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DISINFECTION OF SIMULATED
COOLING TOWER WATER
Larry L. Barton, Ph.D.
Mixed oxidants proved to be a better disinfectant
than sodium hypochlorite at equal dosages.
Cooling tower waters tend to be breeding areas for bacteria that cause human disease, aswell as biocorrosion or biofouling of the cooling tower. A study was conducted to determine the effectiveness of mixed oxidants (MIOX) versus sodium hypochlorite (NaOCl) in the inactivation of Bacillus stearothermophilus, Pseudomonas aeruginosa, and Legionella pneumophila. The two disinfectants were compared with and without addition of bromide, which is frequently added to cooling tower waters:
B. stearothermophilus: At a dosage of 4 mg/L and exposure of only 5 minutes, MIOX was able to achieve total kill, at pH levels ranging from 6.5 to 9.0. NaOCl was unable to achieve total kill at pH 8.0. The addition of bromide had little effect on either disinfectant after 10 minutes of exposure.
P. aeruginosa: At a dosage of only 2 mg/L and exposure of 10 minutes, MIOX achieved 0 levels at pH 8.0, as compared to 1,200 CFU/mL with NaOCl under the same conditions. The addition of bromide did not affect MIOX's effectiveness, but it caused bacteria levels to rise to 1,500 CFU/mL with NaOCl.
L. pneumophila: Again, at a dosage of only 2 mg/L and exposure of 10 minutes, MIOX achieved total kill at pH 8.0, compared to levels > 2 CFU/mL with NaOCl. Addition of bromide did not change MIOX's effectiveness, and had little effect on NaOCl's effectiveness.
The waters associated with cooling towers are important areas for bacterial growth and subsequent dissemination of bacteria into the environment. One important feature is that bacteria in the cooling tower water may be a source of human disease. Additionally, biofilms composed of various species of bacteria may lead to biocorrosion, biofouling, and other detrimental activities.
In an effort to determine the effectiveness of the mixed oxidant solution produced by the
MIOX process, several experimental procedures were followed. One question to be addressed was the effectiveness of MIOX mixed oxidant versus chlorine both in the presence and absence of low levels of bromide. As indicated by Richard Fincke, LFP Systems, Inc., San Antonio, the addition of NaBr at 5-6 mg/L is made frequently to waters in cooling towers. Therefore, the effect of bromide as an additive was examined here in relation to mixed oxidant and chlorine. NaOCl was to be used as the source of chlorine and NaBr was the source of bromide.
Parameters to be addressed included: the concentration of mixed oxidant, the concentration of NaOCl, the addition of bromide with mixed oxidant, the addition of bromide with NaOCl, the effect of pH, and the temperature effect at 50oC or 37oC.
BACTERIOLOGY PROCEDURES
Bacterial Cultures
The following cultures, purchased from ATCC (American Type Culture Collection, Rockville,
MD), were used for the study:
Bacillus stearothermophilus ATCC 9753
Pseudomonas aeruginosa ATCC 7700
Legionella pneumophila subsp. pneumophila ATCC 33152
Cultivation of Bacteria
To culture Bacillus stearothermophilus and Pseudomonas aeruginosa, a rich organic medium was employed. To prepare medium for broth cultures the following was added to 1 liter of deionized water: 5 g yeast extract, 5 g tryptone, and 2 g glucose. To prepare solid medium, 12 g was added to the broth medium. Chemicals for the bacterial growth media were purchased from Difco (Detroit, MI). Sterilization of media was by autoclaving. Incubation of B. stearothermophilus was at 50oC, while P. aeruginosa was at 37oC.
For the culture of L. pneumophila, a special medium was employed (2). The composition of 1 liter of the Legionella medium was as follows: 10 g yeast extract (Difco), 2 g charcoal (Sigma Chemical Co., St. Louis, MO.), 0.4 g L-cysteine HCl.H2 (Sigma), and 0.25 g ferric pyrophosphate (Sigma). To prepare solid media, 12 g/L of agar was added. The basal medium was sterilized by autoclaving, but cysteine and pyrophosphate were sterilized by filtration. For the cultivation of L. pneumophila, a 5% CO2 atmosphere was employed with incubation at 37oC.
Cultivation of broth cultures to be added to the test bottles
An overnight culture of B. stearothermophilus was grown in broth and examined by light microscopy to ensure that there were not bacterial spores produced. A standard was prepared to compare optical density (OD) with viable cell counts using an established procedure (1). This standard was used to estimate the number of bacteria present in the culture to enable dilutions to be made so that the final quantity of bacteria present in the test bottles would be about 105 bacteria per ml. At the start of each day, the OD of the bacterial culture was taken, and appropriate dilutions were made in physiological saline to provide 105 bacteria/ml of test solution.
For experiments employing P. aeruginosa, there was a graph established for OD versus viable cell counts similar to the one established for B. stearothermophilus. An overnight culture of P. aeruginosa was evaluated for OD, and the dilution of cells was to provide about 105 bacteria/ml of test material.
The use of L. pneumophila required the passage through a charcoal free medium to reduce the amount of charcoal that would be carried into the test bottles. To accomplish this, an inoculum of 10 ml from a 2 day culture was introduced into 100 ml of medium that did not contain charcoal. After 3 days, the culture OD was determined and viability of the culture was estimated from the standard prepared for P. aeruginosa.
Sampling and enumeration of bacteria in test bottles
For the recovery of B. stearothermophilus in the test bottles, 1 ml was removed and placed in
18 ml of liquified nutrient medium at 60o C and pour plates were made. For the control bottles where the number of bacteria/ml would be high, two dilutions were made before pour plates were made. This strain of B. stearothermophilus was highly sensitive to cooler temperatures and would die if the culture cooled to room temperature. Thus, we devised a system whereby the entire plating procedure could be accomplished and the culture would never be colder than 40o C.
The recovery of P. aeruginosa from the test bottles was performed in a manner similar to that used for B. stearothermophilus. One ml of the test fluid was placed in a petri plate, and 18 ml of liquified agar medium at 60o C was added in the making of the pour plates. The control test solutions were also diluted before pour plates were made.
A recovery system of L. pneumophila employed the use of most probable number technique (3). The Legionella give colonies on agar plates that are so small that they can be seen only with a microscope. Thus, in order to quantitate the bacteria, a series of dilutions were performed. This involved removing 0.1 ml from the test bottle and making several 1:100 dilutions into sterile physiological saline. Four aliquots of 0.5 ml were removed from each dilution set and placed in 3 ml of the Legionella broth. Growth was determined by an increase in turbidity after incubation had proceeded for 5 days.
PROCEDURES FOR TESTING DISINFECTION
Main Tests with Bacillus stearothermophilus
Four disinfectants at four different concentrations were added to simulated cooling tower waters after B. stearothermophilus cells were added to the water to produce approximately x 105 cells/ml. The additions to the water are as follows:
Mixed oxidant alone
Chlorine added as NaOCL
Mixed-oxidant solution plus 2 mg/L bromide
Chlorine added as NaOCL plus 2 mg/L bromide
The doses of chlorine or mixed oxidant were at several concentrations, including 0, 2, 4, 6, and 8 mg/L as Cl2. The time of contact between the disinfecting agent and the bacterial cells was as follows: 5, 10, 15, 30, 60, and 120 minutes. The temperature was maintained at 50°C throughout the study with B. stearothermophilus by using a series of adjustable water baths and hot air incubators. Three different pH levels were employed, which were 6.5, 8.0 and 9.5.
Specific guidelines to conduct the experiment were given in the "Test Plan" that was provided on December 12, 1995.
Additions to the test mixtures are listed on the tables with the results.
Confirmatory Tests with Pseudomonas aeruginosa and Legionella pneumophila
It was important to broaden the study to include additional bacteria of interest; the organisms selected for study were those that would be found commonly in cooling tower waters. P. aeruginosa is extremely important because it is a primary organism in biofouling and is an initiator of biocorrosion. The Legionella pneumophila strain was tested because cooling towers for various applications have been implicated in harboring bacterial agents that are capable of producing pneumonia in humans. These two bacterial agents are pathogenic for humans. The pH for testing was selected to be 8.0, and the exposure time of bacterial cell and chemicals in the test bottles was selected to be 10 min.
Specific guidelines for conducting this experiment were also given in the "Test Plan" provided on December 12, 1995.
Additions are listed on the tables where the data is presented.
The results are recorded in the following series of tables. The effect of disinfectants on B. stearothermophilus are given in Tables 1-12. The conclusions that can be drawn from theseresults are as follows:
The mixed oxidant kills 105 cells/ml of B. stearothermophilus within 5 min of exposure at pH values of 6.5, 8.0, and 9.5 at chlorine levels greater than 4 mg/L.
The mixed oxidant appeared to be more of an effective disinfectant than NaOCl at an equivalent amount of Cl2 added.
At pH 8.0 the mixed oxidant and the NaOCl were less effective in killing B. stearothermophilus than at pH 6.5 or pH 9.5, especially at the level of 2 mg Cl2 equivalent present.
Bromide, as NaBr, appeared to have a slight interaction with the mixed oxidant and with NaOCl. This effect was generally not seen after 10 minutes of exposure of the cells to the disinfectant.
The results of the confirmatory tests with P. aeruginosa and L. pneumophila are given in Tables 13-20. The conclusions that can be drawn from these confirmatory tests are as follows:
Cells of P. aeruginosa were readily killed at pH 8.0 in 10 minutes with mixed oxidant even at 2 mg equivalent of Cl2.
Cells of L. pneumophila were killed at pH 8.0 in 10 minutes with mixed oxidant even at 2 mg equivalent of Cl2.
Both the cells of P. aeruginosa and L. pneumophila were slightly resistant to 2 mg/L equivalent of Cl2 as NaOCl.
There appeared to be the same general disinfecting activity at 50o C with B. stearothermophilus and at 37o C with P. aeruginosa and L. pneumophila.
Barnett, M. 1992. Microbiology Laboratory Exercises. Wm. C. Brown Communications, Inc. Dubuque, IA. pp. 155-184.
Media formulations number 1088. 1992. IN: Catalogue of Bacteria and Phages, American Type Culture Collection, Rockville, MD. p. 455.
Colwell, R.R. 1979. Enumeration of specific populations by the most-probable number (MPN) method. IN: Native Aquatic Bacteria: Enumeration, Activity, and Ecology (J.W. Costerton and R.R. Colwell, eds.) American Society for Testing and Materials, Philadelphia, PA. pp. 56-61.
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