Biofilm can be removed and/or destroyed by chemical and physical treatments. Chemical biocides can be divided into two major groups: oxidizing and nonoxidizing. Physical treatments include mechanical scrubbing and hot water. An article by Mittelman (1986) has the most comprehensive information on treatment of biofouling in purified water systems. Refer to this article for more information.
Table 4. Typical biocide dosage levels. [NOTE: mg/l = ppm] (Mittelman 1986)
| Biocide |
Dosage Level (mg/l) |
Contact Time (hours) |
|
Chlorine
Ozone
Chlorine dioxide
Hydrogen peroxide
Iodine
Quaternary ammonium cmpds.
Formaldehyde
Anionic & nonionic surfactants
|
50-100
10-50*
50-100
10% (v/v)
100-200
300-1000
1-2% (v/v)
300-500
|
1-2
<1
1-2
2-3
1-2
2-3
2-3
3-4
|
* Ozone dosage is 10-50 mg/l, but the residual levels in water were 1-2 mg/l.
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Oxidizing biocides
Mittelman says the effectiveness of the oxidizing biocides in purified-water systems on an equal milligram-per-liter-dosage basis decreases in the following order:
ozone > chlorine dioxide > chlorine > iodine > hydrogen peroxide
Chlorine
According to Mittelman (1986), "Chlorine is probably the most effective and least expensive of all oxidizing and nonoxidizing biocides." The activity of chlorine against attached biofilms is particularly high; not only are planktonic and biofilm bacteria killed, but chlorine also reacts with and destroys the polysaccharide web and its attachments to the surface. By destroying the extracellular polymers, chlorine breaks up the physical integrity of the biofilm.
Characklis (1990) recommends improving a chlorine treatment program by taking the following measures:
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 Increase the Chlorine Concentration at the Water-Biofilm Interface
As chlorine diffuses into a biofilm, it is used up in reactions with bacteria cells and extracellular materials. At low chlorine levels, biofilm bacteria can produce extracellular material faster than chlorine can diffuse through it so they are shielded in slime. By increasing the concentration, chlorine will diffuse farther into the biofilm. When it comes to disinfection of biofilms, high chlorine concentration for short durations is more effective than low concentration for long durations.
 Increase the Fluid Shear Stress at the Water-Biofilm Interface
- Increased mass transfer of chlorine from the bulk water to the biofilm.
- Disruption of the biofilm during chlorination exposes new biofilm surfaces for chlorine attack. Decreased thickness of viscous or laminar sublayer.
 Use pH Control
High pH favors hypochlorite-ion-promoted detachment of mature biofilms, and low pH enhances hypochlorous acid disinfection of thin films. Characklis proposed an interesting procedure would be to alternate between continuous chlorination at pH 6.5 and shock chlorination at pH 8. He doesn’t imply that this has been tested.
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Chlorine dioxide
Chlorine dioxide has biocidal activities similar to those of chlorine. Because it is unstable, it must be mixed and prepared on-site. Like chlorine, chlorine dioxide is corrosive to metals and must be handled with care.
Ozone
As an oxidizer, ozone is approximately twice as powerful as chlorine at the same concentrations. Like chlorine dioxide, ozone must be generated on-site because of its high reactivity and relative instability. Systems must be designed with appropriate ozone resistant materials.
"Ozone is usually dosed on a continuous basis at 1-2 mg/l. Success in employing higher dosages on a noncontinuous basis has been limited, possibly because of the limited solubility of ozone in purified water; it is difficult to produce high concentrations of ozone in solution." (Mittelman 1986). Although chlorine isn’t as powerful as ozone when you compare 1-2 mg/l of each, chlorine can be used in higher sanitizing concentrations with equal disinfecting strength.
Hydrogen peroxide
"Hydrogen peroxide is frequently used as a biocide in microelectronic-grade purified-water systems because it produces no by-products; it rapidly degrades to water and oxygen. A 10% by volume solution in purified water appears effective in killing planktonic bacteria, but more studies are needed on the effectiveness against attached biofilm" (Mittelman 1986).
Non-oxidizing biocides
Quaternary Ammonium Compounds
In addition to their biocidal activity, quats are effective surfactants/detergents, which may be an important factor in their use for biofilm inactivation and removal from surfaces. Rinseability can be a problem as removal from a purified-water system often requires exhaustive rinsing.
Formaldehyde
Formaldehyde has been applied to pharmaceutical-grade systems. It is relatively noncorrosive to stainless steel. Its effectiveness against biofilm is questionable and it is a toxic carcinogen.
Anionic and Nonionic Surface-Active Agents
These surfactant or detergent compounds have limited biocidal activity against the bacteria in purified water systems. Applications may be found for these detergents in conjunction with other biocides to improve biofilm and other particulate removal.
Physical Treatments
Heat
Pharmaceutical Water-for-Injection systems use recirculating hot water loops (greater than 80°C) to kill bacteria. According to Mittelman (1986), when these systems are used on a continuous basis, planktonic bacteria are killed and biofilm development is reduced. Biofilms are even found in hot water (80°C). Periodic hot water sanitization can also be used to destroy bacteria in biofilm, but according to Collentro (1995) this requires a temperature of 95°C for a period in excess of 100 minutes. This would not be practical in an animal drinking water system!
Mechanical removal
From Mittelman: "Heavy biofilms cannot be removed from storage tank walls by the use of chemicals alone; mechanical scrubbing or scraping, high-pressure spraying, or a combination is also required. Mechanical removal of biofilm from distribution systems is impractical." For RO system maintenance, we don’t routinely scrub storage tanks, but there is usually a continuous low chlorine level in the stored water, so heavy biofilms aren’t allowed to develop.
Biocide resistance
Unlike antibiotics used to fight bacteria associated with human, animal, and plant diseases, bacteria do not develop the same type of resistance to industrial biocides. The difference between antibiotics and industrial biocides is that while an antibiotic may have a small number of target sites on or in the bacterial cell, all oxidizing biocides have a multitude of potential target sites. Chlorine, for example, is thought to have more than a hundred potential target sites on or in microorganisms. It is virtually impossible for microorganisms to develop a general resistance to such compounds (Mittelman 1986). However, bacteria in a biofilm can resist biocides because they are shielded in slime.
Biofilm recovery (Regrowth)
Bacteria associated with biofilms are much more difficult to kill and remove from surfaces than planktonic organisms. According to Characklis (1990), numerous investigators and plant operators have observed "a rapid resumption of biofouling immediately following chlorine treatment." Incomplete removal of the biofilm will allow it to quickly return to its equilibrium state, causing a rebound in total plate counts following sanitization.
Figure 12, below (Mittelman 1986), shows typical regrowth following sanitization. Initially, the bulk water bacteria count dropped to zero after sanitization, but this was followed by a gradual increase in numbers to levels at or below the pretreatment levels. In this example, regrowth started after 2 days and was back up to equilibrium levels after 20 days. This is similar to results seen in in-house sanitization testing at Edstrom Industries.
Figure 12. Example of sanitization followed by biofilm recovery. Bacteria count samples were taken on a daily basis. (Mittelman 1986) |
According to Characklis (1990), biofilm recovery may be due to one or all of the following.
- The remaining biofilm contains enough viable organisms that there is no lag phase in regrowth. Thus, biofilm recovery after shock chlorination is faster than initial accumulation on a clean pipe.
- The residual biofilm on the surface makes it rougher than clean pipe. The roughness of the deposit may provide a stickier surface which adsorbs more microbial cells and other compounds from the water.
- The chlorine preferentially removes extracellular polymers and not biofilm cells, thus leaving biofilm cells more exposed to the nutrients when chlorination ceases.
- Surviving organisms rapidly create more slime (extracellular polymers) as a protective response to irritation by chlorine.
- There is selection for organisms less susceptible to the sanitizing chemical. This is usually the organisms that produce excessive amounts of slime like Pseudomonas.
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Case Study:
Sanitization Selecting
for
Pseudomonas
|
When several mice on long term studies died in 1996, this laboratory animal facility suspected that the cause was Pseudomonas aeruginosa which was found in rack manifold piping. They had never identified Pseudomonas in the water until after they chlorine sanitized their recirculating automated watering system (AWS) for the first time since the system was installed in 1982.
System Description: RO with no chlorine pretreatment followed by a carbon filter and deionization tanks before filling 3 storage tanks. Carbon and DI filters most likely add a lot of bacteria to the RO water. No chlorine is allowed in the animal drinking water due to the nutritional studies being conducted. One tank is non-recirculating and holds more than 2 weeks water supply for non-chlorinated rack manifold flushing. The other 2 tanks are on recirculating loops supplying the AWS. There is no RDS flushing and low turn-over of tank water.
Bacteria Testing: Facility does regular testing of drinking water for bacteria. They regularly got total counts of 10,000-50,000 cfu/ml from the RO storage tanks and never had any animal health problems. When counts exceeded 100,000 cfu/ml, they decided to do the first system sanitization.
Sanitization: 20 ppm chlorine for 4 hour soak with all animal racks removed. The bacteria counts were very low for a couple weeks afterwards, but then increased (typical of regrowth following sanitization).
Speculation: Chlorine sanitization selectively promoted more chlorine resistant organisms like Pseudomonas. It was probably present all along, embedded in the mature biofilm attached to pipe and tank walls. A one-time sanitization with a low concentration like 20 ppm is not going to kill 100% of Pseudomonas.
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Perhaps if sanitization could be automated, a watering system could be easily re-sanitized every few days before biofilm fully recovers.
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