Study Concerning the Inactivation of Microbes by the ...nass-wt.com/englisch/PDF/Study_in_English.pdf · Translation of the Spanish Study – University of Ba rcelona Manufacturer

1

Translation of the Spanish Study – University of Barcelona Manufacturer has the original – 01/2004

Study Concerning the Inactivation of Microbes by th e Device AQUA HP-SYSTEM ® University Barcelona Heck Trade S.L. -Spain

Study Concerning the Inactivation of Microbes by the Device Aqua Hydro Physical System

(AQUA HP-System®)

Barcelona, in July 2003

2

This report corresponds with the final report of the work "Study Concerning the

Inactivation of Microbes by the Device AQUA HP-System®" in accordance with the

agreement for collaboration between the company Heck Trade, S.L., Spain, the

professors of the Microbiological Institute of the University of Barcelona, Joan Jofre

Torroella and Francisco Gutiérrez.

The following also participated in this study:

Dr. Xavi Méndez Dr. Maite Muniesa,

Members in the Microbiological Institute.

Barcelona, in July 2003

3

Study Concerning the Inactivation of Microbes by the Device AQUA HP-SYSTEM®

Preliminary report of the laboratory experiments wi th the device AQUA HP-SYSTEM®

1. Introduction

This report endeavors to define some basic conditions for performing the preliminary

tests and experiments with the Aqua Hydro Physical System (hereafter referred to

as AQUA HP-SYSTEM®), with the goal of being able to assess the inactivation of

microbes during their activity within a closed water circulation system.

The function mode of the device can be looked up on the website of the company

distributing the device.

http://www.aqua-correct.dk/hps-press-uk.html Nielsen Technical Trading

P.O. Box 10, DK-4621 Gadstrup, Danmark

TECHNOLOGY:

The core of the AQUA HP-SYSTEM® technology consists of a high performance

water reactor.

When a corresponding pressure pump is connected upstream (required operating

pressure of 5-8 bar), high overpressure and underpressure conditions as well as

enormous centrifugally and centripetally active water shear forces are generated

within the reactor. Overpressure and underpressure conditions and the occurring

shear forces are so high that bacteria, germs, and other organic compounds are

effectively and thoroughly destroyed.

4

Another step consists of the forced aspiration of ambient air due to the

underpressure conditions within the reactor; this air is supplied to the treatment

process.The dissolved oxygen and the percentage of oxygen (12%) from the

aspirated ambient air are thus available for a natural oxidation process (cold

combustion). The combination of overpressure/underpressure, enormously high

water shear forces, and natural oxygen oxidation successively reduces organic

loads and pollutants and effectively and thoroughly prevents new microbial growth.

Another advantage of the AQUA HP-SYSTEM® technology is that the already

mentioned manipulation of the molecular structure also results in a change of the

surface tension and viscosity of the treated water. The treated water is softer so

that washing and cleaning processes are significantly improved or optimized.

2. Objective of the Experiments:

The objective of the conducted tests and experiments was to proof that the AQUA

HP-SYSTEM® is able to inactivate microorganisms within a closed water circulation

system. Therefore, the function conditions of the AQUA HP-SYSTEM® for realizing

the tests were determined first.

According to the recommendations of the manufacturer, the device was installed in

numerous hot water circuits, cooling towers, decorative fountains, etc. to prevent the

growth of bacteria, spores, fungi, etc. within these systems. If the ability of the dive

to inactivate microorganisms/microbes can be proven after conducting the tests and

analyzing the results, new tests and experiments exceeding the scope of this report

can be planned.

5

3. Test Equipment

The experiments were carried out with the following device: AQUA HP-System®,

Model K0 Junior. The exterior dimensions of this device are as follows: Length 350

mm, width 480 mm, height 620 mm, weight 40 kg.

4. Flow Rate

The device permits a flow of 0.5 to 2.5 m³/h. A flow of 1 m³/h was selected for the

tests to avoid large volumes of water.

5. Water Samples and Specimens

The water samples for the experiments were taken from 200 liters of water from the

Barcelona water network, treated with thiosulfate to neutralize the effect of the

chlorine.

The samples were injected with various bacteria and bacteriophages with a control

sample always being prepared in addition to the sample for the experiment. After

homogenization, a fraction of 1 l of infected sample water was available, which was

stored outside of the water circuit to serve as control sample during the experiment.

Two hundred liters of infected water was routed through the AQUA HP-SYSTEM®

while applying the time programs described in the test protocol.

6

6. Test Circuit

The water circuit in the experiments contained the following:

� Water storage tank with 200 l water.

� Flexible water hose (part of accessories) to aspirate the water at the charging pipe (inlet) of the device AQUA HP-SYSTEM®."

� The device AQUA HP-SYSTEM®, model K0 Junior, incl. high pressure pump.

� Flexible water hose (part of accessories) to drain water from device.

� Metering instrument for water volume.

The following diagram shows the composition of the system while conducting the experiments.

7

7. Used Microorganisms and Microbes

Used bacterial strains, bacteriophages, and nutrient medium

7.1 Bacterial Strains

A gram negative and a gram positive type of bacteria was selected, which represent the most frequently occurring following strains:

x_ Enterococcus faecalis ATCC 29212 ( gram +)

x_ Escherichia coli WG5 (ATCC 700078) (Gravox Cobrough 1986) (gram-)

7.2 Bacteriophages Two different bacteriophages were selected:

x_ MS2 (ATCC 15597-B1) ISO 10805-1 (Anonymous 1995)

x_ B56-3 (ATCC 700786-B1) ISO 10705-4 (Anonymous 2002) 7.3 Nutrient Media

x_ mFC-Agar (Difco, Becton Dickinson, USA). Ref: 267720

x_ Chromocult Coliforme Agar (Merck Darmstadt, BRD). Ref: 1.10426

x_ m Enterococcus Agar (Difco, Becton Dickinson, USA ) Ref: 274620

8

8. Test Protocol

The different experiments were carried out with the following steps:

a) Preparing the device by connecting the flexible water hoses for supplying and

draining the water and connecting to the water tank.

b) Preparing a water volume of 200 liters by adding thiosulfate.

c) Starting the pump and determining water flow rate (1 m³/h).

A test cycle takes only about 12 minutes with this flow rate and a stored water

volume of 200 l.

d) Stopping a lead time used for stabilization of less than 60 seconds. Checking the

flow circuit. cycle. Discard the treated water during this time.

e) Adding the corresponding microbe amount for the experiment at a ratio of 1000

cfu or 1000 pfu per ml water.

(cfu/ml = bacteria titer = concentration indication; pfu/ml= phages titer =

concentration indication)

f) Homogenization

g) Preparing a water sample of 1 l as control sample.

h) The control experiments were conducted with a water storage tank of equal design

with a capacity of 200 liters and injected with the same amount of microbes to prove

that the microbes are not absorbed within the circuit. The water volume in the hoses

and in the HP reactor was less than 4 liters, i.e. no more than 2% of the total volume

of the sample.

i) Taking the same sample amount from the water circuit and the control water at the

start of the experiment ("zero hour") and then at different times.

j) Conducting volumetric analyses / titer analyses of the different microbes acc. to

standardized protocols.

k) The water temperature was continuously checked and remained below 27° Celsius

during the experiments.

l) The experiments were carried out with the following day program: Eight hours of

treatment starting at zero hour, then 16 hours die down (rest). This day program was

repeated several times.

9

9. Results

The results are represented in the form of a chart. The charts depict the

concentrations of the microbes during the test sequences, in the control water as

well as the treated water. The achieved kinetics of inactivation in most cases

approximates a straight line with R² greater than 0.70.

This sensitive model makes it possible to determine an inactivation time of T90. This

is the time in which 90 percent of the microbes in the sample are inactivated. This

value serves to measure the inactivation of the different microbes and amounts to

less than 100 hours for each of the experiments conducted with the HP reactor.

In the control experiments (without HP reactor), a (natural) degradation and

inactivation of the microbes was detected as well but at an inactivation time T90 of

more than 100 hours. This means the natural inactivation of microorganisms was

much lower than the one triggered by the reactor.

10


10. Findings

10.1 Inactivation of the Enterococci

Experiment 1:

Treatment hours: ɿ treated with HP-water reactor

Ŷ Control water without HP-reactor

Experiment 2:



11

Experiment 3:



Experiment 4:



12


10.2 Inactivation of the Escherichia coli

Experiment 1:



Experiment 2:



13

Experiment 3:



14

10.3 Inactivation of the Bacteriophages B56-3

Experiment 1:



Experiment 2:



15

Experiment 3:



16

10.4 Inactivation of the Bacteriophages MS2

Experiment 1:



Experiment 2:



17

Experiment 3:



18

Results:

In addition to the "inactivation kinetics," the inactivation times T90 and T99 of the

different added microbes were calculated (in hours).

Results of the Inactivation Times T90 and T99:

Statistics of the Inactivation Times T90 and T99 of the Used Microorganisms and Microbes:

Est. Des.: Standard deviation I.C.: Confidence range Confidence interval V min: minimum value (minimum) V max: the highest value (maximum)

19


Chart of the Inactivation Times T90 and T99 of the Used Microorganisms and Microbes:

Inactivation Times T90 and T99 of the Test Microbes

1: T90 for Enterococcus faecalis ATCC 29212

2: T99 for Enterococcus faecalis ATCC 29212

3: T90 for Escherichia coli QTCC 7000078

4: T99 for Escherichia coli QTCC 7000078

5: T90 for Bakteriophages MS2 ATCC 15597-B1

6: T99 for Bakteriophages MS2 ATCC 15597-B1

7: T90 for Bakteriophages B56-3 QTCC 700786-B1

8: T99 for Bakteriophages B56-3 QTCC 700786-B1

20


11. Conclusion

This study included experiments concerning the inactivation of microorganisms. The

experiments consisted of a closed water circuit with 200 liters of water from the public water

network, which was injected with various microorganisms and then routed through the

device Aqua Hydro Physical System (AQUA HP-SYSTEM®). Two types of bacteria were

used: Enterococcus faecalis and Escherichia coli as well as two different bacteriophages:

MS-2 and B56-3.

Concerning the test conditions, it was possible to determine the inactivation time T90, i.e.

the time needed to inactivate 90% of the microbe population, or to determine the reduction

of the present pollution according to a logarithmic unit, which has the same result.

The inactivation times T90 depend on the characteristics of the microbes used for the tests.

For example, the median value of T90 amounts to 20 hours for Enterococcus faecalis, 23

hours for Escherichia coli, 6 hours for the bacteriophage MS2, and 51 hours for the

bacteriophage B56-3. The results of the inactivation of the used bacteriophages as virus

indicators shows that the AQUA HP-SYSTEM® also inactivates viruses.

These results prove the inactivation of the microbes under the described test conditions due

to the action of the HP reactor since parallel control experiments yielded inactivation times

T90 with more than 100 hours. The inactivation by the reactor is also significantly higher

than the natural inactivation of the examined microbes in water.

The survival rates determined in the described experiments always yield a straight

reduction line, which seems to be a simplified model of first-order kinetics. This kinetics

reaction makes it possible to determine an inactivation time of T90 and T99. However, we

also cannot exclude the possibility that a comprehensive study would not result in a

different inactivation kinetics.

21

The inactivation times T90 of the bacteria used in the experiment with values of 20 to 23

hours are very close together; the same applies to the inactivation times of T99, which

amount to between 35 and 37 hours for enterococci and E. coli. Since these are

microorganisms typical for water, they are representative for an inactivation of a broad

spectrum of bacteria using the HP reactor. The types of bacteria referred to as Legionella

are also within this spectrum.

This means the information of the manufacturer concerning the inactivation of bacteria and

the use of the AQUA HP-SYSTEM® in different water application areas has been

substantiated.

The result was not the same for the examined bacteriophages, both model viruses. In this

case, the inactivation time T90 of the bacteriophage MS2 of 6 hours stood in contrast with

the inactivation time T90 of 51 hours of the bacteriophage B56-3.

The different results can be the result of the different characteristics of bacteria and

bacteriophages as well as the differences between the two examined bacteriophages

themselves.

In summary, it is possible to say that the stated results prove the effectiveness of the AQUA

HP-SYSTEM® in removing bacteria and viruses under the test conditions. It is also possible

to conclude that the AQUA HP-SYSTEM® minimizes the growth of bacteria in closed water

circulation systems with similar conditions and keeps the concentration of any type of

microorganism at a low level, as described by the manufacturer of the AQUA HP-SYSTEM®

in the associated product information.

12. References

Anonymous (1995) ISO 10705-1: Water quality. Detection and enumeration of bacteriophages. Part 1:

Enumeration of F-specific RNA bacteriophages. Genf, Schweiz, International Standardisation Organisation.

Anonymous (2000) ISO 10705-2: Water quality. Detection and enumeration of bacteriophages. Part

2: Enumeration of somatic coliphages. Genf, Schweiz, International Standardisation Organisation.

Anonymous (2002) ISO 10705-4: Water quality. Detection and enumeration of bacteriophages. Part 4: Enumeration of

bacteriophages infecting Bacteroides fragilis. Genf, Schweiz, International Standardisation Organisation.

Grabow WO & Cobrough P (1988): Practical direct plaque assay for coliphages in 100 ml samples of

drinking water. Appl. Environ. Microbiol. 52: 430-433.

Documents

Study Concerning the Inactivation of Microbes by the ...nass-wt.com/englisch/PDF/Study_in_English.pdf · Translation of the Spanish Study – University of Ba rcelona Manufacturer