When bacteria of one or more species initially come into contact with a surface, they bind to it using extracellular structures such as fimbriae and flagella [1*]. If they enter a sessile (stationary) phase, gene expression is modified and induces the production of extracellular polymeric substances or EPS [2]. The EPS consists mostly of polysaccharides, proteins, and nucleic acids [3]. These substances form a structural matrix, a biological scaffolding that the bacteria use to attach to surfaces. This structure and the bacteria within it constitute what is known as a biofilm.

Biofilms can form anywhere as long as nutrients and a surface for adhesion are present. This includes industrial systems used in wastewater management, food processing, brewing, pulp and paper manufacturing, and dairies. These durable colonies of Bacteria impose an economic burden on companies as they are often responsible for contamination by biofouling- the accumulation of microorganisms in aqueous environments [4*]. Within food industries, concern is even higher as biofilms make treating current contaminations and preventing future ones more difficult. Thus control of biofilms is a crucial requirement for companies to maintain product quality, safety, and efficiency.

Biofilms form in nutrient-poor environments like ultra-pure water systems and are capable of attaching to surfaces commonly found in industrial settings, such as stainless steel [5,6 **]. As a result, prevention of biofilm formation is difficult. Some methods include strict hygiene regimens, using resistant materials, and antibacterial coatings like paints which fight colonization [2]. Therefore, focus has remained on treating or clearing them from systems. Corrosive cleaning agents and disinfectants such as chlorine, chlorine dioxide, or nitric acid or caustic sodas are commonly used. However, these are only effective against planktonic bacteria. Manual scrubbing is required to remove biofilms, and results in damage to equipment over time through corrosion [1*,7].

Microbial enzymes have proven to be safer and more efficient alternatives to traditional chemical means of removing biofilms. Bacterial proteases and amylases can remove biofilms completely, instead of killing only the planktonic organisms [4*]. This solves the major problem of biofilm materials that remain after the bacteria have been killed, which makes recontamination more likely. Yet these methods still focus predominantly on clearing existing biofilms, instead of preventing them. A question posed here is whether or not one can use bacteria that produce and secrete enzymes that can break down biofilms as a defense against harmful biofilm formation.

The structure of the biofilm poses a problem surrounding the prevention of formation and the clearing of existing biofilms. The increased ability to adhere to the surface by new bacteria is not the only advantage of biofilms. It also provides a protective barrier for the bacteria contained within it by providing not only a physical barrier from stresses like fluid flow, but also a chemical barrier. Antimicrobials have a limited effect on bacteria in a biofilm for several reasons centered around its structure and the bacteria themselves. Prakash explains that the EPS is a negatively charged resin which binds to larger antibiotic molecules [8]. Antimicrobials and other chemicals get stuck in the outer regions of the biofilm and are effectively inactivated before they reach the bacteria embedded within. Also, bacteria within a biofilm are typically in a slow/no growth phase, which blocks the inhibitory properties of an antimicrobial [8]. As stated before, a biofilm is a matrix and not a solid mass. It has channels allowing the flow of water which not only brings in nutrients and oxygen to the interior of the matrix, but removes wastes products [9]. These characteristics make controlling biofilms a costly process for many industries. Within the United States alone, biofilms account for roughly $138 billion dollars of damage due to corrosion, product quality, and maintenance including cleaning and replacement of parts [10].

The most effective method for removing a biofilm and reported by Jessen is by the Clean-In-Place (CIP) method in combination with chemicals [11]. This involves manual scrubbing of the affected area. This is expensive in both unwanted downtime of the system and the time of those cleaning the system. It is also impractical for larger machine or plumbing systems where the more afflicted regions like joints, filters, or gaskets are not easily accessible [12].

Enzymes, specifically the group of hydrolases, are an attractive option because they are readily available, as some are already produced at an industrial scale, biodegradable, and generally have a lower toxicity than chemical and enzyme alternatives such as oxidases [3]. Proteases alone account for up to 65% of total enzyme production in the world market overall [13]. The reason for their attractiveness is that they are more than antimicrobials. For many biofilms, proteins are the largest percentage of the total material [1*]. Proteases are able to cleave these proteins at specific amide bonds, effectively destroying the physical structure of a biofilm. Instead of only killing the majority of planktonic bacteria and some of the sessile bacteria, proteases break up the biofilm and allow its removal from the system altogether [14].

An effective and available group of proteases on the market are subtilisins. Subtilisins are serine proteases and cleave proteins in which serine serves as the nucleophilic amino acid [15]. These enzymes are produced and secreted in large quantities by many Bacillus species making them readily available. One approach is to over express aprE, the gene responsible for subtilisin production in Bacillus subtilis [16]. Subtilisins have also been shown to work on a number of different biofilms including those produced by industrially significant problematic species such as Pseudomonas fluorescens, Pseudoalteromonas sp. D41 and Bacillus cereus [1*,3,4*]. Protease treatments have also been shown to prevent biofilm formation of Listeria monocytogenes by removing surface proteins essential for adhesion [17]. While some commercial subtilisins are more effective than others, many non-proprietary subtilisins can still be used effectively in clearing and preventing biofilm formations [18].

Proteases may be the most commonly used of the hydrolases, but others are useful as well. Amylases, when used in conjunction with proteases, help to eliminate existing biofilms and prevent bacteria from adhering to surfaces [18,19]. A mixture of proteases and amylases is used because of the diverse composition of biofilms. Whereas proteases digest the proteins, amylases cleave the carbohydrates associated with the structure. While all amylases have the ability to cleave 1,4-glycosidic bonds, α-Amylases have been found to be more effective than β- and γ-amylases [20*]. α-Amylases are widely used because of their high thermostability, but do not remain active very long because they are calcium metalloenzymes. The activity and structural stability of α-amylases are based on their dependency of calcium ion binding, but calcium may not always be present. Genetic engineering techniques such as site-directed mutagenesis have been used to solve this problem. Gholassi designed the H58I mutation in B. megaterium to create a more thermostable and calcium independent α-amylase [21*].

An alternative to the subtilisins discussed above are subtilisin-like proteases, or SLPs. Like their namesake, these enzymes target serine amino acids in proteins. An advantage that SLPs have is that many are produced by hyperthermophiles. Given the extreme conditions of industrial environments like that of heat exchangers used in wastewater treatment, proteases that work optimally in such conditions are desired. For example, while the maximum temperature for subtilisin 309 is 75 ºC, the SLP, pernisine, from Aeropyrum pernix is produced and functions optimally at 90 ºC [1*,22].

Biofilms are difficult to prevent. Even with surfactants coating stainless steel, Pseudomonas aeruginosa is still able to form a biofilm 10% of the time [23]. In a food industry or medical setting, this remaining 10% would still leave significant chance for contamination, resulting in a public health risk for infection. To combat the last remaining percentage, microbial competition could be useful. Modifying harmless species of microorganisms could provide a constant preventative measure against formation of detrimental biofilms much like bacteria in other systems such as the human gut [24]. Most industrial systems contain bacteria that are both nonpathogenic and are far less harmful to product quality. All of the enzymes discussed in this article could be produced and secreted by microorganisms fitting this description.

Implementation of such an approach may be difficult. There are three ways that may prove to overcome some of the problems. First, and simplest, would be the addition of the beneficial species of bacteria to the system while it is running, with the fluid flowing. This of course poses a problem concerning the ability of the microbes to attach to a surface at the desired location. If a significant number of cells do not attach early enough after application, then the process will likely be unsuccessful. An alternative method would be applying the desired microorganisms before initial system start up. This could eliminate the threat of the beneficial species being washed away and allow for more precise placement at sites likely to become fouled by biofilm producers. This does, however, raise an issue of viability. The organisms would have to be able to live both in pre- and post-start up conditions which may include changes in available oxygen and nutrients, eliminating obligate aerobes and anaerobes. Last is the possibility of including a bioreactor which is connected to the system that could produce a supply of the desired organisms on site and deliver them into the system directly. This approach would remove the concern of viability could allow for a constant presence of the beneficial microorganisms in sufficient number at the precise location where fouling is likely to occur. Without further research and available data, however, predicting the real world practicality of applying these methods is limited.

Species of Bacillus are of the greatest interest in industry in regards to practical applications. The reason for this is their ability to produce both the subtilisins, and α-amylases [25]. Some species, such as B. amyloliquefaciens, are able to produce both. Also, related organisms like Brevibacillus brevis can prevent corrosion by sulfide-reducing bacteria, limiting the surface area available for further biofilm formation [26]. The enzyme production of Bacillus is attractive, as is their diversity. Since the variety of organisms to choose from is large, one could presume that finding a microbe that fits the environment of the system would be reasonable. B. amyloliquefaciens, for example is an aerobic species with optimal growth temperatures between 30 and 40 ºC while B. licheniformis is anaerobic and grows at 50 ºC while surviving at even higher temperatures [27,28]. They are great candidates for use as a result of the extensive knowledge about Bacillus species and the favorable characteristics they share. There are limitations to the application of these bacteria to systems that may include those in the food industry. There have been documented cases of allergic reactions to both subtilisins and α-amylases, however these instances are rare [29,30]. While the patients appeared to only respond to some, but not all, subtilisins and amylases, this possibility should still be seriously considered.

Where conditions are particularly hostile, extremophilic Archaea may be a more viable option. As noted above, thermostability is one area where these excel, producing and secreting subtilisin-like enzymes which function at temperatures far above others. Aeropyrum pernix is capable of functioning at near boiling temperatures, but there are even more thermophilic microbes. Pyrobaculum aerophilum produces another SLP called aerolysin which is active at temperatures up to 130 ºC [31]. However, extremophiles are also found to survive in halophilic, alkaline, and acidic environments increasing their potential as possible biofilm deterrents. Adding to the desirable traits for these microorganisms, there is only one known Archaea to date that may be a human pathogen, though research has not confirmed this [32]. These characteristics make Archaea plausible options to introduce into a system that may be generally tolerable, but contains more sections that represent extreme environments. This would increase the number of areas in which the beneficial organisms could take hold.

Archaea do fall short in one area where the bacteria excel. Cell engineering is difficult among extremophiles, especially among hyperthermophiles [33]. This restricts the ability to not only engineer extremophiles to over express the desired enzymes, but also limits the potential of transferring the genes necessary for production into conventional heterologous hosts. This may be useful if the upstream environment is more suited to a mesophile, but the enzyme must pass an area with more extreme conditions later on. This does not mean that it is impossible, as pernisine has successfully been expressed in Escherichia coli [22].

Many industries including pulp and paper manufacturing and water treatment are still clearing biofilms as they did ten or twenty years ago with CIP methods. Caustic chemicals are still being used because of cost considerations, but the future of biofilm control could lie in preventative measures using microbes to produce and deliver antifouling enzymes. They are not only more efficient at removing biofilms, they provide less threat to the users as well as the products. If these enzymes can be produced and distributed using harmless microorganisms already within the system, running such systems with a more-or-less “hands off” approach to maintenance would be possible. Allowing the microbes to break apart harmful biofilms would reduce equipment downtime costs on solvents and detergents, and time spent by employees cleaning. With the efficiency of existing enzymes, it is clear that interest will not dwindle unless the costs are unfavorable. There is ample room for improvement and incorporation of new enzymes into antibiofouling strategies is likely to continue in the future. Also, Archaea species, while already utilized for enzyme production of pernisine, have a potential that cannot fully be exploited until sufficient cell engineering methods have been developed. Successful methods must also be developed to genetically engineer these species in order to maximize their potential.

I would like to take this opportunity to thank Dr. Garrity and Dr. Waldron of Michigan State University for their editorial guidance in producing this manuscript as well as the assistance of Anna Peters and Alex Hoekstra.