“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, 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)

Oil industry faces with important and costly difficulties due to biofilm-related issues. These include, but are not limited to, pipeline and metallic structure corrosion, porosity clogging in the rock or the tubing, microbial souring, petroleum spoilage during storage. The main consequences of microbial at the global scale are several :
A reduced quality of the final product (microbial alteration), an increase in treatment costs (souring), the reduction of well production (souring, microbial alteration, clogging). Biofilm formation is involved in the corrosion of the metallic structures, including the oil-plateform, but most importantly the pipelines and tubing.

Biofilm-induced alteration will affect the structure, resulting in two effects :
The first one is the clogging of the tubing, which much like cholesterol deposits in our arteries, will reduce the available circulating space, reducing the flux of gas or oil that can be transported through the pipe. Even small amounts of biofilm can negatively

The second is the anaerobic corrosion of the metal from the structure, which will be instrumental in establishing the biofilm and induce clogging, but will also fragilize the structures.

Impact of biofilms and microbiologically influenced corrosion in oilfield. Siri plateform (center) is located in the North sea and flanked by the smaller Cecilie and Nini satellite plateforms. Seafloor lines between the 3 structures and wells carry oil and gas (gas for lift and injection water for pressure support). INSET: in 2007, water injection line ruptured. Subsequent investigation revealed a mixture of iron sulfide and other corrosion by-products plus microbes and polysaccharide slime at the rupture site. These deposits allow sulfate-reducing procaryotes and other troublesome microbes to grow protected from biocides. (Augustinovic et al., Microbes- Oilfield Enemies or Allies? Oilfield Review, Summer 2012, Schlumberger)

Most importantly, failure on the production line can lead to dramatic oil or gas spills that lead to unprecedented environmental risks and damages. Such failure has recently happened on the Elgin Field, located in the North Sea ca. 240 kilometers off Aberdeen (Scotland), on a gas field exploited by the french potroleum company Total. The failure of the production line lead to a gas spill estimated to ca. 1.8 million euro loss per day for the company. Due to the location of the failure at the sea floor, it took several month for the leak to be stopped. In addition to the cost to the company, a maritime exclusion zone had to be created which perturbated maritime traffic, and ca. 20 tons of gas were released in the atmosphere daily. The previous year, in the same region an oleoduc exploited by the Royal Dutch Shell had ruptured also leading to oil spill in the North sea.

The problems with oil or gas production pipe is two folds : they are expected to be in place for decades and they often are difficult to access. Thus cleaning and locating biofilm is no simple task. Biofilms can be in dead zone which make them impossible to clean with mechanical process.

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 exemplified 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 necessary for the exploration, the drilling, and the extraction necessary for the production of the crude oil and natural gas.
  
• The downstream operations deal with the transport and transformation of the product itself takes. 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. 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 or the koweti Kuwait Petroleum Corporation.

The daily production of oil is ca. 13 billion liters. The Oil patch involves long lines of production and a lot of transportation, from the extraction site of oils and gas to their distribution: that is a lot of tanks and pipes to keep clean! Every one of those materials is likely to be contaminated by bacteria and to encounter microbial-induced deterioration.


This logo means that our solution “Biofilm Killer” can be used in this part of the oil process toremove bacteria and clean pipes or tanks and save the company from much bigger issues such as a hole in a pipe letting gas or oil flow out in the deep of an ocean, meaning loss of millions dollars.