Once a biofilm has been initialized, that is, once bacteria have attached and begun to produce EPS, the bacteria begin to multiply. At this stage the biofilm begins to grow and develop. It becomes convenient to describe the development at a larger scale. The description of this part of the life-cycle is very different depending on the bacterial type, the location of the biofilm, and other external environmental parameters. Typically, the biofilm begins to become heterogeneous, and form complex structures such as towers, clusters and plumes [19]. Often there are large scale detachment events, called sloughing, where a large piece of the biofilm detaches and reenters the bulk fluid [19]. These chunks will sometimes re-attach further downstream or break up and revert to individual planktonic bacteria. The biofilm will typically grow to a quasi-steady state. That is, the thickness of the biofilm will tend toward a constant average thickness generally less than 300 microns[19].
Many of the fundamental questions about biofilms are related to the properties of the structure of biofilms. For example, it is not well understood why biofilms are heterogeneous, and to what extent the environment causes the heterogeneity. The mechanisms that govern detachment and sloughing events are also not well understood. Below is a discussion of several other research areas.
There are several important biological properties that have been alluded to above which are of interest to researchers. One area of research is in the relationship between the structure and function of a biofilm. The complexity of the physical space occupied by the biofilm has been demonstrated experimentally. However, less is known about the reason for the complexity. There are essentially two views that have been taken. The first explains the heterogeneities as resulting from environmental factors. Thus, the reason a biofilm has towers because forces exerted by the fluid flow has caused detachment events on either side of the remaining tower. This explanation is not satisfactory in general because biofilms in drastically different environments have many levels of heterogeneities.
Another explanation of the structures holds that a biofilm is healthier due to these structures. Clearly if there are water channels throughout the biofilm, nutrients are more able to penetrate the biofilm. Towers and clusters also have the effect of reducing the likely-hood that all of the substrate will be used by the neighboring bacteria. In a sense this explanation claims that the structures are there in order to keep a biofilm well fed and reproducing. This idea has led to further investigation of dispersion events and EPS production. One of the outcomes of this investigation is a description of the phenomena of quorum sensing.
In general quorum sensing refers to the process of regulating the expression of certain genes in the bacteria within a biofilm. This regulation is governed by products released by the bacteria. So the process of auto-induction depends strongly on the immediate environment including the local density of bacteria. In general, chemicals which are produced by planktonic bacteria are able to diffuse rapidly away. When biofilm forming bacteria have sufficient space between them many of the chemicals can also diffuse away. However, when the density of bacteria grows, due to rapid growth, for example, the chemicals cannot diffuse away. There is a build up of chemical within the biofilm, but in the exterior of the cell walls of the bacteria. Since the production of the chemical by the bacteria is done by diffusion of the product through the cell walls, if the concentration of the product is higher on the exterior of the cell walls, than on the interior, the chemical can build up within the bacteria, causing different genes to be expressed. It has been demonstrated that bacteria undergo a phenotypic change from a planktonic bacteria to a biofilm forming bacteria under certain circumstances. It has also been shown [5] that the bacteria produce chemicals, which, when a threshold is reached causes the biofilm to produce a substances that breaks the EPS strands that hold the biofilm in place. This can cause large dispersement events that are different from sloughing events, in that the bacteria become motile and reenter the bulk fluid under their own power, rather than being passively swept downstream. It has been hypothesized that there is another auto-inductive signal that causes the bacteria to begin increasing the production of EPS. This complex behavior has many impacts on both the theoretical and experimental knowledge of biofilms. It indicates an important area of biofilm control since it is the EPS which locks the bacteria together and to the substratum.
Another fundamental property of biofilms is the property of resistance that is unique to sessile (biofilm) bacteria, rather than planktonic bacteria. It has been demonstrated [24] that the bacteria in a biofilm are remarkably hard to kill. A typical bacteria that is studied is Pseudomonas aeriginosa. This bacteria is often found in infections found in hospitals. The planktonic type are susceptible to anti-microbial agents such as chlorine. However, if one subjects a biofilm made up of Pseudomonas aeriginosa bacteria to a concentration of an anti-microbial agent that would be sufficient to kill a population of the same number of planktonic bacteria, one finds that the biofilm bacteria are essentially unharmed. Further, the bacteria recover very quickly from exposure to anti-microbial agents [24]. Here anti-microbial agents can range from antibiotics to chlorine. Therefore it is not possible to alleviate a biofilm infiltration by large quantities of chemicals. In fact, in water treatment plants most of the removal of impurities is done physically, by filtering, because it is one of the most effective way to remove biofilm impurities. Much of biofilm research being done concerns this resistance. See [24] and references therein.