Comparison of three laboratory devices for UV-inactivation of microorganisms

e>

Pergamon

0273-1223(95)00256-1

Wat Sci Tech. Vol. 31, No. S-6, pp. 147-IS6, 1995. CopyrigblC I99S IAWQ PriDted it! Great Britain. All rigbts res«Ved. 0273-1 22319S $9'SO + 0-00

COMPARISON OF THREE LABORATORY DEVICES FOR UV-INACTIVATION OF MICROORGANISMS R. Sommer*, A. Cabaj**, D. Schoenen***, J. Gebel***, A. Kolch***, A. H. Havelaart and F. M. Schetst .. Hygiene-Institute, University of Vienna, A-1095 Vienna, Austria .... Institute of Medi1:al Physics, Veterinary University of Vienna, A-J030 Vienna, Austria .."'.. Hygiene-Institute, University ofBonn. D-5300 Bonn I, Germany t Laboratory of Water and Food Microbiology, National Institute of Public Health and Environmental Protection, 3720 BA Bilthoven, The Netherlands

ABSTRACT UV inactivation experiments of microorganisms have been performed and published by various workers for decades. Resulting data even of the same species of microorganisms may show important differences in UV-susceptibility. The reasons for these varying results could be found either in different biological conditions like culturing methods for preparing the test organisms or in technical problems regarding UV-irradiation equipment and dose measurement. Therefore three groups working on UV inactivation performed a collaborative study to find out which influences could be responsible for varying results in laboratory UV experiments. Each working group had developed a laboratory UV irradiation apparatus, which differed in technical construction and method for UV dose measurement. For our study we used as a test organism spores of Bacillus subtilis ATCC 6633 which were cultured in large quantity. freeze-dried and stored for all following experiments. Thereby we established controlled biological conditions. The first series of experiments in 1992 showed that differences in inactivation curves did occur, related especially to dose distribution in irradiation vessels, in irradiation geometry and in partial shadowing of UV light. Subsequently the irradiation procedure and methods for dose measurement were improved resulting in consistent, reproducible and comparable results. The equation of the regression curve was: log (N/NOI -0.013 0 + 0.18. A 2109 reduction would require a dose of 169 ± 11 J/m 2 , a 0.05 I. 3 log reduction 241 ± 9 J/m 2 , respectively (level of significance: a In recent years bioassay methods have been suggested in order to evaluate UV• disinfection plants. Therefore it will be of increasing public health interest to ensure the quality of laboratory UV irradiation devices used for calibration of test-organisms for these bioassays.

=

=

147

R. SOMMER tl al.

148

KEYWORDS

Ultraviolet irradiation; laboratory device; water disinfection; Bacillus subtilis spores INTRODUCTION

UV-inactivation experiments on microorganisms have been performed and published

by

various investigators

for

decades.

Due to various test

conditions, the results are often not similar and differ from one another even regarding the same species of test organism. Particularly

earlier

publications

lack

precise

descriptions

of

irradiation

arrangement and dose measurement. Therefore no conclusions can be drawn. The necessary information comprises the type of irradiation vessel, depth of the layer irradiated, starting concentration of the suspension of

test organisms,

transmittance at 254 nm of the suspension, type of UV lamps and arrangement, method of dose measurement, irradiation time, etc. In addition the use of various measurement units confuses the situation. Even in recent research, data on UV susceptibility of microorganisms investigated by various working groups conspicuously diverge from one another. Some examples shall be given. A 5 log inactivation of E. coli needs a UV dose of 350 J/m 2 on the one hand (Zemke and Schoenen, 1989) or 150 J/m 2 (Sommer et al., 1989t on the other hand. 2 log UV inactivation of MS2 phage occurs with doses of 400 J/m 2 (Havelaar et al., 1991) and 184 J/m 2 (Wiedenmann et al., 1993) respectively, whereas far more than 250 J/m 2 for 1 log reduction is

reported by Battigelli et al. (1993). A 3 log reduction of Bacillus subtilis spores ATCC 6633 requires 240 J/m 2 (Qualls et al., 1983), 630 J/m 2 (Chang et al., 1985) and 250 J/m 2 (Sommer et al., 1989). These deviations in UV susceptibility can not just

be explained by biological test conditions, for

example the use of different culturing methods for preparing the test organisms. Therefore three groups working on UV inactivation performed a collaborative study to find out which influences could be responsible for varying results in laboratory UV experiments.

MATERIALS AND METHODS

TEST ORGANISM Spores of Bacillus subtilis ATCC 6633 were cultivated by method A as described elsewhere (Sommer and Cabaj,

1993a). Spore suspension was

149

Three laboratory devices for UV-inactivation of microorganisms

ultrasounded and aliquots of 3 ml were filled into glass vials and freeze-dried (EF03, Edwards High Vacuum, Ltd). The content of each vial was about 1.0x 109 spores. A number of vials sufficient to perform all experiments were sent by special delivery to the other two laboratories and stored at room temperature. For the irradiation experiments one vial each was diluted in 750 ml sterile, distilled water. About ten thousand cells of the spore solution each were microscopically

checked

for

single

cells

before

freeze-drying

and

after

reconstitution. The concentration of the test suspension was approximately 1.3x 106 spores per ml, the transmittance at 254 nm was 93% (depth 1 em). Pour plating method was used to measure the spore concentration before and after UV irradiation. The medium for measuring the spore concentration was Plate Count Agar (CM 325, Oxoid) from the same batch and was delivered to the other two laboratories as well. Experiments were performed in duplicate, and curves of reduction (log NINo) versus UV dose were then computed. The tests for examining the influence of the transmittance of the suspension being irradiated and the different irradiation geometries were carried out with

B. subti/is spores ATCC 6633 cultivated by method C (Sommer and Cabaj, 1993a). The transmittance of the spore suspension was reduced by adding sodium thiosulfate. UV-IRRADIATION Experimental design A A bank of 10 low pressure mercury UV lamps (EK 36, Katadyn, length 500 mm) were horizontally suspended over the irradiation vessel; 25 ml suspension each were irradiated in plastic petri dishes (diameter 90 mm) and were stirred on a magnetic stir plate during irradiation. The UV dose was varied by increasing UV intensities, at a constant exposure time of 30 s each. UV irradiance was measured by a selective detector (SED 240, International Light) at a reference point during the experiments and integrated over the exposure time by a dosemeter (lL 1700, International Light). UV dose as space exposure was calculated taking into consideration the transmittance at 254 nm and irradiation with parallel and divergent rays (Sommer et a/., 1989; Cabaj, in preparation). The experiments using irradiation of the vessel with parallel beams were done after increasing the distance between lamps and vessel and fixing a diaphragm (50x10 mm) 10 mm below the lamps. Experimental design B One low pressure mercury UV lamp (Sterisol NN 30/89, Hereus) was vertically situated

at

a distance of 500 mm from the irradiation vessel. The vessels were

quarz glass cuvettes (149x29x5 mm) containing 30 ml suspension. The

R. SOMMER tt al.

150

irradiation dose was varied by increasing exposure time and determined by a modified potassium ferrioxalate actinometer (Zemke and Schoenen,

1989.

Zemke et al.• 1990). For the second trial the UV irradiation apparatus was rotated 90 0 and the cuvette as irradiation vessel was replaced by a petri dish (diameter 90 mm) containing 25 ml test suspension (Schoenen et at, 1993). Experimental design C Two low pressure mercury UV lamps (TUV 40W, Philips) were situated above the Irradiation vessel. At a distance of 0.05 m below the lamps a stainless steel plate with holes of 2 mm at a spacing of 4 mm was placed to intercept non· vertical UV beams. A two litre volume of suspension was irradiated in a perspex vessel (380x230x50 mm) with rounded corners while being stirred by six magnetic stirring devices simultaneously (depth of the suspension 25 mm) as described elsewhere (Nieuwstad et al., 1994). The irradiated suspension was sampled as a function of time. The average UV intensity was measured with a calibrated UVX radiometer using a UVX-25 sensor (UVP, Inc.). The UV dose was calculated by summation of the products of the time intervals between sampling and the average UV intensity during that period regarding the transmittance at 254 nm and the thickness of the layer (Havelaar et al.• 1990).

RESULTS The results of the first trial (1992) given in figure 1 showed great differences in the inactivation data for the test organism, a 3 log reduction required doses of 200. 400 or 600 J/m 2 . In particular, the inhomogeneous results given by experimental design 8 were conspicuous. Moreover we observed at the end of the inactivation curves a plateau with device A and a slower inactivation rate with device C. In

figure

2 the

reduction data

with

and

without consideration

of the

transmittance at 254 nm of the testing solution for the dose calculation are presented. These experiments were performed with experimental design A and yielded

different

inactivation curves of the same test organism. A 3 log

reduction was reached either with 380, 460 or 620 J/m 2 . If the transmittance has been taken into account a correct reduction curve resulted. In figure 3 inactivation data obtained with divergent and parallel UV irradiation are compared. It can be seen that there was a significant difference in inactivation curve, if non-parallel rays have not been' taken into account in calculating the UV dose.

151

Three laboratory devices for UV-inactivation of microorganisms

o reduction log (NINo) -1

*A (Vienna) *A (Vienna) +B (Bonn) +B (Bonn)

-2

+C (Bilthoven) +C (Bilthoven) - - detection limit --- 3 log reduction

-3 -4

-5

-6

+----r--_r_-~--_r_-~-______<

o

200

400

600

800

1000 1200

UV dose (J/m 2 ) Figure 1. UV inactivation of B. subtilis spores in three different laboratory irradiation devices (first trial, 1992).

reduction log (NINo)

o....-==::-----=------------------, ,...------..., +T (90%) -&T (51%)

-1

....T (14%1

-2

+inactivation curve - - 3 log-reduction

-3 -4 -5 -6

+-----..--...--.....-.........,r---.,.--......,..--....- - l 300 400 500 600 700 800 o 100 200 UV dose (J/m 2 ) Figure 2. UV inactivation of bacterial spores with and without taking into consideration the transmittance T of the testing solution (T 254 nm; 10 mm) to the calculation of the UV dose.

R. SOMMER et al.

152

reduction log (NINo)

o ,.....~~~---;:;::::::;:;:;::::::;=.=========:;-, e-parallel irradiation -+-without consideration of divergent rays .with consideration of divergent rays - - 3 log-reduction

-1 -2 -3 -4

-5 -6

+----r----...---r---r---r---~--_r_-___l

o

100

200

300

400

500

600

700

800

UV dose (J/m 2 ) Figure 3. Inactivation of bacterial spores by irradiation with parallel and non parallel UV light, with and without taking into consideration the divergent rays to the calculation of the UV dose.

o reduction log (NINo) -1 *A lVienna) *A (Vienna) .B IBonnl .B IBonnl +C (Bilthoven) +C (Bilthoven) - - detection limit ·_·3 log-reduction

-2

-3 -4

-5 -6 +---.----.-----,---...--r---I

o

200

400

600

800 1000 1200

UV dose (J/m 2 ) Figure 4. UV inactivation of 8. subtilis spores in three different laboratory irradiation devices (second trial, 1993).

Three laboratory devices for UV-inactivation of microorganisms

IS3

Figure 4 shows the results of the second collaborative trial (1993). The inactivation data with all three devices were consistent, reproducible and comparable. After a shoulder the inactivation rate was constant following first order kinetics. A decreasing inactivation rate obtained with device C could be observed after 4 log reduction. Out of the data of the second trial the equation of the regression curve for the test organism B. subtilis was: log (NINO)

=-

0.013 0 + 0.18. Therefore a 2 log reduction required a dose of 169 ± 11 ± 9 J/m 2 , respectively (95 % confidence

J/m 2 , a 3 log reduction 241 interval). DISCUSSION

Inactivation data of microorganisms causing waterborne diseases have to be investigated to establish the required UV dose for a safe water disinfection. These experiments must be done in laboratory devices by irradiation in batch. Testing of microorganisms in UV plants for water disinfection is not useful, because there is no possibility to measure UV dose physically or chemically in flow through reactors (Sommer and Cabaj, 1993 a; 1993 b). Although

biological

differences occur even

within

the

same

species

of

microorganisms, for example due to cultivating methods (Sommer and Cabaj, 1993 al. this paper demonstrates that also an inadequate experimental design can be a major reason for diverging UV inactivation data. As shown in the presented data it is not .simple irradiation apparatus. Sources of error include

to get correct results in laboratory UV

unsuitable irradiation vessels as shown for quartz

glass cuvettes by Schoenen et al. (1993). Of special interest is the partial shadowing appearing in irradiation vessels resulting in a remaining concentration of test organisms, therefore no total inactivation in batch irradiation can be reached. Menisci of the solution can cause areas of total or partial shadowing of the UV light as demonstrated schematically in figure 5. These insufficiently irradiated volumes can result in no further (trial 1, data Al or slower (data C) decrease of the inactivation rate at the end of the inactivation curve. To avoid this problem it is useful in irradiation experiments carried out in petri dishes to take samples of small volumes from the centre of the vessel (Schoenen et al., 1993). To calculate the applied UV dose the 254 nm transmittance of the test solution and the irradiation geometry have to be taken into consideration. As shown in the presented data with artificially reduced transmittance of the test solution, correct data can only be reached including the influence of transmittance into dose calculation.

v: ~

I

1'y

~tl_J~_LJ

I

I

I

I

AIR

___ ~ _____ ~x I I 1 1 I

-.I -.I

~

m ~ ~

~ 0

I I I

-.I -.I

AIR

~

1- mm I

WATER

I I I I

m ~

WATER

~

CI>

0

:z:

~

0 0

I l...2mm

I I I I I 1

I I -!--3mm I I

(a) Concave meniscus (hydrophilic material of the vessel): the pencil of rays coming from above is, due to refraction, the below divergent in meniscus resulting space decreasing irradiance with increasing the below depth meniscus.

?'

Convex meniscus (b) (hydrophobic material of the vessel): the pencil of rays coming from above is, due to refraction, near the convergent meniscus inside the liquid. Additionally, a completely unirradiated volume inside exists solution the (hatched).

Figure 5. Schematic drawing of the influence of the meniscus of the water at the border of a container wall on the radiation-field inside the liquid.

.,....~

Three laboratory devices for UV -inactivation of miaoorganisms

By using the whole length of a UV source higher intensities and therefore shorter irradiation times can be obtained, though a complex calculation of the space irradiance due to divergent rays has to be applied (Cabaj, in preparation). Under these circumstances stirring of the test solution is essential to ensure that each microorganism receives the same irradiation dose. If the non-parallel light, which irradiates the test vessel, is not taken into consideration false and too susceptible inactivation curve results. This effect will be intensified if the test solution has a low transmittance at 254 nm. Following these requirements our investigation demonstrated that it is possible to get correct results even with non-parallel light. Moreover, the necessity of continuous control and registration of the UV intensities during irradiation experiments has to be mentioned. The lack of an on line measurement could be another element of uncertainty due to variation in the lamps output during Irradiation tests. Reliable UV inactivation data are not only important regarding pathogenic and indicator microorganisms but also in view of microorganisms used for bioassay methods in order to test UV-disinfection plants (Qualls and Johnson, 1983; Havelaar et al., 1990; 1991; Nieuwstad and Havelaar, 1993; Sommer and Cabaj, 1993). The resulting laboratory inactivation data are fundamental to the design of water disinfection plants and to the evaluation of disinfecting capacity of UV plants. Therefore more attention has to be paid to the quality of laboratory UV irradiation devices; this has to be ensured as for example by collaborative trials.

REFERENCES Battigelli, D.A., Sobsey, M.D. and Lobe, D.C. (1993). The inactivation of Hepatitis A virus and other model viruses by UV irradiation. Wat.Sci. Tech., 27 (3-4), 339-342. Cabaj, A. Calculation of the space irradiance for UV irradiation device (in preparation) . Chang, J.C.H., Ossof, S.F., Lobe, D.C., Dorfmann, M.H., Dumais, C.M., Qualls, R.G. and Johnson, J.D. (1985). UV inactivation of pathogenic and indicator microorganisms. Appl.Environ.Microbiol., 49, 1361-1365. Havelaar, A.H., Meulemans, C.C.E., Pot-Hogeboom, W.M. and Koster, J. (1990). Inactivation of bacteriophage MS2 in wastewater effluent with monochromatic and polychromatic ultraviolet light. Wat.Res., 24, 1387• 1393. Havelaar, A.H., Nieuwstad, Th.J., Meulemans, C.C.E. and van Olphen, M. (1991). F-specific RNA bacteriophages as model viruses in UV disinfection of wastewater. War. Sci. Tech., 24(2),347-352. Nieuwstad, Th.J. and Havelaar, A.H. (1994). Kinetics of batch UV inactivation of bacteriophage MS2 and microbiological calibration of a UV pilot plant. Toxic Hazard.Subst.Contr. in press. Qualls, R.G. and Johnson, J.D. (1983). Bioassay and dose measurement in UV disinfection. Appl. Environ. Microbiol., 45, 872-877.

ISS

156

R. SOMMER el al.

Schoenen, D., Kolch, A. and Gebel, J. (1993). Influence of geometrical parameters in different irradiation vessels on UV disinfection rate. Zbl.Hyg., 194, 313-320. Sommer, R., Weber, G., Cabaj, A., Wekerle, J., Keck, G. and Schau berger, G. (1989). UV-inactivation of microorganisms in water. Zbl.Hyg., 189, 214• 224 Sommer, R. and Cabaj, A. (1993 a). Evaluation of the efficiency of a UV plant for drinking water disinfection. War. Sci. Tech., 27.(3-4),357·362. Sommer, R. and Cabaj, A. (1993 b). Prototype Testing: a Promising Tool to proof the Safety of UV Disinfection Plants. In: Safety of Water Disinfection: Balancing Chemical and Microbial Risks, ILSI Press, Washington D.C. 569-572. Wiedenmann, A., Fischer, B. Straub, U., Wang, C.-H., Flehmig, B. and Schoenen, D. (1993). Disinfection of Hepatitis A virus and MS-2 coliphage in water by ultraviolet irradiation: comparison of UV-susceptibility. War. Sci. Tech., 27 (3-4), 335-338. Zemke, V. and Schoenen, D. (1989). UV disinfection experiments with E.coli and actinometric determination of the irradiation intensity. Zbl.Hyg., 188, 380-384 Zemke, V., Podgorsek, L. and Schoenen, D. (1990).Ultraviolet disinfection of drinking water. 1.communication: inactivation of E.coli and coliform bacteria. Zbl.Hyg., 190,51-61