Q. How do you remove biofilm? (The featured image is sourced from here.)
A. In order to “kill off” any microbial residue, a cleaning and removal of inanimate and organic matter is a must. This would follow with the disinfecting, “kill” step if you will, with an EPA registered disinfectant.
Low foaming (CIP) applications: A reliable way to prepare stainless steel process piping and tank surfaces for the killing of thermophilic bacilli and similar biofilm is to use a two-step process entailing an aqueous alkaline cleaner, such as our Solujet® Low-Foaming Phosphate-Free Liquid, followed by an aqueous acidic cleaner, such as Citrajet® Low-Foam Liquid Acid Cleaner/Rinse. Note that, as compared to solvent use, aqueous detergent use also avoids flammability and disposal concerns associated with solvents and other hazardous biofilm cleaners.
For heavy biofilm applications, we see the best results when the first 3% Solujet detergent (alkaline cleaner) at 75C is applied for 30 minutes and is followed by a cursory rinse. The second step of 3% Citrajet detergent (acidic cleaner) at 75C applied for 30 minutes is followed with a final thorough rinse with water. This two-step process produces a very clean result on stainless steel. And then, and only only then, per CDC guidelines, we can proceed with killing of the biofilm with a registered disinfectant.
Some applications and facilities are limited in the amount of heat that can be imparted to a cleaning process. For PVC or other piping types, lower temperatures may be needed. (Check with your manufacturer of the substrate in question.) Others would include modular and other small scale-type facilities where the infrastructure for heating water for cleaning is simply not available in sufficient quantities.
If you are not able to achieve the desired 75C temperature for the cleaning cycle, then it is possible to compensate for reduced temperature by using longer cleaning times. Since the surfactant accelerated alkaline hydrolysis reactions follow first order reaction kinetics, every 10C decrease in temperature corresponds to a doubling of the time to get the same amount of cleaning reaction. (The Arrhenius equation.) Consequently, in theory, if you go down to 35C (circa 90F), you would have to clean for 480 minutes instead of 30 minutes at 75C (doubling the time 4x for a 40C temperature reduction). In practice, you will almost certainly see efficacy in a shorter time – on the order of a couple hours for even moderate to heavy biofilm buildup. The theory nonetheless provides excellent guidance in demonstrating the power of this principle: Heat is a good thing in cleaning organic residues.
The above time and temperature discussion would then come in to play with the Solujet and Citrajet CIP detergents. Thus allowing for a method to clean and prepare tanks, centrifuges and the like, for killing biofilm with a registered disinfectant. And then a final thorough rinse, as described above. (Note that Keylajet detergent can certainly be considered as a more powerful alternative to Solujet cleaner.)
Of course this long a cleaning regimen and may not be practical in a manufacturing environment. Whatever can reasonably be achieved with regard to raising temperature will shorten the cleaning times. Also note, once again, these times (30min at 75C) are a conservative, heavy biofilm scenario. It can also be considered routine, periodic “deep cleaning” between day-to-day regimens. Subsequent cleaning, prior to a robust biofilm forming can be far shorter cycles. As stated, we often see in practice cycles of approximately one hour each for the alkaline and acidic steps.
Manual and higher foaming detergent applications: Where scrubbing, soaking, sonciation, or recirculation applications that will avoid higher pressure/air entrainment (ex. filter cleaning) are in use, we recommend an enzymatic cleaner like Tergazyme® Enzyme-Active Powdered Detergent that contains both protease and surfactants. Furthermore, and as discussed in more detail further below, it is effective at more mild temperatures. The protease addresses both the cells and any proteins bound up in the polysaccharide biofilm, whereas the surfactants improve wetting and penetration through the hydrophobic polysaccharide biofilm. The result is thorough and complete removal of the inanimate portions of the biofilm.
This is advantageous because efficiently removing the proteinaceous, organic and inanimate residues of the biofilm will lengthen the amount of time until it returns. We recommend that Tergazyme detergent be used as a maintenance step between alkaline/acidic cleaning described above. This intermittent use as a maintenance procedure will completely remove all dead cells and traces of polysaccharide biofilm portions. This in turn slows down the formation of biofilm and allows the regular cleaning cycle to clean effectively for more cycles.
Also, please note that Tergazyme detergent is a powder that you typically mix 1-3% in cool to warm water (35C would be fine – please do not exceed 55C to ensure integrity and activity of the enzyme). Tergazyme detergent is a high foaming cleaner and would not be suitable for a spray-in-air clean-in-place system. You can pump and gently agitate Tergazyme solutions; however you cannot employ high agitation at an air/solution interface without getting excessive foam. If you have an old biofilm, you might want to do a 30 minute 1-3% Tergazyme clean (soak/recirculation) at 35C before the 3% Solujet/3% Citrajet cleaning above.
This approach with Tergazyme detergent can also be reliably used in manual surface cleaning and cleaning preparation for subsequent disinfectant-based biofilm removal, such as in cleanrooms.
Certainly we believe that incorporating an alkaline cleaner and/or an enzymatic cleaner will improve the results as compared with some that try just bleach. In our experience, bleach alone when used at concentrations that are suitable for food contact surface does have some efficacy on thermophilic bacilli and similar biofilms. But the efficacy is not persistent. Possibly this is because the bleaching mechanism breaks down membranes at the sulfhydryl groups and unsaturated side chains. This mechanism can often fail as you use up all exposed labile sites to attack. The biofilm might then persist underneath. Furthermore, many prefer to minimize the use of bleach to minimize chloride stress cracking on stainless steel.
Biofilms can be extremely tough. We are always pleased to discuss your particular application further. Contact us any time!
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