and the formation of polysaccharide-like materials that act as glue to “cement” the cells to the surface and to one another. This polysaccharide or polymer will also serve to trap other cells and debris. Within 24 hours, the organisms making up the film are firmly entrenched.

In an environment in which there are nutrients being continuously supplied to the film, the biofilm develops at a steady rate up to a point where the film is continuous and stable (Figure 2).

Once the surface has been colonized, the film is irreversible and special protocols are required to fully clean and sanitize the surface (Figure 3).

A mature biofilm is a system that reaches an equilibrium of sorts. The flowing product delivers nutrients, oxygen and other elements necessary for growth, and carries away fermentation products and cells or debris that have been sloughed from the surface. Once equilibrium is reached, the film reaches a certain thickness and remains as such.

There are researchers who have broken biofilm formation into five distinct phases:

• Transport of nutrients, inorganic and organic material to the surface

• Adsorption of a conditioning film containing inorganic and organic nutrients

• Attachment of microbial cells to the wetted surface and growth initiation

• Bacterial metabolism within the film

• Cell disruption and detachment from the biofilm

Biofilms may form on all kinds of surfaces, from stainless steel to plastic (Figure 4). Researchers have shown that films will even form on materials such as copper, rubber and lead; therefore, processors can reasonably assume that films can form anywhere in the plant. Biofilms can form on surfaces within a range of processing operations. A great deal of research has focused on what occurs in the meat and poultry industries. Why? Perhaps one reason is that meat and dairy products provide a rich substrate for growth, and that there are many pathogenic organisms associated with these products. Biofilms are also a concern with chilled foods, juice processing operations and water systems.

Figure 4. Biofilms form on many types of surfaces, from stainless steel to plastic.

Biofilm Prevention and Removal Strategies
Good prevention of biofilm formation is a function of implementing several measures. Obviously, the first step in good prevention is the development, implementation and adherence to a good cleaning and sanitizing program. Studies have shown that organisms in flow can begin to attach to a surface within 30 minutes, and that within eight hours, a stable film can begin to develop. As stated in previous Food Safety Magazine articles on cleaning and sanitation protocols, the most important part of your sanitation program is cleaning because that cleaning prepares the surface for sanitizing. If the surface is not clean, it cannot be properly sanitized. In other words, if the surface has not been properly cleaned, it is dirty, which will provide an excellent enviornment on which biofilms can more easily form. The application of sanitizers to dirty surfaces is both ineffective and a waste of money since the efficacy of that sanitizer will be reduced by the presence of residual soil. The best bet is to match proper sanitation programs and measures to the types of equipment and soils typically found in your specific type of processing operation and make sure that the sanitation program requires the appropriate use of both mechanical and chemical removal treatments.

The use of well-designed and properly installed processing equipment is another good biofilm prevention measure. Poorly designed or installed systems can be difficult to clean for a number of reasons. For example, parts of the line can be hard to reach; hence, workers often do not take the time or expend the necessary effort to clean the hard-to-reach parts. Installation of oversized pipes or closed lines may be good for moving product, but may not be easily cleanable. For example, if an operation is using a 12-inch-diameter pipe to convey product from one place to another. If the pumps cannot develop enough pressure to completely fill the line, cleaning will be very difficult. Maintenance of equipment also can affect biofilm formation. Damaged lines or steel that is badly abraded or scratched create surfaces where organisms can more easily attach. Instituting best practices based on principles of sanitary equipment and facility design is one of the most effective ways to prevent biofilms from taking hold on equipment surfaces.

Of course, the greatest issue where biofilms are concerned is that they are very difficult to eliminate once they have been established. One of the tools that researchers have used to evaluate biofilm formation and control is the insertion steel chips in food processing equipment. The chips are exposed to the food and the materials applied during cleaning and sanitizing. The Food Product Association (formerly National Food Processors Association) is one of the organizations that has conducted research using this method. In one study, FPA researchers conducted a two-year project in which stainless steel chips were placed in food processing equipment for up to seven weeks. They found that not only were biofilms formed, but they formed in spite of their exposure to plant cleaners and sanitizers.

Further, biofilms act almost like insulators, which adds to the difficulty encountered in their removal from surfaces. They not only protect the organisms within the matrix from cleaners and sanitizers, but organisms within the film are also more heat resistant. As these films get older, they become more resistant to cleaners and sanitizers. [2,3] It has been reported by LeChevallier et al that attached cells may be 150-3,000 times more resistant to hypochlorous acid than unattached cells.[4] Frank and Koffi have found that attached colonies of Listeria monocytogenes survived exposure to benzalkonium chloride for 12-20 minutes, whereas free cells were destroyed within 30 seconds.[5] In another study, researchers determined that biofilm removal was dependent upon the type of culture, the strain of the organism forming the film and the length of cleaning. In other research, Krysinski et al evaluated the effects of a variety of cleaning and sanitizing compounds.[6] They found that it was essential that cleaners and sanitizers be used in combination for more effective removal of biofilms. This helped affirm the work of Stone and Zottola, who recommended that proper use of cleaners and sanitizers was necessary to minimize biofilm formation in milk pipes.[7]

If a biofilm problem does develop, a cleaning program that is focused on biofilm removal needs to be developed and implemented. Remember, the biofilm consists of soil and other materials on the surface and a matrix of organisms, polymers and soil below that surface. The program measures need to both remove the surface soil and the sub-surface film. The application of a detergent followed by a rinse will remove the surface soil, which exposes the subsurface. The theory is that the exposed surface is now more susceptible to the action of the sanitizer. In reality, the cells are still attached to the surface and still much more resistant than unattached cells.

According to Kramer, proper removal of biofilms requires the use of a material that can penetrate and solubilize the polymers making up the biofilms.[8] This observation is supported by the Humm’s work.[9] This study found that exposing biofilms to chlorine at levels of 25, 50 and 200 parts per million (ppm) and iodine at 25 ppm resulted in survival for contact times as long as five minutes. The study also stated that the compound that performed best was a hydrogen peroxide/peroxyacetic acid-based compound, which apparently has the ability to penetrate the film, facilitating removal of the film.

If biofilms are a concern in your operation, work with your chemical supplier to select and evaluate a system to eliminate the problem. To properly evaluate the anti-biofilm system you have elected to test, a seven-step program should be used as follows:

1. Identify an area to test the system.

2. Select what variable will be tested to evaluate the efficacy of the system. Examples of the types of variables to select include high counts, high or

3. Determine the sample size. Identify how large an area will be evaluated and how much product will be required.

4. Establish a control mechanism. Will measurements be taken before and after, or will side-by-side studies be conducted?

5. Determine how length of the study.

6. Establish microbiological sampling programs.

7. Evaluate the results.

Reducing the Problem
The problem of biofilms is not going to go away. In fact, with the food industry moving towards longer processing runs with minimal cleanups between runs, the problem may only get worse. Biofilms can form on any surface that is exposed to non-sterile water or other liquids. This includes process lines, pipes and even surfaces on refrigerators or the refrigeration units themselves. The biofilm acts as a shield and will protect the organisms within that film, rendering them more resistant to heat and sanitizers. If the biofilm contains pathogenic organisms, such as Salmonella or Listeria monocytogenes, the film can seed the food flowing by it with the pathogens.

Further, since organisms within the biofilm (attached to the surface) are more resistant than those that are unattached, biofilm formation can pose a significant threat to food safety. Sanitary equipment design and maintenance, development of and adherence to proper cleaning and sanitizing methods and programs and intelligent production scheduling all will help minimize the potential for biofilm formation, and any problems they may cause.

Richard F. Stier is a consulting food scientist with international experience in food safety (HACCP), food plant sanitation, quality systems, process optimization, GMP compliance and food microbiology. He has worked with a wide range of processing systems and products, including canning, freezing, dehydration, deep-fat frying, aseptic systems, and seafood processing. At every position he has worked, he has shown a unique ability to work with companies at all levels, from top management, including marketing, to line workers, to understand, develop and implement systems to enhance these companies’ operations. Previously, Stier served as Director of Quality Assurance for Dole Packaged Foods North American operations. He can be reached at rickstier4@aol.com.

References
1. Marriott, N.G. Principles of Food Sanitation, 3rd Edition. Chapman and Hall, New York and London. 1995.
2. Wirtanen, G. and T. Mttila-Sandholm. Effect of growth phase of foodborne pathogens on their resistance to a chlorine sanitizer, Part II. Lebensm.-Wiss. U Technol., 25:50-54. 1992.
3. Mustapha, A. and M.B. Liewen. Destruction of Listeria monocytogenes by sodium hypochlorite and quaternary ammonium sanitizers. J. Food Protection, 52:306-311. 1989.
4. LeChevallier, W.M., C.D. Cawthon and R.G. Lee. Inactivation of biofilm bacteria. Appl. Environ. Micro., 54:2492-2499. 1988.
5. Frank, J.F. and R.A. Koffi. Surface adherent growth of Listeria monocytogenes is associated with resistance to heat and sanitizers. J. Food Protection, 53:550-554. 1990.
6. Krysinski, E.P., L.J. Brown and T.J. Marchisello. Effects of cleaners and sanitizers on Listeria monocytogenes attached to product contact surfaces. J. Food Protection, 55:246-251. 1992.
7. Stone, L.S. and E.A. Zottola. Effect of cleaning and sanitizing on the attachment of Pseudomonas fragi to stainless steel. J. Food Science, 50:957-960. 1985.
8. Kramer, D.N. Myths: Cleaning, sanitation and disinfection. Dairy, Food and Envtl. Sanitation, 12, 507-509. 1992.
9. Humm, B. A research update on the effects of cleaners and sanitizers on food processing biofilms. Food Protection Report, 8:2, 5. 1992.