Purified water is a key reagent that laboratories depend on. The most common types of purified water can be seen in Table 2 of this article, with Type I being pure and Type I* being the purest with the lowest levels of bacteria and endotoxins.
Therefore, to achieve the low levels of bacteria and endotoxins in Type I and Type I* water, removal of bacteria and endotoxins is key to keeping lab water purified. When bacteria are not removed from the water, they begin to group up, adhere to surfaces, and form biofilms. Biofilms are multispecies structures encased in an extracellular polysaccharide matrix. They provide physical protection and adhesion to the bacteria, and, if left undisturbed, biofilms can release populations of bacteria into the water, leading to prolonged microbial colonization. In the case of gram-negative bacteria, biofilms contain endotoxins in their outer membrane that are also released when the bacterial cell dies.
Not only are bacteria and endotoxins harmful to humans, but their presence in laboratory water can lead to the generation of erroneous data. Therefore, preventing these organisms from forming, and trying to remove them if they do form, are critical to maintaining the purity of lab water.
Microbial removal techniques
There are several purification techniques for the removal of bacteria and endotoxins. They are listed below in order of effectiveness.
Distillation
Distillation is a process that involves heating water to a volatile vapor phase in order to leave nonvolatile impurities behind. Through multiple distillation steps, the cooling of the volatile vapor phase back to a liquid water phase effectively removes bacteria and endotoxins.
Submicron filtration
Purification water systems with an ultrafiltration component utilize submicron water filters that act like a sieve or a pore size-based physical barrier that prevents the passage of particles. Ultrafiltration uses submicron filters such as micro-, ultramicro-, and ultrafilters in the 1–200 nm range. The pores of submicron filters are smaller than their intended target and capture the impurity while allowing water to pass through. Submicron filtration can remove such impurities as bacteria, colloids, enzymes, endotoxins, and particulates.
Endotoxins, which are negatively charged at a pH greater than 2, can be effectively removed with positively charged filters.
Submicron filtration can be used as part of a so-called polishing loop or at the point-of-use.
Ultraviolet radiation
Two UV wavelengths—254 and 185—can be used to effectively remove bacteria and endotoxins to purify lab water.
Use of UV oxidation with UV 254 effectively breaks down carbon, nitrogen, and hydrogen bonds, which disrupts the DNA of living microorganisms. At this wavelength, microorganisms are rendered ineffective as they lose their ability to replicate and form colonies, and can be removed. Because of the possibility of some bacteria leaching from deionization into the water tanks, a UV lamp with a wavelength of 254 nm should be used after the tanks.
A UV lamp with a wavelength of 185 nm carries more UV energy than a lamp with the longer 254-nm wavelength. It is able to effectively break down organic bonds and generate free radicals. These free radicals are effective at oxidizing organic and inorganic molecules and producing ionized organic molecules, which can be removed using ion-exchange resins used downstream from the UV lamp. This wavelength also oxidizes endotoxins.
The UV lamp can form part of a polishing treatment loop, including ion exchange, through which water is repeatedly circulated to maintain quality. If using deionization tanks, a UV 185 lamp should be placed before deionization tanks to lower the conductivity of the water. Use of a 185-nm UV light increases the water’s conductivity and should be polished back up by a deionization system when ultrapure water is needed.
The size of a UV lamp is an important consideration, with oversizing more of a problem than undersizing. An oversized UV light can heat the water, decrease contact with the bacteria, and encourage growth in the bacteria not inactivated.
Reverse osmosis
In reverse osmosis, water is forced via hydraulic pressure through a membrane that excludes ionized species and materials with molecular weights above 100–200 Daltons. The pressure applied to the water must be significantly greater than the opposing osmotic pressure generated by the concentration gradient across the reverse osmosis membrane.
Reverse osmosis removes 90–99% of particles, ions, organics, and microorganisms. However, due to the small pore size of reverse osmosis membranes, the flow rate across the membrane is a rate-limiting step in the purification process. Therefore, purification systems coupled with reverse osmosis typically require use of a storage reservoir to supply immediate demands for large volumes of water. Reverse osmosis membranes require pretreatment with particle filtration and chlorine removal plus periodic maintenance to remove biofilms, mineral scale, and organic accumulation.
Ion exchange
Ion exchange involves the removal of ions from water using an ion-exchange resin consisting of charged chemical functional groups attached to a polymer backbone. The charged groups attached to the resin remove certain ions in the bulk water phase, chemically bonding them to the resin and liberating the counter ion into solution.
On their own, ion-exchange resins do not effectively remove bacteria and endotoxins. They are most effective when used in a combination with a UV lamp to remove the ionized organic molecules produced as a result of UV oxidation. Bacteria and endotoxins can be removed using ion-exchange resins used downstream from the UV lamp.
Implementing microbial removal
Employing a combination of purification technologies in integrated purification systems is beneficial because bacteria or endotoxins may be further filtered. Some of these systems are described in this article.
When stored in purified tanks, static water may degrade and become susceptible to the formation of biofilms and bacterial growth. Therefore, in addition to selecting the appropriate purification technology, in most instances maintaining microbial removal requires recirculation of the purified water and sanitizing the water purification system itself.
Water recirculation disrupts the formation of biofilms and bacterial colonies. To sanitize the water purification system, an oxidant such as chlorine or peracetic acid, ozone, and heat sanitization can be used. The sizing of the water tank will be a large part of selecting which method will be the most effective application to disrupt the formation of biofilms and bacterial colonies.
Above all, successful microbial control in purified water systems requires diligence on the part of team members in establishing and implementing a planned schedule of maintenance procedures.
For more information, visit http://www.elgalabwater.com.
Lina Genovesi, Ph.D., JD, is a technical, regulatory, and business writer based in Princeton, NJ, U.S.A.; e-mail: [email protected]; www.linagenovesi.com.