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Microsoft powerpoint - simmons - final - 2010 asm - cu disinfection.ppt

P-2310 Copper Ionization for Inactivating E. coli, Coliphage MS2, and
Bacillus Atrophaeus Spores in Irrigation Water for Fruits and Vegetables
Otto "Chip" D. Simmons, III and Jose Garzon, Department of Biological and Agricultural Engineering ,NC State University
Food borne outbreaks of gastroenteritis occur despite extensive measures by the
Test Microbial Indicator Organisms
Figure 2. Stability of Copper Ion Solution at High (4 mg/L) and Low (1 mg/L)
Table 2. Log
Microbial Indicator Reductions Achieved by Free Copper at Low
industry to ensure a food supply free of microbial contaminants. Water used for
Bacteria (E. coli K011)
(1 mg/L) Concentration (25oC)
irrigation in fields may be an important route for microbial contamination. A
Propagated in tryptic soy broth supplemented with chloramphenicol
variety of chemical disinfectants can be used on farms, including copper
10 microbe inactivation
Assayed by spread plating appropriate ten-fold dilutions on tryptic soy
ionization. Copper ionization uses a low voltage current to release positively
agar supplemented with chloramphenicol
charged copper atoms from electrodes suspended in water and is not harmful to
Virus (Coliphage MS2)
0 min (Initial)
1.4 ± 0.1
humans, is relatively inexpensive, and requires little maintenance. However,
Propagated on tryptic soy agar using the "sloppy agar" method and
1.3 ± 0.0
-0.1± 0.0
1.4 ± 1.4
there are few studies to determine the efficacy of copper for inactivating
purified by organic solvent extraction
1.3 ± 0.0
0.0 ± 0.0
1.8 ± 1.7
0.1 ± 0.1
microbial contaminants in environmental waters. Therefore, the objective of
Assayed by the Single Agar Layer (SAL) method with E. coli C3000 host
1.3 ± 0.0
0.2 ± 0.1
0.3 ± 0.3
0.0 ± 0.1
these studies was to determine the kinetics of ionized copper for inactivating a
cells (EPA Method 1602)
1.3 ± 0.1
0.7 ± 0.2
4.8 ± 0.4
0.0 ± 0.1
suite of indicator organisms spiked into environmental water potentially used for
Bacterial Spores (B. atrophaeus)
1.3 ± 0.1
1.1 ± 0.2
3.8 ± 2.2
0.2 ± 0.2
irrigation on farms. E. coli, coliphage MS2, and Bacillus atrophaeus spores were
Obtained from a commercial source (SGM Biotech, Inc., Bozeman, MT
propagated using standard methods then seeded into test waters at levels
All trials were conducted at 25oC
sufficient to follow a 99.99% reduction. Experiments were conducted at 25oC with
Appropriate ten-fold dilutions heated shocked at 70oC for 10 minutes then
Sufficient concentrations of microbial indicators were added to follow 4
two copper concentrations (1 and 4 mg/L). Samples were analyzed initially then
Tim e (hrs)
assayed by spread plating on AK2 agar
over a 4 hour period. Copper concentrations remained constant providing a
Free copper was stabile over the entire 2 hour experimental trials
stabile residual in test waters. Greater than 99% E. coli reductions were achieved
Test Waters (Table 1)
Moderate reduction (1.1 log ) was achieved for E. coli K011 at 2 hrs
with the low and high copper concentrations at 2 hours and at 30 minutes,
Representative surface water from research farm in eastern North
Significant reduction (4.8 log ) was achieved for coliphage MS2 at 1 hr
respectively. Coliphage MS2 reductions of 98% and greater than 99.9% were
Free copper concentration measured at 15 min, 1 hr, 2 hr, 3 hr, and 4 hr
No reduction was observed for the B. atrophaeus spores
achieved by low and high copper concentrations at 30 minutes and at 5 minutes,
Both high and low concentrations stabile in test waters over 4 hr period
respectively. Bacillus atrophaeus spores were not reduced with the low or high
Table 1. Physical Parameters of Test Waters used for Experiments
Three trials completed on different days; copper concentrations measured in
copper concentrations over the 4 hour trials. In conclusion, copper ionization
Parameter Range
duplicate for each sample
Table 2. Log
Microbial Indicator Reductions Achieved by Free Copper at High
appears to be effective for reducing bacteria and viruses at concentrations of 1
Variability in high concentration due to slightly different starting
(4 mg/L) Concentration (25oC)
Physical Parameter
and 4 mg/L in water but is not effective for bacterial spores at either copper
concentrations on different days
concentration. Copper provides a stable disinfectant residual in water and
10 microbe inactivation
appears to be relatively effective for bacteria and viruses. Therefore, copper
Chlorine (mg/L)
0.0 ± 0.0
Figure 3. Stability of Microbial Indicator Organisms in Test Water at 25oC
ionization may be a suitable alternative to other chemical disinfectants for
7.3 ± 0.2
0 min (Initial)
4.0 ± 0.2
inactivating microbial contaminants in irrigation water used on farms.
Turbidity (NTU)
19.6 ± 0.8
4.0 ± 0.2
0.8 ± 1.4
0.8 ± 1.3
3.9 ± 0.2
1.4 ± 1.5
1.2 ± 1.8
0.0 ± 0.1
TDS (mg/L)
41.2 ± 0.5
3.9 ± 0.2
2.8 ± 1.0
0.1 ± 0.2
Average pH measured for test water was 7.3
3.9 ± 0.2
3.2 ± 1.6
3.7 ± 0.4
0.1 ± 0.2
Average turbidity measured for test water was 19.6 NTU
3.9 ± 0.2
4.3 ± 0.7
4.3 ± 0.9
0.0 ± 0.0
Average total dissolved solids measured for test water was 41.2 mg/L
All experimental trials were conducted at 25oC
All trials were conducted at 25oC
Food borne outbreaks of gastroenteritis continue to occur despite extensive
Sufficient concentrations of microbial indicators were added to follow 4
Copper Ion Solution (Figure 1)
measures by the industry to ensure a food supply free of microbial
Copper solution generated by an Aqua-Hort Copper Ionization unit
Free copper was stabile over the entire 2 hour experimental trials
Two concentrations tested to represent high (4 mg/L) and low (1 mg/L)
Significant reduction (4.3 log ) was achieved for E. coli K011 at 2 hrs
concentrations that may be expected for use on farms
Disease outbreaks occur that can be linked to contaminated fresh produce
Significant reduction (4.3 log ) was achieved for coliphage MS2 at 2 hrs
Fresh produce important because it is often consumed raw
No reduction was observed for the B. atrophaeus spores
Time (hrs)
Fruits and vegetables generally have uneven surfaces making it difficult to
Figure 1. Aqua-Hort Copper Ionization Equipment
remove or inactivate microbial pathogens on their surface
Waters used for irrigating fruits and vegetables can become contaminated
Microbial indicator concentration measured at 15 min, 1 hr, 2 hr, 3 hr, and 4 hr
Free copper at low (1 mg/L) and high (4 mg/L) concentration and microbial
with pathogenic bacteria, viruses, or parasites
E. coli K011, coliphage MS2, and spores of B. atrophaeus stabile in test
indicator organisms were stabile in test water over four hrs at 25oC
In addition to protecting source water, measures may be necessary for
waters at 25oC over 4 hr period
inactivating microbial pathogens in waters used for crop irrigation
Three trials completed on different days; microbial indicator concentrations
Given that both copper and microbes were stabile in water, inactivation
measured in duplicate for each sample
of microbial indicators in experimental trials are attributable to the
A variety of methods may be used to disinfect water used for fresh produce
microbiocidal effects of free copper
Figure 4. Neutralization of Free Copper by Sodium Thioglycolate
chemical disinfectants (i.e. chlorine, hydrogen peroxide, etc.)
Sodium thioglycolate did not appear to quench the free copper in test water;
physical measures (i.e. ultraviolet irradiation)
dilution appears to be sufficiently effective for reducing free copper in
New/novel disinfectants (i.e. copper ionization)
Copper ionization
Moderate (1.1 log ) and significant (4.3 log ) reductions were achieved for
electronic release of positively charged copper atoms by application of a
Copper solution generated in aliquot of test water (2 L beaker) then diluted
E. coli K011 at 2 hrs with low (1 mg/L) and high (4 mg/L) concentrations of
low voltage current to copper electrodes suspended in water
with untreated test water to achieve "High" (4 mg/L) or "Low" (1 mg/L)
free copper, respectively
Copper ions in water can pierce the protective outer membrane of cells
concentrations used for experimental trials
thereby disrupting enzyme balance leading to cell death
Free copper concentrations measured with a Hach Pocket Colorimeter II Test
Significant (4.8 log
and 4.3 log ) reductions were achieved for coliphage
Proven effective for inactivation of algae
Kit (Hach Company, Loveland, CO, Product # 5870019)
MS2 at 1 hr and 2 hrs with low (1 mg/L) and high (4 mg/L) concentrations of
Provides a disinfectant that is not harmful to human consumers
free copper, respectively
Is relatively inexpensive
Microbial indicators spiked to half volume of test water
Requires little maintenance
"Initial" sample collected to determine copper and microbe concentrations
There was little to no reduction of B. atrophaeus spores by free copper at low
Available to plants as a micro-nutrient
Double strength (i.e. 8 mg/L or 2 mg/L) copper solution added to initial spiked
(1 mg/L) or high (4 mg/L) concentration
test water
Neutralization of free copper by sodium thioglycolate reported in literature
Samples collected at appropriate time points to follow disinfection kinetics –
Although bacteria and virus reductions were achieved with free copper, long
1:100 dilution of a 10% sodium thioglycolate solution
copper and microbial concentrations measured at each time point
contact times were required to achieve these reduction
Experimental trials conducted with high (4 mg/L) copper solution in test water
to determine the inactivation efficacy of
Sodium thioglycolate did not appear to effectively quench, or significantly
copper ions for a suite of microbial indicator organisms representative of the
Copper neutralized in samples using sodium thioglycolate
These results demonstrate that both bacterial and viral indicators can be
reduce, the free copper solution (Initial vs. Quenched)
major classes of microbial pathogens in spiked test waters at 25oC
Dilutions made immediately after samples collected for each time point
reduced by free copper and, given the stability of copper in water, this may
Dilution appeared to effectively reduce the free copper concentration to
Time points: 1 min, 5 min, 30 min, 1 hr, and 2 hr
be a suitable disinfectant that can be applied to irrigation water for control
effectively quench the disinfection reaction (1:10 and 1:100 dilutions)
of microbial contaminants on farms.
This research was supported by a research and development grant from the Agricultural Advancement Consortium
of the North Carolina Rural Economic Development Center, Inc. Authors wish to thank Lars Marohn of Lauren Hailey Technologies
for providing the Aqua-Hort Copper Ionization Equipment for testing.


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