Water filter test : Bacterial stocks, bacteriophages inactivation

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Physical Water Conditioning Technic

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AQUA Hydro Physical Systems Test

Preampel
Below represent a translation in English of the original report edition by
The University of Barcelona, Spain, Department of Microbiology.


This Internet translation relates to the information of the original report in Spanish and the content here, any terms or disputes are as such limited and not claimed to being approved or fully corresponding to wordings in the officially report. Only the original report by the University of Barcelona, Spain, Department of Microbiology represent the official valid content, which may be downloaded here ): http://www.aqua-correct.com\docs\hp-test-university-barcelona.pdf
(require Adobe Reader 5.0 or later)

The measured values and graphics results of the experiment tests with different bacterial stocks, bacteriophages and average cultures however, are the same as being presented in the original university report in Spanish.

Study of the micro biological inactivation
by the equipment
Aqua Hydro Physical System™




Barcelona, July, 2003

University of Barcelona, Spain, Dpt. of Microbiology

This refer to the final report of the work "Study of the microbiological inactivation by the equipment Aqua Hydro Physical System ™ ", corresponding to investigation agreement entered between the company Heck Trade, LMT. COMPANY and Mr. Joan Jofre Torroella and Francisco Lucena Gutiérrez, university professors at the Department of microbiology of the University of Barcelona.

In addition participating in the accomplishment of the study:

- Dr Xavi Méndez

- Dra. Maite Muniesa


Both assigned to the Dpt. of Microbiology

Barcelona, July of 2003

PRELIMINARY REPORT OF TESTS WITH THE EQUIPMENT
AQUA-HP-SYSTEM UNDER LABORATORY CONDITIONS

1 INTRODUCTION.
The present report serve to define the basic conditions for the accomplishment of tests and preliminary tests with the equipment Aqua Hydro Physical System™ (in the following Aqua HPS), for the purpose of being able to evaluate the microbiological inactivation due to its operation function in a closed water circuit.

The principle of operation of the equipment is described in the manufacturer / supplier Web page :

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


2 SUBJECT / PURPOSE OF THE TEST.

The purpose as well as the subject of the tests was to verify the capacity of inactivation of present microorganisms in a closed water circuit by the equipment Aqua HPS.

According to the references of the manufacturer the equipment has been installed in numerous circuits of sanitary hot water, cooling towers, air washers, fountains, ornamental ponds and other applications where the elimination of bacteria, fungi, etc. is required.

In case that the capability of inactivation is verified, further tests can be planned, beyond the scope of this preliminary report.

3 THE TEST EQUIPMENT.

The tests was performed with the Aqua HPS™ type K0 Junior, which consists of a compact equipment with a circulation pump to the HP reactor, mounted in a supported cabinet, to which pipes for the in- and outlet of the water circulation are connected.

4 WATER VOLUMES AT TEST.

The equipment allows a treatment volume of 0.5 to 2.5 m3/h-1. For the test it was chosen to operate with volume of 1 m3/h-1 for the purpose of avoiding excessive great water volumes.

5 THE TEST SAMPLES.

The test samples was made with 200 liters of water from the Barcelona supply network, pre-treated with tiosulphate to neutralize the effect of added chlorination.

Separately for each test, the samples "was inoculated" with different types of microorganisms (bacteria's and bacteriophages). After homogenization a liter of the inoculated sample is being separated and implemented parallel to the water circuit where it serves as control reference of the test.

The water samples, of 200 liters, was passed through Aqua HPS™ equipment in step sequences as indicated in the "Test Protocol " (Item 8).

6 THE TEST CIRCUIT.

The test is performed in a circuit consisting of the following elements:

Flexible hose with connections of the water to the inlet of the Aqua HPS™ equipment.
The Aqua HPS™ type K0 Junior with integrated pressure pump.
Flexible hose with connection to the water outlet of the Aqua HPS™ equipment.
Volume measureing meter.



7 THE APPLIED, DILUTED MICROORGANISMS .

Bacterial stocks, bacteriophage and average used.

7.1.1 Bacterial stocks. Gram+ and other equal was chosen as bacterium gram- as these represent the most frequent "structural" types.

7.2 Bacteriophage. Two bacterial virus with very well differentiated structures was chosen .

7,3 The culture average


8 TEST PROTOCOL.

The different tests was made by the following steps:

a) Preparation of the equipment with inlet- and outlet hoses properly connected to the container.

b) Preparation of a volume of 200 liters of water with added tiosulphate.

c) Start up of the pump and adjusting of the flow volume (1 m3/h). By this flow and with a volume of 200 liters, the maximum time of test will be of 12 minutes/cycles.

d) To start a run cycles time of at least 60 seconds. Verifying the circulating volume. To reject the water treated during this period.

e) To add the microorganism for test at the rate of 1000 ufc/ufp per milliliter of sample water.

f) Homogenize.

g) Then take a sample of one liter for test control reference.

h) In parallel with the start of the tests the container holding 200 liters, i.e. equally employed to the reactor                  which is inoculated with same nº of microorganisms, in order to making tests control with the purpose of              verifying there is no adsorption of the microorganisms in the circuit. The volume of water that is contained in the tubes and the reactor does not surpass the 4 L/s, which for the total volume of the sample at the most represents 2%.

i) Realizing the taking samples, at time zero or on the start of test and at different times, as well as of the water in the circuit, like of the control water.

j) To notice the degrees of the different microorganisms according to standardized protocols.

k) Controls of temperature of the water are made at any moment. The ºC during the tests is maintained below 27.


l) During the accomplishment of the tests, the treatment sequence has be the following: 8 running hours as of time zero, followed by 16 hours of rest. This repeated sequence h, each 24 hours.


9 EXPRESSIONS OF THE RESULTS.

For the purpose of facilitating the understanding of the results of each test made, these have imagined by means of the use of graphs. In these graphs one imagines the results of the concentrations of microorganisms throughout time as well of the control water as of the water circuit that has passed through the reactor.

The "kinetic of inactivation" obtained, come near to straight lines, with R2 greater to 0,70; in most occasions.

This simple model, allows us to calculate the T90, the time it takes to eliminate the 90% of the present microorganisms in the sample. This value serves us to evaluate the inactivations of the different microorganisms, in our case all less than 100 hours.

In the different tests of the control water also a reduction or inactivation of the microorganisms has taken place, the T90 obtained has been superior to 100 hours, that is to say, a reduction has occurred due to
natural inactivation, but this has been much smaller than the one caused by the effect of the reactor.


10 RESULTS.

10.1. - Inactivation of enterococos.

Experiment 1



Hours of treatment

Treated - Control - Linear (Control) - Linear (Treated)



Experiment 2



Hours of treatment

Treated - Control - Linear (Control) - Linear (Treated)



Experiment 3




Hours of treatment

Treated - Control - Linear (Control) - Linear (Treated)

Experiment 4



Hours of treatment

Treated - Control - Linear (Control) - Linear (Treated)



10.2.- Inactivation of Escherichia coli

Experiment 1



Hours of treatment

Treated - Control - Linear (Control) - Linear (Treated)


Experiment 2



Hours of treatment

Treated - Control - Linear (Control) - Linear (Treated)


Experiment 3



Hours of treatment

Treated - Control - Linear (Control) - Linear (Treated)



10.3.- Inactivation of bacteriófago B56-3.

Experiment 1



Hours of treatment

Treated - Control - Linear (Control) - Linear (Treated)


Experiment 2


Hours of treatment

Treated - Control - Linear (Control) - Linear (Treated)


Experiment 3



Hours of treatment

Treated - Control - Linear (Control) - Linear (Treated)



10.4.- Inactivation of the bacteriófago MS2.

Experiment 1




Hours of treatment

Treated - Control - Linear (Control) - Linear (Treated)



Experiment 2


Hours of treatment

Treated - Control - Linear (Control) - Linear (Treated)



Experiment 3



Hours of treatment

Treated - Control - Linear (Control) - Linear (Treated)


Results

From the kinetic values of inactivation, the T90 and T99 was calculated (in hours) of the different tested microorganisms.

Results of the T90 and T99 for the different experiments:







Des.est.: Standard deviation
I.C. Intervals of confidence
V. mín: Minimum value
V.máx.: Maximum value



Diagrams in graphics of the T90 and T99 for the different microorganisms tested:
Hours


T90 and T99 tested microorganisms

1: T90 for Enterococcus faecalis ATCC 29212
2: T99 for Enterococcus faecalis ATCC 29212

3: T90 for Escherichia coli ATCC 7000078
4: T99 for Escherichia coli ATCC 7000078

5: T90 for Bacteriófago MS2 ATCC 15597-B1
6: T99 for Bacteriófago MS2 ATCC 15597-B1

7: T90 for Bacteriófago B56-3 ATCC 700786-B1
8: T99 for Bacteriófago B56-3 ATCC 700786-B1


11 Discussion

In the present work inactivation experiences have been made, with different microorganisms, (two bacteria:
'Enterococcus faecalis and Escherichia coli; and two bacteriophage ones: MS-2 and B56-3)

In the tested conditions it was possible to calculate the T90, is to say time necessary to inactivate 90% of the population, or what is the same to reduce the present contamination in a logarithmic unit.

The T90 depends on the characteristics of the tested microorganism, thus in average we have a T90 value of 20 hours for Enterococcus faecalis; of 23 hours for Escherichia coli, of 6 hours for the bacteriophage MS2 and of 51 hours for the bacteriophage B56-3. The results of inactivation of proposed bacteriophage as virus indicating, this indicates to us that virus are also inactivated by Aqua HPS.

These results show evidence of the microbial inactivation, under test conditions, achieved by the action of the reactor, since all controls being made in parallel in the experiences presented/displayed leave a T90 superior to 100 hours. It is to say, the inactivation achieved by the reactor is significantly greater than the inactivation the micro organism suffer naturally when staying in the water.

The experiences made under the described conditions, materials and methods give survival data that in each case give a straight line of regression, simplifying the model would indicate a kinetic type of first order.
This allows calculating the T90 and T99. The possibility that cannot be excluded in a study much more detailed, was it possible to be link to another kinetic type of inactivation.

The T90 of the tested bacteria's are very close to each other and near to 20 hours (20 and 23 hours), like it happens with the T99, which is 35 and 37 hours for enterococos and E.coli respectively. As they are
microorganisms model, these can be indicative for the processes of inactivation of an ample spectrum of bacteria, which by the action of the reactor will act similar. Within this spectrum it is possible to think that they would be of the bacteria Legionella type. The indications of the manufacturer would therefore be fulfilled in whatever to the inactivation of
this type of bacteria and its application in diverse types of water installations.

This does not happen equally for the bacteriophage types, both microorganisms model, tested. Thus, the T90 of MS2 is of 6 hours as opposed to the 51 hours of the T90 of bacteriophage B56-3. This disparity of the results can be due to different characteristics as well by the bacteria applied to the bacteriophage types, as well as these to each other.

In summary, the presented/displayed results gives evidence to the effectiveness of the apparatus to elimination of bacteria and virus under this test conditions. From the results the supposition corresponds that this equipment (Aqua Hydro Physical System™) will have the capability, under suitable conditions of operation, to diminish bacterial increases in closed circuits of water and to keep the concentrations of any microorganisms at a low level, as it is expressed in the manufacturer product information.


12 REFERENCES

Anonymous (1995) ISO 10705-1: Water quality. Detection and enumeration of bacteriophages - part 1: Enumeration of F-specific RNA bacteriophages. Geneva, Switzerland: International Standardisation Organisation.

Anonymous (2000) ISO 10705-2: Water quality. Detection and enumeration of bacteriophages -part 2: Enumeration of somatic coliphages. International Organisation for Standardisation. Geneva, Switzerland: International Standardisation Organisation.

Anonymous (2002) ISO 10705-4: Water quality Detection and enumeration of bacteriophages -part 4: Enumeration of Bacteriophages infecting Bacteroides fragilis Geneva, Switzerland: International Organisation for Standardisation.

Grabow WO & Cobrough P (1986) Practical direct plaque assay for coliphages in 100-ml samples of drinking water. Appl. Environ. Microbiol. 52: 430-433.

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University of Barcelona, Spain, Dpt. of Microbiology