Many compendial water systems, particularly purified water systems, have distribution systems with a biofilm. Detection, removal, and control of biofilm in compendial water systems are extremely important. The microbial quality of delivered compendial water requires elimination of biofilm. A technique for effective removal of biofilm is discussed. Design, operating, monitoring, and preventative maintenance operations to control biofilm are presented. This article does not address the theory associated with biofilm formation but presents information from numerous “case histories” where “hands on” effective biofilm removal and control have been achieved.

Identification of the Presence of Biofilm

Total Viable Bacteria Enumeration Technique – Many Purified Water Systems have established Total Viable Bacteria “Alert” and “Action” levels expressed in units of cfu/ml. Point-of-use Total Viable Bacteria enumeration methods often use a Heterotrophic Plate Count of a 1 ml sample1. In fact, the United States Pharmacopeia General Information Section <1231> indicates that the suggested enumeration method for Purified Water is by Heterotrophic Plate Count (Pour Plate)or Membrane Filtration of a 1.0 milliliter sample2. A typical example of bacteria attachment, accumulation, and replication within a biofilm is presented in Figure A3. Bacteria associated with biofilm are often released in periodic “burst” into the flowing water stream. The actual time for sampling collection and frequency of sample collection are very small when compared with total time. Subsequently, bacteria may not be detected in a 1 milliliter sample or may be detected at specific points-of-use on some sample days and not on others as exhibited in Table 1.


Figure A: Biofilm formation





Trending of data is difficult. Increasing the sample frequency when each point-of-use is not sampled each day does not necessarily confirm the presence or absence of bacteria associated with biofilm in the system. Increasing the sample volume and frequency of sampling should be considered. The use of membrane filtration4 of a 10 to 100 milliliter sample volume coupled with increase sampling frequency will often identify the presence of biofilm before bacteria levels increase beyond established “alert” and “action” levels.

Rapid Increase in Bacteria Levels – As indicated above, once biofilm becomes well established and the sampling frequency has been increased bacteria will be noted in samples from every point-of-use, often exceeding both “Alert” and “Action” limits. Further, highly undesirable species of bacteria, such as Pseudomonas aeruginosa, may be noted upon identification. This generally results in termination of production while system sanitization is performed. Further, a thorough investigation of the “excursion” is required. Finally, systems with a biofilm will often exhibit poor ability to recover from introduction of bacteria from extraneous sources such as membrane filter “grow through” of bacteria or back contamination at a point-of-use.

Removal of Biofilm

Ineffective Techniques – Storage and distribution loop sanitization with both hot water (65 - 90?C) and steam may destroy bacteria but are not effective in removing biofilm. In particular, loop sanitization with steam is ineffective for sanitization of purified water systems. The use of USP Pure Steam provides the potential for cross contamination from the Purified Water distribution loop with biofilm. Further, two phase flow (steam and water) associated with condensation of pure steam, can result in significant temperature variation throughout the storage and distribution system. The use of either technique will not remove an established biofilm.

Chemical Sanitization – Proper sanitization with select chemical agents can provide highly effective removal of biofilm. This technique may be used periodically (such as once every six months) to “supplement” periodic hot water sanitization of Purified Water Storage and Distribution Systems. The technique may also be used with other bacteria removal techniques such as cartridge membrane filtration. The selection of sanitizing agent and, more importantly, the method and exposure time, are extremely critical.


Figure B: In the dynamic mode, the recirculating Hydrogen Peroxide/Peracetic Acid
flows through the interior of the distribution tubing, remaining in the “flowing stream”.



Figure C: "Static" Exposure of biofilm to sanitizing agent

The most effective and practical (personnel exposure and disposal considerations) chemical sanitizing agent is a mixture of Peracetic Acid and Hydrogen Peroxide. A concentrated mixture of the two chemicals is commercially available. The concentrated solution contains about 1% Hydrogen Peroxide and 0.08% Peracetic Acid. Effective sanitization can be achieved with a 1% solution of the sanitizing agent at ambient temperature. However, manufacturer’s information for the chemical sanitizing agent implies that a six log reduction of the “bacteria population” can be achieved with a “soak” time of 36 minutes (5). Unfortunately, when used for effective removal of biofilm from the interior of Purified Water Storage and Distribution Systems, the information is misleading. Effective sanitization with biofilm removal/destruction is a function of two major variables; concentration of sanitizing agent and exposure time. Extensive field experience indicates that the following procedure will remove biofilm:

• An adequate volume of Hydrogen Peroxide and Peracetic Acid is added to the storage and distribution system (into the stored water) to obtain a 1% concentration of the mixed sanitizing agent at all points-of-use. The loop distribution pump should be operational at this time. It is important to remember that any dead legs will suppress the effect of the sanitizing agent. Subsequently, zero dead leg type valves should be considered for any “branch” valves from the distribution loop. If system design does not include zero dead leg valves, it is important that each “branch” valve be opened to allow the 1% solution of Hydrogen Peroxide and Peracetic Acid to slowly flow through the valve to a waste collection container with “air break”. If there are other dead legs in the system, a similar procedure should be performed to insure that the concentration of sanitizing agent at all interior surfaces in contact with liquid are exposed to the 1% solution.

• Once the concentration of the 1% sanitizing agent is verified, the time of day is recorded. The 1% solution is allowed to recirculate for about 15 minutes. If necessary, additional concentrated sanitizing agent may be added to the tank to insure that a 1% solution is present. After the 15 minute recirculating time period, the distribution pumps are turned off. The 1% solution of Peracetic Acid and Hydrogen Peroxide is allowed to “sit” in a stagnant condition for 20-24 hours.

• At the end of the 20-24 hour time period the distribution pump is restarted. Recirculation of the 1% solution proceeds for a time period of about 15 minutes. At this time, the 1% solution of sanitizing agent is removed by a “non-intrusive” method, displacement of the sanitizing agent with bacteria-free water. If the presence of bacteria at a level ≥ 1 cfu/100 milliliters is suspected, a technique such as cartridge ultrafiltration with a micron rating ≤ 0.05 should be employed to insure that the displacement operation does not introduce bacteria to the freshly sanitized storage and distribution system. Each point-of-use and any dead legs should be flushed to verify that disinfecting agent is not present. The use of temporary “rechargeable ion exchange canisters” for providing “flush” water during this operation is inappropriate. Once all Hydrogen Peroxide and Peracetic Acid have been removed normal system operation may resume. If available, online conductivity and Total Organic Carbon (TOC) instruments may be used to verify chemical tests, confirming the absence of disinfecting agent.

• The procedure presented above will not effectively remove/destroy biofilm if dynamic flow exists for the indicated 20-24 hour time period. In a dynamic mode, the recirculating Hydrogen Peroxide/Peracetic Acid flow through the interior of the distribution tubing, remaining in the “flowing stream” as shown in Figure B above. The amount of sanitizing agent that physically reaches bacteria and other material in biofilm is extremely small.

When the sanitizing agent is allowed to sit in a stagnant condition there is no hydraulic force to move the water with sanitizing agent. The sanitizing agent will move, by diffusion, to and into the biofilm. The driving force for “movement of Hydrogen Peroxide/Peracetic Acid is concentration difference, increasing as material in the biofilm is oxidized and the sanitizing agent “depleted”. Figure C contains depicts the “diffusive” process.

While this sanitization method is extremely effective, several factors should be considered to eliminate/minimize biofilm. These factors are presented below.

Control of Biofilm

Elimination of Dead Legs – There are several definitions of a “dead leg”. The definitions include a specified number of “pipe diameters” and L/D ratio. When biofilm removal is considered with a chemical sanitizing agent, any dead leg ultimately determines the effectiveness of the operation. The historical “six pipe diameter rule” only applies to thermally sanitized system. It is based on conduction of heat to the “branch” fitting, valve, etc. The use of zero dead leg valves and short outlet tubing tees must be considered during the design and installation of any system that will be chemically sanitized.

Loop Recirculation – Recirculation of the distribution loop during normal operation should be such that turbulent flow is maintained at all times. This includes return line tubing to the storage tank at maximum draw-off from the loop. Modulating-type return loop back pressure regulating valves and distribution pump motor variable frequency drives provide flexibility in controlling loop flow rate particularly when processing operations include large volume “batching” requirements.

Proactive Preventative Maintenance Program – A proactive preventative maintenance program should be established for every compendial water system. This program should include all components in the system from raw water feed to points-of-use. Items such as filter media replacement, ultraviolet lamp and sleeve replacement, cartridge filter replacement, reverse osmosis membrane replacement loop valve diaphragm replacement, and tank vent filter replacement should be adequate to avoid “system excursions” between schedule operational shutdown periods. A “reactive” maintenance program can result in conditions contributing to biofilm.

Microbial Monitoring Program – A comprehensive sampling and bacteria monitoring program should be established. The program should include product water Total Viable Bacteria measurement for each component in the system. Identification with appropriate response of a bacteria excursion in the pretreatment system for a reverse osmosis unit, as an example, can prevent an increase in downstream Total Viable Bacteria levels.

Elimination of Distribution Loop Back Contamination – Point-of-use “delivery” accessories should not introduce bacteria to the compendial water system. Accessories of concern include, but are not limited to, lab sink hoses, larger diameter hoses used for feeding processing operations, any pressurized operation, and pressurized washers.

In summary, control and removal of biofilm require extensive effort and specific procedures. When effective biofilm control is not implemented, complete removal of the resulting established biofilm requires use of a chemical sanitizing agent considering “effective” contact time and concentration.



(1) Standard Methods for the Examination of Water & Wastewater, Edited by: Eaton, A.D., Clesceri, L.S., Rice, E.W., and Greenberg, A.E., ISBN: 0-87553-047-8, APHA, AWWA, and WEF, Section 9215, pp 9-34 to 9-40, 2005

(2) United States Pharmacopeia 35, National Formulary 30 with First Supplement, General Information Section <1231>, “Water for Pharmaceutical Purposes”, The United States Pharmacopeial Convention, Rockville Maryland, August 1, 2012

(3) Collentro, W.V., “Pharmaceutical Water - System Design, Operation, and Validation”, Second Edition Informa Healthcare, ISBN 9781420077827, New York, New York, January 2011, pg. 327

(4) Standard Methods for the Examination of Water & Wastewater, Edited by: Eaton, A.D., Clesceri, L.S., Rice, E.W., and Greenberg, A.E., ISBN: 0-87553-047-8, APHA, AWWA, and WEF, Section 9215D, pp 9-50 to 9.53, 2005

(5) Minncare® Cold Sterilant, MAR COR Purification, A Cantel Medical Company, Technical Literature, Bioscience Products, 2011