“Biofilm Killer” is designed to meet several industrial needs, because although almost every industry has problems dealing with biofilm development, cleaning biofilms does not mean the same in the different industries.
These applications can be briefly categorized in three different needs :

1. The need for a clean, microorganism-free and chemical-free surface. The industrial fields requiring this level of protection are basically those dealing with Human and Animal Health issues, in which the presence of contaminating microorganisms, whether good or bad, prevent certification and/or use of the instruments. This may as well concern the food industry because of health and hygiene related issues as well, although the use of a probiotic bacterium as a host for Biofilm Killer may be acceptable in certain conditions or countries.
   
   
2. The need for a clean deleterious microorganism-free, with a protective bacterial barrier. Two main application domains are concerned. First, in farm animal breeding, protective biofilm barriers are already in use (ex. poultry), and has application in other animal system to prevent pathogen infection and excessive mortality. The second application domains is phytoprotection, e.g. biological control of plant pathogens in agriculture. Several microbial agents have been used successfully for year to control plant diseases. In many cases the biocontrol agent is used to replace the local flora and create a protective barrier against the pathogens to prevent infection.
   
   
3. The need for a clean, deleterious microorganism-free but biofilm colinization-protected surface. This may apply to certain areas in which long term protection would be useful, but the presence of the protective biofilm is inadequate, e.g. food-industry, cosmetics, and other soft-chemical industry. This also apply to refrigeration systems and air-conditioning units.

The beauty of Bioflm Killer resides in its versatile design that allows the user to choose the best combination of actions for his needs. The 3 modules of Biofilm Killer are independent, and can be used efficiently in any combination with the use of non toxic inducers in very limited quantities.
Basically with regards to the above classification of needs the following options are suggested.

• Health and food industry: Use of only Module 1 (Kill and Scatter) to facilitate the complete removal of the biofilm bacteria and accomplish a surface 100% free of bacteria after a regular cleaning procedure steps.
  
  
• Poultry and Animal raising, Oil industry : Use of Module 1 (Kill and Scatter) to facilitate the complete removal of the biofilm bacteria and induction of Module 3 (STICK) to establish a protective B. subtilis biofilm to create a barrier flora in the intestine of the animal or on the surface of tubing to achieve long term protection against pathogen recolonization or corrosion.
  
  
• Food industry, Soft chemical industry or the treatment of refrigeration units, air-condition modules : In other applications where the establishement of a bacterial biofilm cannot be envisionned, but long term protection against microbial recolonization needs to be achieved, the users can use Module 1 (Kill and Scatter) to remove the biofilm in combination with Module 2 (COAT) to generate a protective, peptide-based protective coating of the surface to protect.
  
  

To make Biofilm Killer compatible for most industrial applications, we further propose to introduce our genetic constructions in a NON-sporulant Bacillus subtilis strain to prevent the release and survival of this strain in the environment. ( See safety page)

affect flow of hydrocarbons, as can be seen on the figure on the right showing the results of an an experiment performed on gas fluxes in presence or absence of only 8% of biofilm coverage. As a consequence of biofilm formation, we can see that about 50% of the gas flux is lost (from Z. Augustinovic et al.).

In the above example of biofilm-induced pipe corrosion, while a significant part of the corrosion occurs on the outside of the pipe, it is estimated that more than half of the corrosion is due to microbial growth on the inner surface of pipelines. Internal and external metallic corrosion contributes significantly to the risk of oil and gas pipeline deterioration and failure (see below), causes well and reservoir souring and plugging, and results in billions of dollars in annual costs to the oil and gas industry.

Our Biofilm Killer construction in Bacillus subtilis 168 is designed to help address both the ease of delivery of the cleaner as well as induce a long term protection on the inner surface of the tubing. Biofilm Killer can be applied following the procedure which is already accepted in the industrial processes where Clean In Place procedures are performed. In this protocole, the process features 3 tanks usually filled with sodium carbonate, nitric acid and sodium hypochlorite. These three chemicals are used in sequence to remove the biofilm by inducing an alkaline and acidic treatment to destabilize the biofilm and a sterilizing treatment with hypochlorite. Refinements to the CIP procedure include the use of the use of specific enzymes targeting the biofilm, e.g. dispersin (Realco).

In order to specifically remove biofilms and to lower amount of toxic chemicals used, we propose to fill one of the CIP bulk with “Biofilm Killer”. “Biofilm Killer” will be targeted to the place to clean by the flow of water delivered in the pipe. It will swim inside the biofilm and produce and deliver in situ inside the biofilm both the biocide and the scattering agent. “Biofilm Killer” will have an action on both the target strain to kill it and on the exopolysaccharide matrix of the biofilm to dissolve it. Our physiological tests show that after 1 hour there are already significant effects of lysostaphine and dispersine. The impressive effect of the combined action of the two proteins on a staphylococcal biofilm is shown HERE. To maximise the dispersing effect of the construction, we recommend a 5 hour duration of “Biofilm Killer”. The scattered and killed biofilm will then be eliminated by subsequent acid, caustic and sanitizing treatment as in classical CIP, if no recolonization is required. A significant decrease in the needed amount of these chemical is expected. Alternatively, “Biofilm Killer” will be induced to colonize the surface or produce surfactin in order to form a long-term protection against deleterious recolonization of the surface.
Preparation and storage of Biofilm Killer is not an issue since the very same organism is already produced and stored for poultry breeding or crop plant phytoprotection. In our case, it will be most convenient to prepare Biofilm Killer as a lyophilisate since it can be stored for months until use.

The oil patch is a worldwide industry and moreover a multi-billion dollars activity. It has ramifications worldwide. One can find at least one step of the production or the distribution line in every country. From the exploration to the distribution or the transformation in different products and to the consumer, this system is quite linear and can be schematized as follows :

This work flow can be divided in two types of activities :

• The upstream operations concerns the production sensu largo of the resource itself. It includes all the operations needed for the exploration, the drilling, and the extraction necessary for the crude oil and the natural gas production.
  
• The downstream operations deal with the transport and transformation of the product itself. It includes transporting the oil or gas by pipes or Tankers, the refining, the retailing and the consuming, until it finally reaches the consumer, which can be the car owner at the gas station or a company using petroleum-derived products.

The gas and oil patch is the biggest industry in the world. The daily production of oil is ca. 13 billion liters. It is dominated by 5 or 6, extremely big companies called “Supermajors” which rule the system and have control over it: among them are BP, Total or Shell. Despite their gigantic size, these companies control only ca. 5% of the oil reserves worldwide. The large majority of the reserve is controlled by local and national companies such as the Saudi Aramco, the iranian National Iranian Oil Company or the koweti Kuwait Petroleum Corporation.

Oil or gas production involves millions of kilometers of tubing at the production site or for transportation, tanks and tankers for storage and oversea transportation. Moreover, oil extraction often involves the use of large amounts of water, which create a very favorable environments for microbes to grow in. This oil and water mixture travels thourgh the pipe until it is finally separated at the surface. Thus, oil propduction represent a vast numbers of tanks and kilometers of pipes in which micrbial biofilms are likely to grow and induce corrosion, and which need to be cleaned!