Compendial Water Systems: Biofilm

Remediation and Control

Thu, 10/25/2012 - 8:01am 1 Comment
by William V. Collentro
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.

References:
(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