If a water purification system is designed, operated, and maintained properly, it will not be plagued by microbial contamination. However, if special attention is not dedicated to the control of microbial growth, problems will very likely result. Neither the proposed (USP 23) nor the current (USPXXII) specifications for USP Purified Water have established a limit for microbial concentration. However, the General Information chapter in both cases gives a "recommended action limit" of 500 colony-forming units per milliliter (cfu/mL) for the feedwater and 100 cfu/mL for the final product water. Despite the fact that these action limits are not necessarily required, most pharmaceutical manufacturers strive to maintain microbial concentrations below these recommended action limits. This article will address the methods for operating and maintaining a USP Purified Water system to continuously meet the objective of 100 cfu/mL.

Typical Purified Water Systems.

A typical USP Purified Water system may consist of several processing steps, each designed to provide further purification of the water. The first step of a typical system, based on two-pass reverse osmosis (RO) technology, is usually a multi-media filter followed by a water softener to remove insoluble particles and hardness ions, respectively. Next, a bisulfite (HSO₃) injection system may be used to remove chlorine. The central purification process is then the two-pass RO system. The final step is to pump the purified water to a heated or ozonated recirculating loop (see Figure1).

Feedwater. The first task of system design is to evaluate the feedwater. When microbial contamination is a concern, it is important to measure the residual disinfectant in the feedwater and obtain a baseline microbial count. This baseline microbial count and disinfectant concentrations should be monitored over each season of the year to determine the possible range encountered from season to season. The disinfectant residual should be high enough to maintain a microbial concentration at or below the USP-recommended action limit of 500 cfu/mL. The typical city water supply contains between 0.2 and 1.0 parts per million (ppm) of free chlorine (or chloramines) and, in most cases, is adequate to control microbial concentrations in the feedwater to the USP Purified Water system.

Pretreatment steps.  As mentioned above, the first processing step is usually filtration with a multi-media filter containing gravel, manganese greensand, and anthracite. The primary purpose of the manganese greensand is to remove iron, but it also serves as a very good particle filter. The anthracite provides a "light" layer that is easily backwashed, alleviating much of the load from the greensand, allowing the sand to perform more effectively. These two media types together are effective at removing suspended solids at sizes as small as 5 to 10 micron (m).

The second processing step is typically water softening, using ion-exchange softening. The water softener is used to remove hardness (calcium and magnesium) from the water, replacing these with sodium ions. Removing hardness protects the RO system by keeping hardness scale from forming on the membrane surface. It is best to not control scale by acid addition, which has the disadvantage of increasing the free carbon dioxide (CO₂) by shifting the bicarbonate to carbonic acid that, in turn, dissociates into CO₂ and water. The resulting carbon dioxide will pass through the membrane, producing a high-conductivity product water as the CO₂ re-associates with the water to reform ionic bicarbonate. Acid addition creates a problem in meeting the proposed pH specifications (5 to 7) for USP 23 Purified Water, since an RO system will inherently reduce the pH because of the shift in alkalinity/carbon dioxide ratio.

The next processing step is dechlorination with either activated carbon (AC) filtration or bisulfite injection. The advantage of using bisulfite is that it does not facilitate microbial growth as does an AC filter. It is also less costly than AC. When bisulfite is injected into the process stream, it is oxidized to sulfate (SO₄²-), and it also reduces free chlorine to the chloride ion. The by-products, sulfate and chloride, are easily rejected by the two-pass RO system.

While activated carbon beds reduce chlorine levels, the carbon bed provides an environment along with food sources that fosters growth of micro-organisms. The output from an AC bed will typically show a microbial concentration that has doubled over a 24-hour period. However, if dechlorination by activated carbon is felt necessary, it is wise to at least sanitize the complete bed daily. At a minimum, the bed should be steam-sanitized weekly.

After dechlorination, the pH is raised to the alkaline range by the addition of sodium hydroxide. This converts remaining carbon dioxide in the water to carbonate and bicarbonate ions, both of which are removed by the two-pass RO. Without adequate rejection of these ions, it is very difficult to meet the USP's proposed conductivity specification.

Reverse osmosis system. After dechlorination and/or pH adjustment, the water is filtered through a two-pass RO system to remove dissolved solids. It is important to note that the RO system must contain high-rejection thin-film type membrane elements. Preferred are sanitary-design polyamide (PA) membranes that reject more than 99.0% sodium chloride and have stainless steel permeate tubes.

A two-pass RO provides "double barrier" removal of microbes because the product water, or permeate, from the first pass is used as feed for the second pass. However, an RO system does not offer complete assurance that the product water will be totally free of bacteria. Even when designed. operated, and sanitized on a regular basis, some microbial growth is likely to occur on the permeate or product side of the membrane. The process should be viewed as a biological reduction process and not necessarily a "sanitization process" in itself. Thus, it is important to either use the purified water immediately, or send it to a distribution system that controls microbial growth, using either heat or some form of chemical sterilant (e.g., ozone).

Bacterial Reduction by RO

Bacteria levels of pharmaceutical and dialysis systems should be monitored in the permeate and in the system downstream of the RO. If installed and operated correctly, RO membrane elements should provide a 3-log reduction (99.9%) in bacteria. If the bacteria levels in the product water of the RO are higher than the bacteria levels in the feed, it is likely that bacteria are multiplying on the surface of the membrane and downstream. Table A shows the results of a study conducted on a 7.5-gallons-per-minute (gpm), two-pass RO in a controlled environment. ln this study, feedwater was seeded with Brevundimonas diminuta (formerly known as Pseudomonas diminuta) to measure the reduction potential of a two-pass RO system over time.

There are several ways to reduce potential microbial growth in a membrane system: minimize available nutrients; reduce water stagnation; and provide continuous chemical control and/or intermittent chemical control.

RO Design

The two-pass RO system should be designed to prevent areas where microbial growth may occur. Since bacteria cells are as small as 0.2 m in diameter, they may grow in minute crevices and cracks in a water purification system. Bacteria will also adhere to most surfaces, and a colony or biofilm will form within 48 hours. Bacteria multiply rapidly, and within days noticeable levels may appear at sample locations and in the purified water.

Some typical places conducive to bacterial growth include threaded connections, ball valves, imperfection pores in polyvinylchloride (PVC) piping, dead leg piping (noncirculating lengths of pipe greater than three to six pipe diameters), nonsanitary instruments, and areas behind seals and nonsanitary sampling valves. These areas are typically difficult to sanitize because they are stagnant, and chemical cleaning and sanitizing agents cannot reach the area of microbial growth. If a fluid is flowing across the internal surfaces of the machine, it is easier to destroy bacteria with sanitization. The design of the RO machine is critical; the machine design must minimize the number of dead legs or stagnant areas, use sanitary design membrane elements and instruments, and employ sanitary piping for the second-pass permeate.

Storage and Distribution

There are two common options for controlling microbial growth in a distribution and storage system: ozonation and heat. There is a growing trend toward using ozone in storage and distribution systems because of its relatively low capital and operating costs compared to hot-water generation and storage. It also has the added value of reducing the total organic carbon (TOC) to levels well below those proposed for USP 23 Purified Water (500 ppb). Finally, it may be removed by ultraviolet (UV) light at 254-nanometers, reducing the ozone to oxygen.

System Operation and Monitoring

To minimize the potential for bacteria growth, a USP Purified Water system should be operated continuously. When water is not in demand. the system should be kept in operation by either recirculating the final product water or diverting the water to drain or other uses. This avoids stagnation and the resulting microbial proliferation.

When the system is installed, baseline operating parameters should be taken for all critical parameters, including daily microbial counts from the feedwater. A microbial count should also be obtained after the dual-media filter, the water softener, the dechlorination device, after each pass of the RO system, and at each point-of-use (POU).

After a baseline performance is established, a regular monitoring schedule should be implemented, at each point-of-use on a daily basis, and weekly at other critical points in the system.

Microbial counts at each sample location should be plotted on a control chart. Action limits should be established for each sample point. Since the USP suggests 500 cfu/mL as the microbial limit for the system feedwater, it is reasonable to assign that limit at every point in the pretreatment system, although a lower limit may be selected if desired.

Microbial levels should be plotted on the control chart whenever samples are obtained. When there is a noticeable increase or when any level exceeds its particular action limit, the action plan should be implemented.

Action Plans for Microbial Control

The appropriate action plan for the control of micro-organisms is sanitization. The sanitization procedure for each component follows:

Sanitization of dual-media filter.  Media filters typically do not require sanitization if the feed and effluent from the media filter contain free chlorine or other residual disinfectant. If chlorine use is intermittent, it is wise to augment the city water supply with additional chlorine. Most media filters are designed to be backwashed when they become loaded with silt or other particles. This backwashing step also helps to reduce biological growth in the media. However, if an analysis of the effluent water from the multi-media filter shows that bacterial contamination is present. it may be necessary to sanitize the multi-media filter.

There are two common approaches for sanitizing multi-media filters. One is to chemically sanitize with 200 to 2,000 ppm of chlorine. To accomplish this type of chemical sanitization, close the inlet and outlet valves of the filter, then release the pressure from the filter using the air bleed valve. Remove the manway and add the proper amount of concentrated chlorine (bleach will suffice) in order to bring the concentration of the water in the dual-media filter to the 200 to 2,000 ppm level. Next, open the drain valve until the water level falls to two to three inches above the media. After the media soaks overnight, refill the tank and bleach the air from the vessel, then backwash until the chlorine levels in the backwash water (to drain) are at typical residual levels.

The second option is to use hot water or steam sanitization. In order to conduct this type of sanitization, it is necessary to design the filter to withstand high temperatures. The typical hot-water sanitization temperature is 80C (176F), maintained for 30 to 60 minutes; and the typical steam-sanitization temperature is 120C (250F), maintained for 15 to 30 minutes.

Sanitization of Softeners

As with multi-media filters, softeners typically do not require sanitization. Efforts should be made to maintain some residual chlorine in the effluent water from the softener if possible, although chlorine is usually lost as the water flows through the resin. If the softener becomes contaminated with microbes, it may signal that the residual chlorine entering the softener has been completely consumed.

It is not difficult to chemically sanitize a water softener with chlorine, but resin life may be reduced. To minimize the impact on resin life, one can regenerate the water softener prior to sanitization, putting the resin in the sodium form. In order to sanitize a water softener, first add bleach to the brine tank to a level of 100 ppm of free chlorine. Begin a normal regeneration cycle. Interrupt the cycle immediately after the brining step, before the fast rinse, and let the softener soak for two to eight hours. Then continue the regeneration cycle, flushing to drain with clean water until the chlorine residual levels are below 1 ppm.

It is also possible to hot-water sanitize a softening system with 80C (176F) water for 30 to 60 minutes, working with certain resins, piping, and tanks that are designed to tolerate these high temperatures.

Microbial Considerations for Chemical Addition Systems

Most chemical solutions injected into the water before the two-pass RO are not susceptible to microbial growth at typical feedstock levels. The osmotic pressures simply limit the growth potential in the solution itself. Typical chemicals include acids and caustics such as sodium hydroxide, which will be beyond the pH tolerance of most micro-organisms. However, bisulfite and other reducing agents that are used for chlorine removal may be susceptible to microbial growth, particularly molds, at the solution/air interface. Thus, it is important to monitor the microbial concentration in the chemical feed tank on a regular basis. Any of these solutions should be replaced if contaminated.

Chemical Sanitization of RO Systems

To control microbial growth, RO systems must be chemically sanitized on a regular basis. Prior to sanitization, it is important to chemically clean the first-pass RO system. This will help to disrupt any biofilm that protects viable bacteria from contact with the sanitant. It also removes foulants that will react with and chemically deplete the sanitizing agent. Typically this is done in a two-step process. The first step commonly involves the use of an acid cleaner such as citric acid to remove the inorganic foulants. Next, a high-pH cleaner such as sodium hydroxide is used in order to remove organic foulants. Then the system is sanitized with one of the following agents:   formaldehyde, hydrogen peroxide, or peracetic acid/hydrogen peroxide. It is important to consult the manufacturer of the RO system to determine the correct concentrations of the chemicals that are compatible with the membranes in the system, and always rinse with Purified Water before changing chemicals.

Both cleaning and sanitization processes consist of four steps. First, the cleaning chemical is mixed with permeate water in a clean-in-place (ClP) tank. Second, the chemical solution is recirculated through the RO system for 15 to 30 minutes. Then, the system is left to soak for 20 to 30 minutes. The system should be started once every 5 to 10 minutes for a short time to allow fresh solution to contact the membrane. Finally, the system should be rinsed with permeate water until the residual cleaning and/or sanitization chemicals have been removed.

Although the second pass of the RO does not typically require cleaning as frequently as the first pass, a regular cleaning and sanitization schedule should be maintained. This schedule should be based on the microbial concentrations and samples collected from the permeate water from the second-pass RO machine. When microbial concentrations in the permeate begin to rise steadily, sanitization should be conducted.

If peracetic acid or hydrogen peroxide are used to sanitize PA membranes, the membrane must be cleaned with an acid cleaner in order to remove free iron and any other transitional metals; otherwise the membrane may be chemically damaged by the sanitization procedure. It is also important to make certain that the sanitant is mixed with water that is free of chlorine. The membrane's exposure to the sanitant should not exceed one hour per week at the recommended concentration level, or reduced membrane life may occur.

Biofilm Removal

Biofilm is a common term used to describe the accumulation of micro-organisms and their by-product excretions onto surfaces of a water treatment system. Because most micro-organisms prefer to become attached to a surface, more and more micro-organisms will adhere to the surfaces of a water system. As micro-organisms die, they become nutrient sources for other micro-organisms. Over time, a film consisting of living and dead organisms will form. A slimy cover called a glycocalyx surrounds the organisms and serves to trap nutrients from the water source and protect the organisms from chemical destruction (see Figure 2).

To remove a biofilm, it is necessary to conduct a series of sanitization and cleaning steps. First, inorganics should be removed using a low-pH cleaner. Second, any organic compound or dead micro-organisms should be removed using a high-pH cleaner such as sodium hydroxide. Third, a sanitization should be conducted using a common sanitizing agent such as formaldehyde, chlorine, or peracetic acid/hydrogen peroxide. Then conduct a series of organic cleaning cycles followed by sanitization cycles. Prior to changing chemicals, always flush with purified water. For severe, established biofilms, it may be necessary to repeat this process 5 to 10 times. During each step, the sanitization agent should be in contact with the system for 15 to 30 minutes.

Conclusion

In order to minimize the potential for microbial contamination of pharmaceutical water systems, a system must be designed, operated, and maintained properly. Routine monitoring and analysis are critical elements to controlling the microbial count. A sanitization schedule should be established early, evaluated for effectiveness, and followed rigorously.

Reference

1. United States Pharmacoepia 23, General Information chapter 1231, "Water For Pharmaceutical Purposes', United States Pharmacopeial   Convention Inc., Rockville, Md. (1995).

Author Angela K. Weitnauer can be reached at Scimed, One Scimed Place, Maple Grove, MN5531 1; 612/494-2648 She was employed by Osmonics when this paper was written.

Key words: BIOFILMS, HYDROGEN PEROXIDE, OZONE, PHARMACEUTICAL, REVERSE OSMOSIS, SANITIZATION, USP