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Inactivation of bacteriophage MS2 in wastewater effluent with monochromatic and polychromatic ultraviolet light
War. Res. Vol. 24. No. I 1. pp. 1387-1393. 1990 Printed in Great Britain.All rights reserved
0043-1354/90 $3.00 + 0.00 Copyright C) 1990PergamonPress plc
INACTIVATION OF BACTERIOPHAGE MS2 IN WASTEWATER EFFLUENT WITH MONOCHROMATIC AND POLYCHROMATIC ULTRAVIOLET LIGHT A. H. HAVELAAR l~),C. C. E. MEUL~dANS 3,W. M. POT-HOOlmOOM~ and J. KosTI~ 2"* Laboratory of Water and Food Microbiologyand 'Centre for Mathematical Methods, National Institute for Public Health and Environmental Protection, P.O. Box I, 3720 BA Bilthoven and 3Laboratory Lighting Technique and Application, Philips Lighting B.V., P.O. Box 80020, 5600 JM Eindhoven, The Netherlands (First receired January 1990; accepted in revised form June 1990) A~tract--A model using bacteriophage MS2 for the calculation of the effective dose in u.v.-disinfection by polychromatic light from a medium pressure mercury lamp was studied. The model was based on first-order inactivation kinetics, the u.v.-absorption spectrum of MS2-RNA as the action spectrum and Lambert-Beers' law for light absorption by the effluents. The u.v.-sensitivity of the phage was calibrated with a low pressure mercury lamp; the inactivation rate constant (k) was 0.0106 m2/J, which corresponds well with previously reported data. The model fitted actual data in most experiments, although tailing effects were observed. In some experiments a different value ofk was necessary to fit the actual inactivation curves, varying between 0.0077 and 0.0121 m2/J. The MS2 phage was found suitable for calibration of u.v. sources. Key words--u.v.-radiation, disinfection, bacteriophage MS2, mathematical model, kinetics
INTRODUCTION The use of u.v. radiation for the disinfection of wastewater effluents is an emerging technology. In the past 15 years it has been developed and applied on a practical scale particularly in North America (Anon., 1986). Improvements in reactor design and lamp performance have resulted in a more reliable and economical operation of u.v. disinfection units, which are now feasible for wastewater effluents. The" presently used installations are predominantly equipped with low pressure mercury vapour lamps. These lamps have a high output of germicidal u.v. radiation per watt of electrical energy consumed, but have a low field-intensity. Medium pressure mercury lamps (or others such as antimony lamps) produce a higher u.v. output per lamp but are less efficient because a substantial part of the total energy is emitted as visible radiation (Fig. 1), and energy losses by heat production are greater than for low pressure lamps. Nevertheless, there is a growing popularity of the medium pressure lamps because they require smaller reactors, which is an advantage in larger installations. It has recently been shown for drinking water applications that reduced costs on capital investment and lamp replacements counterbalance the increased energy costs (Kruithof et ai., 1989). Similar calculations have not yet been made for wastewater applications. *Present address: ICIM, P.O. Box 5809, 2280 HV Rijswijk, The Netherlands.
Dose assays for medium pressure lamps are difficult because polychromatic radiation is emitted instead of the essentially monochromatic radiation of a low pressure lamp. The disinfection efficiency of u.v. radiation and u.v. absorption by effluents are dependent on wavelength. A model for the calculation of the effective u.v. dose has been proposed (Meulemans, 1987). In this paper we present an experimental calibration of this model using the F-specific R N A bacteriophage MS2. Reasons for choosing this organism are the following: (1) MS2 is a single-strand R N A virus and consequently its dose-response curve for u.v. inactivation follows first-order kinetics (Harm, 1980). (2) The structure and size of MS2 is similar to that of the human enteroviruses. (3) MS2 has a relatively high u.v. resistance, comparable to that of bacterial spores (Harm, 1980). (4) MS2 is not pathogenic to man, so it can be used for calibration of full-scale reactors without additional safety measures. (5) The organism is easily cultivable in titres up to 10~2pfu/ml, which makes its use for calibration of full-scale reactors advantageous over presently recommended spore suspensions of Bacillus subtilis, which can only be cultured up to 10s cfu/ml (Quails and Johnson, 1983). The model calculations were carded out in the spectral region between 240 and 300 nm, thus partly including both the u.v.-C region (200-280 nan) and the u.v.-B region (280-315 nm). The lower limit was
1387
A. H. HAVELAARet al.
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MATERIALS AND METHODS LOW PRESSURE MERCURY LAMP
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In the course of the experiments, two versions of the irradiation apparatus were used. The units (Fig. 2) consisted of lamp housing A and collimating tube B, on which quartz cell C was mounted. The lamp housing was provided with either a low or a medium pressure mercury lamp. and with an adjustable cooling device consisting of a fan and thermistor for temperature control and recording, to assure that the lamps were burning at their optimal temperature. The bottom of quartz cell C, which was filled with water or secondary effluent, was a window of optical quality in order to improve the uniformity of the u.v. radiation passing through this window, where a Pyrex vessel D (dia 3 cm) with phage suspension could be positioned. During exposure, the phage suspension was constantly stirred by a Teflon-coated spinning bar driven by spinning device E. The first version of the apparatus was constructed with a simple tubular collimator (length 25 cm); in order to improve the uniformity of the irradiation field a second version was constructed in which unwanted reflections from the collimator surface were intercepted by several diaphragms. Two different u.v. sources were used. The low pressure lamp was an M-shaped 15 watt version made in quartz (arc length 26 cm), only available for use within the Philips Company. The medium pressure lamp was an HPK 125 watt lamp with an arc length of 3 cm. The relative spectral distributions of the radiation emitted by these two lamps are presented in Fig. 1. In both versions of the apparatus a flat reflector was mounted behind the low pressure lamp. In version 1 there was a curved reflector behind the medium pressure lamp, but this was omitted in version 2 because of unacceptable reflections and too high irradiance levels giving too short an interval between withdrawal of samples from vessel D.
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Fig. 1. Irradiation spectra of a low and a high pressure mercury lamp. chosen because at wavelengths below 240 n m the t r a n s m i s s i o n o f secondary effluents decreases very rapidly ( u n p u b l i s h e d data) a n d the u p p e r limit was chosen because at wavelengths a b o v e 300 n m the inactivation efficiency of u.v. is negligible.
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Fig. 2. Schematic drawing of irradiation apparatus, version 1 (left) and version 2 (right). A, lamp housing with M-lamp or HPK-lamp; B, collimator tube with diaphragms in version 2; C, quartz cell; D, irradiation vessel with MS2 suspension and spinning bar; and E, magnetic spinning device.
Modelling polychromatic u.v.-disinfection Calibration of the irradiation units was performed with a CARY 17D double monnchromator calibrated by reference to standard lamp, from the National Physical Laboratory (U.K.). According to the calibration methods used, the absolute irradiance levels had a standard deviation of 10%. In order 'to facilitate the flexibility during the experiments a UVX radiometer (UVP Inc., U.S.A.) equipped with a UVX-25 sensor was calibrated simultaneously.
Effluents Secondary effluents were obtained from STP De Bilt and the pilot plant of the Technical University Delft (Laboratory of Sanitary Engineering) and were filtered through 0.22/~m membranes before use. Transmission spectra of the effluents between 240 and 300 nm were measured at 5 nm intervals in a Perkin-Elmer 550S two-beam spectrofotometer in I em quartz cells against Millipore Super-Q water. Transmission at 255 nm ranged between 50--60% cm-I for the STP effluent and 40-50% for the pilot plant effluent.
Microorganisms and microbiological methods Bacteriophage MS2, its host strain E. coli WG21 and the culturing methods were previously described (Havelaar and Hogeboom, 1984). For irradiation experiments, phages were suspended in M9-buffer (Mattern et al., 1965) which was freshly prepared before each experiment as follows: 50 ml vol of two separately autoclaved stock-solutions (I: NH4CI 20g, NaC! 100g, MgSO4.7HzO 2 g in 1000ml water; II: KH2PO 4 60 g, Na 2 HPO 4 120 g in 1000 ml water) were mixed and diluted to a final volume of 1000 ml; by following this procedure it was possible to have a u,v. absorbance which was consistently less than 2% cm-m; pH was 7.0 _+0.05.
Experimental design The general model of the survival curves was based on the assumption of first order kinetics as expressed in the following formula:
N t i S o = e - t " ~ = e -kE'~'
(1)
where N, = number of survivors at time t (pfu/ml) N o = number of bacteriophages at time 0 (pfu/ml) k = rate constant (m2/J) Hat= effective u.v. dose (J/m 2) Eat = effective u.v. irradiance (W/m 2) t = time (s).
Co)Low pressurelamp; effluentin lightpath;MS2 inbuffer. In the following two sets of three parallelexperiments, cell C was filled with 2 cm of Super-Q water and Eo was measured as described above. Subsequently, cellC was filled with 2 and 4 c m of membrane filtered(0.22#m) effluent, resix~tively. The intensity of u.v. radiation pas,~ng through the effluent was estimated by measuring the irradiance at the position of vessel D. This value was used to estimate the irradiation times required for each experiment to produce a dose range similar to that in the experiments under (a). The measured irradiance through the effluent was not used in further calculations; these were based on Eo measurements through Super-Q water. The model used was:
Ed = Eoe-"-~a f .
(3)
"2..q = - In ('c~)
(4)
where ~q~ -- extinction coefficient at 254 nm (cm- J) r2~ = transmission at 254 nm (cm- J) as measured in a two-beam spectrophotometer d = depth of effluent (cm).
(c) Medium pressure lamp; Super-Q in light path; MS2 in buffer, The model used was: 30O
Eat ---Eo fp Z R(A2)S(A~)
(5)
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where fp = correction factor for UVX meter response to polychromatic radiation R(AA)=fraction of total radiation between 240 and 300 tun, emitted in a 5 nm interval S(A2) = sensitivity of MS2 to u,v. radiation in a 5 nm interval, relative to the sensitivity at 254 nm A2 = integration interval (5 nm). S(A~) was taken from the u.v. absorption spectrum of MS2 RNA (Strauss and Sinsheimer, 1963). Data for multiples of 5 nm were used as central values, e.g. at 250 nm for the 247.5--252.5 nm interval.
(d) Medium pressure mercury lamp; effluent in light path; MS2 in buffer. The experiments described above under (b) were repeated with the medium pressure HPK-lamp mounted instead of the M-lamp. The model used was: 3OO
Eat = Eo f , ~ R(AA)S(A2) e - * ~
Eat was calculated according to the experimental set-up.
Irradiation apparatus, version 1 (a) Low pressure lamp; Super-Q in light path; MS2 in buffer. A first series of experiments was carried out with the low pressure M-lamp and a layer of 2 em Millipore Super-Q water in cell C. After stabilization of the u.v.-source, the irradiance was measured at the position of vessel D at nine different measuring points using the UVX radiometer. The average Jr'radiance Eo was calculated as the average of these data. Subsequently, vessel D was filled with 20 ml MS2 suspension in M9-buffer at a titer of approx. 5 × l0 s pfu/ml and was placed under the lamp, while being covered with a piece of carton. Samples (100/~1) were withdrawn before the vessel was placed under the lamp and after exposure for 10, 20, 30, 40 and 50 s, and were assayed for surviving phage in duplicate. This experiment was repeated three times. The model used was:
Eat ffi Eof -
1389
(2)
where E 0 = average irradiance through Super-Q water as measured at the position of vessel D with the UVX meter (W/m 2) f ~ = correction factor for UVX meter response with monochromatic radiation at 254nm.
(6)
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where ct(AA) = extinction coefficient (crn-') in a 5 nm interval.
Irradiation apparatus, version 2 The above experiments [(a)--(d)] were repeated (in singular) with the second version of the apparatus, using the formulas as indicated. The second version, equipped with the M-lamp or the HPK-lamp was also used to measure the inactivation of MS2 suspended in filtered effluent. In these experiments, cell C was filled with 4 cm Super-Q, and vessel D with 20 ml (1.7 cm) of effluent, seeded with MS2 to a level of approx. 106 pfu]ml. The models used were:
(e) Low pressure mercury lamp; Super-Q in light path; MS2 in effluent. Integration of Lambert-Beer's law over the depth of the irradiated effluent (Roeber and Hoot, 1975). e-=~d
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( f ) Medium pressure mercury lamp; Super-Q in light path; MS2 in effluent. Eat = Eof p ~" R(AA)S(AA) 1 2,0
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A. H. HAVELAAR et al.
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Statistical analyses The three sets of parallel experiments described in the section on Experimental design sub-sections (a) and (b) were used to estimate the value of the inactivation rate constant k in equation (1) by finear regression analysis after logarithmic transformation of the inactivation ratios. In the second stage of the regression analysis, the condition was impfied that at t = 0 the inactivation ratio should also be 0, i.e. the regression was forced through the origin. The computed value of k was then used to predict the inactivation curves for the experiments subsections (c), (d), (e) and (f). The actual data of the fourteen experiments were compared with these predictions by a goodness-of-fit test. For each of the five data points in each inactivation curve, the squared difference between the measured and the predicted inactivation ratio was divided by the variance of the predicted value, computed from the regression analysis. The test parameter was calculated by adding these five values and follows a X2"distribution with 5 d.f. If the value of the test parameter is less than the critical Z 2 value at a size of 5%, it may be concluded that the actual data significantly fit the predicted values (Draper and Smith, 1981).
Results with irradiation apparatus, version I(MS2 in buffer)
RESULTS
Calibration of light sources and UVX meter The correction factors for the spectral response o f the UVX radiometer were determined for the spectral region between 240 and 300 rim. This was rather simple for the low pressure lamp because the 254 nm mercury line is the only emission in this region, but more complicated for the polychromatic radiation from the medium pressure lamp. Furthermore, the correction factor found for the medium pressure lamp can only be applied for the spectral energy distribution as emitted by the lamp. When radiation passes through effluent, the spectral distribution is modified and consequently this radiation cannot be measured directly with the UVX meter. Therefore the irradiance was always measured with Super-Q water in cell C and the irradiance passing through the effluent was calculated as described above. 10 o
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In order to find the absolute value of the u.v. irradiance, the radiometer readings were multiplied by a correction factor f = = 1.1 (SD = 0.03) for the low pressure lamp and f p = 1.6 ( S D = 0 . 1 ) for the medium pressure lamp. The diameter of the irradiation field at a distance of 5 cm below the optical window of cell C was 6 cm. The uniformity of this field was poor near the margins and only the central area with a diameter of 3 cra was used. Uniformity of this field was measured with 2 or 4 cm Super-Q water in cell C in order to have similar reflections and refractions to those in the inactivation experiments. The results of the uniformity measurements indicate an average SD of 5% with the low pressure lamp. With the medium pressure lamp the SD was 17% in version 1 of the apparatus, which led to the construction of version 2. In this version the SD with 4 cm Super-Q was 4%.
Table I. Regression analysis of data from experiments with MS2 in M9-buffer and u.v. radiation from a low-pressure (M) lamp (irradiation apparatus version I)
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Results of experiments with a low pressure lamp and Super-Q or effluent in the light path are shown in Fig. 3. Linear regression analysis showed that all inactivation curves had a negative y-intercept of 0.07-0,20 log,-units, indicating that inactivation proceeded more rapidly in the first few seconds of the experiments than later. The y-intercept of experiments with 2 cm Super-Q and 4 cm effluent were significantly different from 0. The regression analysis was repeated with the condition that all inactivation curves should start in the origin (no inactivation at t ffi 0), and the correlation coefficients were found to increase slightly. This approach was chosen for further analysis of the data. The results of this regression analysis are summarized in Table 1. The average value of the inactivation rate constant k, calculated from this series of nine experiments was found to be 0.0106 _+ 0.0005 m2/J, and was used in all further calculations as described below. Inactivation data with a medium pressure lamp and Super-Q in the light path could not be determined accurately in the first version of the apparatus because the very high irradiance resulted in more than 3 log-units inactivation within 20s; hence sampling time could not be controlled with an accuracy which was comparable to that of other experiments. The validation of this part of the model will be described in the section on experiments with the second version of the apparatus.
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Fig. 3. Inactivation of bacteriophage MS2 in M9-buffer by u.v. radiation from a low-pressure (M) lamp after passage through 2 cm Super-Q (O, (D and 0), 2 cm (A, A and A ) or 4 cm ( + , x and ,) effluent (3 separate experiments each, irradiation apparatus version I). , Regression line.
n.y. Absorption medium 2cm Super-Q 2 cm Effluent 4 cm Effluent All experiments
Rate constant and 95% conf. int. (k, m:/J) 0.0104 0.0103 0.0109 0.0106
_ 0.0010 + 0.0008 ± 0.0010 -L-0.0005
Correlation coefficient (r) 0.99 1.00 1.00 1.00
Modelling polychmmatic u.v.-disinfection Input data as used in the calculation of the effective dose with medium pressure lamps and effluent in the light path are shown in Fig. 4. This figure shows the spectral distribution of the emitted radiation and the sensitivity coefficient of MS2 as used in all calculations. The absorption spectrum of a particular sample of effluent is also shown; although actual data differed between experiments, the general shape of the spectra was relatively constant. Figure 5(a) and (b) show data from experiments with 2 and 4 crn effluent in the light path, respectively. The predicted inactivation curve, as indicated by the solid line, fitted the actual data significantly with 4 c m of effluent [Fig. 5(b)], but was found to tail off with 2 cm of effluent [Fig. 5(a)]; only one experiment out of three showed a good fit and one experiment showed a barely significant fit. While performing the experiments we noted that, despite the use of the long collimating cylinder, the u.v.-intensity was not equally distributed over the irradiation vessel, which was particularly obvious with 2 crn of effluent. Results with irradiation apparatus, version 2 (MS2 in buffer)
1391
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Fig. 4. Spectral distribution o f (A) emitted radiation from a medium pressure (HPK) lamp; (B) extinction coefficient of secondary effluent; (C) relative u.v. sensitivity o f bacteriophage MS2; and (D) calculated effective u.v. irradianee. Note the scale difference between (A) and (D). Experimental data: E 0 = 8.04 W/m2; d = 4.0 cm; E~r = 0.367 W/m s. For explanation o f symbols, see text.
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Fig. 5. Inactivation of bacteriophage MS2 in M9-buffer by u.v. radiation from a medium.pressure (I-IPK) lamp after passage through effluent (irradiation apparatus version 1). (A) 2cm effluent; (O) Z2=3.1; (A) Z:=I0.3; ( + ) g2=34.4; (B) 4cm effluent; (O) X2=3.0; (A) Z2=3.0; ( + ) g2 = 4.2. Critical value for X2 = 11.1 (5 d.f., P = 5%). , Predicted inactivation curve. pressure lamps, respectively. Data with the low pressure lamp were evaluated against the predicted inactivation curves using the average value of k =-0.0106 J/m 2 found with the first apparatus. With Super-Q in the light path, a statistically significant fit was found, although the inactivation curve again showed a tendency to tail off; with effluent in the light path all data points were below the predicted line indicating a rapid inactivation during the first few moments of the experiments. By means of linear regression a value of k = 0.0121 + 0.0017 J/m: was calculated. With the medium pressure lamp, all sets of data showed good fits with the prediction based on k = 0.0106 J/m, 2 and less tailing was observed. Results with irradiation apparatus, version 2 (MS2 in e~uenO If MS2 is to be used for biological calibration of u.v. reactors, dose-response curves should also be known for phage suspended in effluent. Two series of
1392
A. H. HAv~.7o~ et al.
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Fig. 6. Inactivation of bacteriophage MS2 in M9obuffer by u.v. radiation from a low pressure (M) lamp after passage through 4cm Super-Q (O) X2=6.6 or 4cm effluent (A) Z2= 11.7 (irradiation apparatus version 2). , Predicted inactivation curve.
Fig. 8. Inactivation of bacteriophage MS2 in 1.7 cm secondary effluent by u.v. radiation from a low pressure (M) lamp after passage through 4 cm Super-Q (irradiation apparatus version 2); (O) X2__ 2.1; (A) X2= 1.4. - - , Predicted inactivation curve.
experiments were done, with low and medium pressure lamps, respectively, and with 4 cm Super-Q in the light path in all cases to assure the same distribution of irradiance over vessel D as in other experiments. Results are shown in Figs 8 and 9. A good correlation between predicted and actual data was found with the low pressure lamp; the actual reduction was significantly less than predicted with the medium pressure lamp. These data fitted a regression model with k -- 0.0077 -t- 0.013 J/m 2. In both cases, the correspondence between two separate experiments, run on different days, was very good.
secondary effluent by polychromatic u.v.-radiation from a medium pressure mercury lamp can be reasonably accurately modelled by a stepwise approach. Lambert-Beer's law was used to describe the absorption of radiation by the effluent and the absorption spectrum of the MS2-RNA was assumed to be equivalent to the action spectrum of the u.v. radiation. In view of the uncertainties involved in measuring u.v. irradiance and in calibrating measuring equipment this approach was tested using 5 nm intervals rather than I nm intervals or a continuous approach. Besides being more realistic, this model also has the merit of simplicity. For MS2, which is a singlestrand RNA-bacteriophage not subject to host-cell reactivation, first-order inactivation kinetics was to be expected (Mattern et aL, 1965; Harm, 1980). By regression analysis it was shown that none of
DISCUSSION
The experiments described in this paper have shown that inactivation of bacteriophage MS2 in 10o
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Fig. 7. Inactivation of bacteriophage MS2 in M9-buffer by u.v. radiation from a medium pressure (HPK) lamp after passage through 4 cm Super-Q (O) x 2= 9.5 or 4 cm effluent; (~) ~ 2 = 1.2 (irradiation apparatus version 2). Predicted inactivation line.
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Fig. 9. Inactivation of bacteriophage MS2 in 1.7 cm secondary effluent by u.v. radiation from a medium pressure (HPK) lamp after passage through 4cra Super-Q (irradiation apparatus version 2); (O) Z2=28.0, (/%) Z2 = 38.3. , Predicted inactivation curve.
Modelling polychromatic u.v.-disinfection the sets of inactivation data exactly followed this model. In fact all measured curves had negative y-intercepts. Several explanations for this phenomenon can be offered. The phage suspension used was a relatively crude preparation and may have contained virions with different degrees of protection from radiation by association with organic matter or cell debris. Alternatively, the upward concave shape of the inactivation curves may have been the result of inhomogeneous distribution of irradiance or from insufficient stirring of the irradiated solution (Budowsky et al., 1981). The practical significance of this observation is relatively small, because most inactivation curves could be forced to fit a firstorder model passing though the origin with high statistical validity. Most experiments could be fitted with a model using k =0.0106m2/L This value is very close to data originally reported by Mattern et aL (1965), who used the same experimental conditions. Using original laboratory notes (personal communication), a value of k = 0.0098 m2/j could be calculated. It should also be noted that in these experiments a significantly negative y=intercept of 0.09 log~-units was found. Severin et al. (1983) reported a value of k =0.0072m2/j for the closely related phage f2 in experiments carried out at 20°C. Hence, the repeatability of inactivation experiments with RNA=bacteriophages appears to be good, which is an advantage when proposing these organisms as biological standards for calibration of u.v.=lamps and disinfection units. Acknowledgements--Calibration of the irradiation units was performed at the Quality Department of Philips Lighting B.V. We thank Mrs I. E. Mattern (TNO, Rijswijk, The Netherlands) for making available original data. The work at the National Institute of Public Health and Environmental Protection was carried out as a part of project 148506 on behalf of the Department-General for Environmental
1393
Protection (Ministry of Public Housing, Physical Planning and Environmental Protection). REFERENCES
Anon. (1986) Municipal wastewater disinfection. EPA/ 625/1-86/021, U.S. Environmental Protection Agency, Cincinnati, Ohio. Budowsky E. I., Kostyuk G. V., Kost A. A. and Savin F. A. (1981) Principles of selective inactivation of viral genome II. Influence of stirring and optical density of the layer to be irradiated upon u.v.-induced inactivation of viruses. Arch. Virol. 68, 249-256. Draper N. R. and Smith H. (1981) Applied Regression Analysis, 2rid edition. Wiley, New York. Harm W. (1980) Biological Effects of UItraciolet Radiation. Cambridge University Press, Cambridge, Mass. Havelaar H. H. and Hogeboom W. M. (1984) A method for the enumeration of male-specific bacteriophages in sewage polluted waters. J. appl. Bact. 56, 439-447. Havelaar A. H., Pot-Hogeboom W. M., Koot W. and Pot R. (1987) F-specific bacteriophages as indicators of the disinfection efficiency of secondary effluent with ultraviolet irradiation. Ozone Sci. Engng 9, 353-368. Kruithof J. C., Van der Leer R. C., Hijnen W. A. M., Huhn P. A. N. M., Houtepen F. A. P. and Feij L. A. C. (1989) Ultraviolet disinfection of carbon filtered drinking water. In Ozone and UV in the Treatment of Water and Other Liquids (Edited by Masschelein N.), pp. III-3-I-III-3-15. International Ozone Association, Paris. Mattern I. E., Van Winden M. P. and R6rsch A. (1965) The range of action of genes controlling radiation sensitivity in Escherichia coli. Mut. Res. 2, 111-131. Meulemans C. C. E. (1987) The basic principles of u.v. sterilization of water. Ozone Sci. Engng 9, 299-314. Quails R. G. and Johnson J. D. (1983) Bioassay and dose measurement in u.v. disinfection. Appl. era'it. Microbiol. 45, 872-877. Roeber J. A. and Hoot F. M. (1975) Ultraviolet disinfection of activated sludge effluentdischarging to shellfishwaters. EPA/600/2-75/060, U . S . Environmental Protection Agency, Cincinnati, Ohio. Severin B. F., Suidan M. T. and Engelbrecht R. S. (1983) Effects of temperature on ultraviolet light disinfection. Envir. Sci. Technol. 17, 717-721. Strauss J. H. and Sinsheimer R. L. (1963) Purification and properties of bacteriophage MS2 and of its ribonucleic acid. J. molec. Biol. 7, 43-54.
0043-1354/90 $3.00 + 0.00 Copyright C) 1990PergamonPress plc
INACTIVATION OF BACTERIOPHAGE MS2 IN WASTEWATER EFFLUENT WITH MONOCHROMATIC AND POLYCHROMATIC ULTRAVIOLET LIGHT A. H. HAVELAAR l~),C. C. E. MEUL~dANS 3,W. M. POT-HOOlmOOM~ and J. KosTI~ 2"* Laboratory of Water and Food Microbiologyand 'Centre for Mathematical Methods, National Institute for Public Health and Environmental Protection, P.O. Box I, 3720 BA Bilthoven and 3Laboratory Lighting Technique and Application, Philips Lighting B.V., P.O. Box 80020, 5600 JM Eindhoven, The Netherlands (First receired January 1990; accepted in revised form June 1990) A~tract--A model using bacteriophage MS2 for the calculation of the effective dose in u.v.-disinfection by polychromatic light from a medium pressure mercury lamp was studied. The model was based on first-order inactivation kinetics, the u.v.-absorption spectrum of MS2-RNA as the action spectrum and Lambert-Beers' law for light absorption by the effluents. The u.v.-sensitivity of the phage was calibrated with a low pressure mercury lamp; the inactivation rate constant (k) was 0.0106 m2/J, which corresponds well with previously reported data. The model fitted actual data in most experiments, although tailing effects were observed. In some experiments a different value ofk was necessary to fit the actual inactivation curves, varying between 0.0077 and 0.0121 m2/J. The MS2 phage was found suitable for calibration of u.v. sources. Key words--u.v.-radiation, disinfection, bacteriophage MS2, mathematical model, kinetics
INTRODUCTION The use of u.v. radiation for the disinfection of wastewater effluents is an emerging technology. In the past 15 years it has been developed and applied on a practical scale particularly in North America (Anon., 1986). Improvements in reactor design and lamp performance have resulted in a more reliable and economical operation of u.v. disinfection units, which are now feasible for wastewater effluents. The" presently used installations are predominantly equipped with low pressure mercury vapour lamps. These lamps have a high output of germicidal u.v. radiation per watt of electrical energy consumed, but have a low field-intensity. Medium pressure mercury lamps (or others such as antimony lamps) produce a higher u.v. output per lamp but are less efficient because a substantial part of the total energy is emitted as visible radiation (Fig. 1), and energy losses by heat production are greater than for low pressure lamps. Nevertheless, there is a growing popularity of the medium pressure lamps because they require smaller reactors, which is an advantage in larger installations. It has recently been shown for drinking water applications that reduced costs on capital investment and lamp replacements counterbalance the increased energy costs (Kruithof et ai., 1989). Similar calculations have not yet been made for wastewater applications. *Present address: ICIM, P.O. Box 5809, 2280 HV Rijswijk, The Netherlands.
Dose assays for medium pressure lamps are difficult because polychromatic radiation is emitted instead of the essentially monochromatic radiation of a low pressure lamp. The disinfection efficiency of u.v. radiation and u.v. absorption by effluents are dependent on wavelength. A model for the calculation of the effective u.v. dose has been proposed (Meulemans, 1987). In this paper we present an experimental calibration of this model using the F-specific R N A bacteriophage MS2. Reasons for choosing this organism are the following: (1) MS2 is a single-strand R N A virus and consequently its dose-response curve for u.v. inactivation follows first-order kinetics (Harm, 1980). (2) The structure and size of MS2 is similar to that of the human enteroviruses. (3) MS2 has a relatively high u.v. resistance, comparable to that of bacterial spores (Harm, 1980). (4) MS2 is not pathogenic to man, so it can be used for calibration of full-scale reactors without additional safety measures. (5) The organism is easily cultivable in titres up to 10~2pfu/ml, which makes its use for calibration of full-scale reactors advantageous over presently recommended spore suspensions of Bacillus subtilis, which can only be cultured up to 10s cfu/ml (Quails and Johnson, 1983). The model calculations were carded out in the spectral region between 240 and 300 nm, thus partly including both the u.v.-C region (200-280 nan) and the u.v.-B region (280-315 nm). The lower limit was
1387
A. H. HAVELAARet al.
1388
MATERIALS AND METHODS LOW PRESSURE MERCURY LAMP
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In the course of the experiments, two versions of the irradiation apparatus were used. The units (Fig. 2) consisted of lamp housing A and collimating tube B, on which quartz cell C was mounted. The lamp housing was provided with either a low or a medium pressure mercury lamp. and with an adjustable cooling device consisting of a fan and thermistor for temperature control and recording, to assure that the lamps were burning at their optimal temperature. The bottom of quartz cell C, which was filled with water or secondary effluent, was a window of optical quality in order to improve the uniformity of the u.v. radiation passing through this window, where a Pyrex vessel D (dia 3 cm) with phage suspension could be positioned. During exposure, the phage suspension was constantly stirred by a Teflon-coated spinning bar driven by spinning device E. The first version of the apparatus was constructed with a simple tubular collimator (length 25 cm); in order to improve the uniformity of the irradiation field a second version was constructed in which unwanted reflections from the collimator surface were intercepted by several diaphragms. Two different u.v. sources were used. The low pressure lamp was an M-shaped 15 watt version made in quartz (arc length 26 cm), only available for use within the Philips Company. The medium pressure lamp was an HPK 125 watt lamp with an arc length of 3 cm. The relative spectral distributions of the radiation emitted by these two lamps are presented in Fig. 1. In both versions of the apparatus a flat reflector was mounted behind the low pressure lamp. In version 1 there was a curved reflector behind the medium pressure lamp, but this was omitted in version 2 because of unacceptable reflections and too high irradiance levels giving too short an interval between withdrawal of samples from vessel D.
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Modelling polychromatic u.v.-disinfection Calibration of the irradiation units was performed with a CARY 17D double monnchromator calibrated by reference to standard lamp, from the National Physical Laboratory (U.K.). According to the calibration methods used, the absolute irradiance levels had a standard deviation of 10%. In order 'to facilitate the flexibility during the experiments a UVX radiometer (UVP Inc., U.S.A.) equipped with a UVX-25 sensor was calibrated simultaneously.
Effluents Secondary effluents were obtained from STP De Bilt and the pilot plant of the Technical University Delft (Laboratory of Sanitary Engineering) and were filtered through 0.22/~m membranes before use. Transmission spectra of the effluents between 240 and 300 nm were measured at 5 nm intervals in a Perkin-Elmer 550S two-beam spectrofotometer in I em quartz cells against Millipore Super-Q water. Transmission at 255 nm ranged between 50--60% cm-I for the STP effluent and 40-50% for the pilot plant effluent.
Microorganisms and microbiological methods Bacteriophage MS2, its host strain E. coli WG21 and the culturing methods were previously described (Havelaar and Hogeboom, 1984). For irradiation experiments, phages were suspended in M9-buffer (Mattern et al., 1965) which was freshly prepared before each experiment as follows: 50 ml vol of two separately autoclaved stock-solutions (I: NH4CI 20g, NaC! 100g, MgSO4.7HzO 2 g in 1000ml water; II: KH2PO 4 60 g, Na 2 HPO 4 120 g in 1000 ml water) were mixed and diluted to a final volume of 1000 ml; by following this procedure it was possible to have a u,v. absorbance which was consistently less than 2% cm-m; pH was 7.0 _+0.05.
Experimental design The general model of the survival curves was based on the assumption of first order kinetics as expressed in the following formula:
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(1)
where N, = number of survivors at time t (pfu/ml) N o = number of bacteriophages at time 0 (pfu/ml) k = rate constant (m2/J) Hat= effective u.v. dose (J/m 2) Eat = effective u.v. irradiance (W/m 2) t = time (s).
Co)Low pressurelamp; effluentin lightpath;MS2 inbuffer. In the following two sets of three parallelexperiments, cell C was filled with 2 cm of Super-Q water and Eo was measured as described above. Subsequently, cellC was filled with 2 and 4 c m of membrane filtered(0.22#m) effluent, resix~tively. The intensity of u.v. radiation pas,~ng through the effluent was estimated by measuring the irradiance at the position of vessel D. This value was used to estimate the irradiation times required for each experiment to produce a dose range similar to that in the experiments under (a). The measured irradiance through the effluent was not used in further calculations; these were based on Eo measurements through Super-Q water. The model used was:
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(c) Medium pressure lamp; Super-Q in light path; MS2 in buffer, The model used was: 30O
Eat ---Eo fp Z R(A2)S(A~)
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where fp = correction factor for UVX meter response to polychromatic radiation R(AA)=fraction of total radiation between 240 and 300 tun, emitted in a 5 nm interval S(A2) = sensitivity of MS2 to u,v. radiation in a 5 nm interval, relative to the sensitivity at 254 nm A2 = integration interval (5 nm). S(A~) was taken from the u.v. absorption spectrum of MS2 RNA (Strauss and Sinsheimer, 1963). Data for multiples of 5 nm were used as central values, e.g. at 250 nm for the 247.5--252.5 nm interval.
(d) Medium pressure mercury lamp; effluent in light path; MS2 in buffer. The experiments described above under (b) were repeated with the medium pressure HPK-lamp mounted instead of the M-lamp. The model used was: 3OO
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Irradiation apparatus, version 1 (a) Low pressure lamp; Super-Q in light path; MS2 in buffer. A first series of experiments was carried out with the low pressure M-lamp and a layer of 2 em Millipore Super-Q water in cell C. After stabilization of the u.v.-source, the irradiance was measured at the position of vessel D at nine different measuring points using the UVX radiometer. The average Jr'radiance Eo was calculated as the average of these data. Subsequently, vessel D was filled with 20 ml MS2 suspension in M9-buffer at a titer of approx. 5 × l0 s pfu/ml and was placed under the lamp, while being covered with a piece of carton. Samples (100/~1) were withdrawn before the vessel was placed under the lamp and after exposure for 10, 20, 30, 40 and 50 s, and were assayed for surviving phage in duplicate. This experiment was repeated three times. The model used was:
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1389
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where E 0 = average irradiance through Super-Q water as measured at the position of vessel D with the UVX meter (W/m 2) f ~ = correction factor for UVX meter response with monochromatic radiation at 254nm.
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Irradiation apparatus, version 2 The above experiments [(a)--(d)] were repeated (in singular) with the second version of the apparatus, using the formulas as indicated. The second version, equipped with the M-lamp or the HPK-lamp was also used to measure the inactivation of MS2 suspended in filtered effluent. In these experiments, cell C was filled with 4 cm Super-Q, and vessel D with 20 ml (1.7 cm) of effluent, seeded with MS2 to a level of approx. 106 pfu]ml. The models used were:
(e) Low pressure mercury lamp; Super-Q in light path; MS2 in effluent. Integration of Lambert-Beer's law over the depth of the irradiated effluent (Roeber and Hoot, 1975). e-=~d
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A. H. HAVELAAR et al.
1390
Statistical analyses The three sets of parallel experiments described in the section on Experimental design sub-sections (a) and (b) were used to estimate the value of the inactivation rate constant k in equation (1) by finear regression analysis after logarithmic transformation of the inactivation ratios. In the second stage of the regression analysis, the condition was impfied that at t = 0 the inactivation ratio should also be 0, i.e. the regression was forced through the origin. The computed value of k was then used to predict the inactivation curves for the experiments subsections (c), (d), (e) and (f). The actual data of the fourteen experiments were compared with these predictions by a goodness-of-fit test. For each of the five data points in each inactivation curve, the squared difference between the measured and the predicted inactivation ratio was divided by the variance of the predicted value, computed from the regression analysis. The test parameter was calculated by adding these five values and follows a X2"distribution with 5 d.f. If the value of the test parameter is less than the critical Z 2 value at a size of 5%, it may be concluded that the actual data significantly fit the predicted values (Draper and Smith, 1981).
Results with irradiation apparatus, version I(MS2 in buffer)
RESULTS
Calibration of light sources and UVX meter The correction factors for the spectral response o f the UVX radiometer were determined for the spectral region between 240 and 300 rim. This was rather simple for the low pressure lamp because the 254 nm mercury line is the only emission in this region, but more complicated for the polychromatic radiation from the medium pressure lamp. Furthermore, the correction factor found for the medium pressure lamp can only be applied for the spectral energy distribution as emitted by the lamp. When radiation passes through effluent, the spectral distribution is modified and consequently this radiation cannot be measured directly with the UVX meter. Therefore the irradiance was always measured with Super-Q water in cell C and the irradiance passing through the effluent was calculated as described above. 10 o
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Table I. Regression analysis of data from experiments with MS2 in M9-buffer and u.v. radiation from a low-pressure (M) lamp (irradiation apparatus version I)
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Results of experiments with a low pressure lamp and Super-Q or effluent in the light path are shown in Fig. 3. Linear regression analysis showed that all inactivation curves had a negative y-intercept of 0.07-0,20 log,-units, indicating that inactivation proceeded more rapidly in the first few seconds of the experiments than later. The y-intercept of experiments with 2 cm Super-Q and 4 cm effluent were significantly different from 0. The regression analysis was repeated with the condition that all inactivation curves should start in the origin (no inactivation at t ffi 0), and the correlation coefficients were found to increase slightly. This approach was chosen for further analysis of the data. The results of this regression analysis are summarized in Table 1. The average value of the inactivation rate constant k, calculated from this series of nine experiments was found to be 0.0106 _+ 0.0005 m2/J, and was used in all further calculations as described below. Inactivation data with a medium pressure lamp and Super-Q in the light path could not be determined accurately in the first version of the apparatus because the very high irradiance resulted in more than 3 log-units inactivation within 20s; hence sampling time could not be controlled with an accuracy which was comparable to that of other experiments. The validation of this part of the model will be described in the section on experiments with the second version of the apparatus.
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Fig. 3. Inactivation of bacteriophage MS2 in M9-buffer by u.v. radiation from a low-pressure (M) lamp after passage through 2 cm Super-Q (O, (D and 0), 2 cm (A, A and A ) or 4 cm ( + , x and ,) effluent (3 separate experiments each, irradiation apparatus version I). , Regression line.
n.y. Absorption medium 2cm Super-Q 2 cm Effluent 4 cm Effluent All experiments
Rate constant and 95% conf. int. (k, m:/J) 0.0104 0.0103 0.0109 0.0106
_ 0.0010 + 0.0008 ± 0.0010 -L-0.0005
Correlation coefficient (r) 0.99 1.00 1.00 1.00
Modelling polychmmatic u.v.-disinfection Input data as used in the calculation of the effective dose with medium pressure lamps and effluent in the light path are shown in Fig. 4. This figure shows the spectral distribution of the emitted radiation and the sensitivity coefficient of MS2 as used in all calculations. The absorption spectrum of a particular sample of effluent is also shown; although actual data differed between experiments, the general shape of the spectra was relatively constant. Figure 5(a) and (b) show data from experiments with 2 and 4 crn effluent in the light path, respectively. The predicted inactivation curve, as indicated by the solid line, fitted the actual data significantly with 4 c m of effluent [Fig. 5(b)], but was found to tail off with 2 cm of effluent [Fig. 5(a)]; only one experiment out of three showed a good fit and one experiment showed a barely significant fit. While performing the experiments we noted that, despite the use of the long collimating cylinder, the u.v.-intensity was not equally distributed over the irradiation vessel, which was particularly obvious with 2 crn of effluent. Results with irradiation apparatus, version 2 (MS2 in buffer)
1391
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Fig. 5. Inactivation of bacteriophage MS2 in M9-buffer by u.v. radiation from a medium.pressure (I-IPK) lamp after passage through effluent (irradiation apparatus version 1). (A) 2cm effluent; (O) Z2=3.1; (A) Z:=I0.3; ( + ) g2=34.4; (B) 4cm effluent; (O) X2=3.0; (A) Z2=3.0; ( + ) g2 = 4.2. Critical value for X2 = 11.1 (5 d.f., P = 5%). , Predicted inactivation curve. pressure lamps, respectively. Data with the low pressure lamp were evaluated against the predicted inactivation curves using the average value of k =-0.0106 J/m 2 found with the first apparatus. With Super-Q in the light path, a statistically significant fit was found, although the inactivation curve again showed a tendency to tail off; with effluent in the light path all data points were below the predicted line indicating a rapid inactivation during the first few moments of the experiments. By means of linear regression a value of k = 0.0121 + 0.0017 J/m: was calculated. With the medium pressure lamp, all sets of data showed good fits with the prediction based on k = 0.0106 J/m, 2 and less tailing was observed. Results with irradiation apparatus, version 2 (MS2 in e~uenO If MS2 is to be used for biological calibration of u.v. reactors, dose-response curves should also be known for phage suspended in effluent. Two series of
1392
A. H. HAv~.7o~ et al.
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Fig. 8. Inactivation of bacteriophage MS2 in 1.7 cm secondary effluent by u.v. radiation from a low pressure (M) lamp after passage through 4 cm Super-Q (irradiation apparatus version 2); (O) X2__ 2.1; (A) X2= 1.4. - - , Predicted inactivation curve.
experiments were done, with low and medium pressure lamps, respectively, and with 4 cm Super-Q in the light path in all cases to assure the same distribution of irradiance over vessel D as in other experiments. Results are shown in Figs 8 and 9. A good correlation between predicted and actual data was found with the low pressure lamp; the actual reduction was significantly less than predicted with the medium pressure lamp. These data fitted a regression model with k -- 0.0077 -t- 0.013 J/m 2. In both cases, the correspondence between two separate experiments, run on different days, was very good.
secondary effluent by polychromatic u.v.-radiation from a medium pressure mercury lamp can be reasonably accurately modelled by a stepwise approach. Lambert-Beer's law was used to describe the absorption of radiation by the effluent and the absorption spectrum of the MS2-RNA was assumed to be equivalent to the action spectrum of the u.v. radiation. In view of the uncertainties involved in measuring u.v. irradiance and in calibrating measuring equipment this approach was tested using 5 nm intervals rather than I nm intervals or a continuous approach. Besides being more realistic, this model also has the merit of simplicity. For MS2, which is a singlestrand RNA-bacteriophage not subject to host-cell reactivation, first-order inactivation kinetics was to be expected (Mattern et aL, 1965; Harm, 1980). By regression analysis it was shown that none of
DISCUSSION
The experiments described in this paper have shown that inactivation of bacteriophage MS2 in 10o
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Fig. 9. Inactivation of bacteriophage MS2 in 1.7 cm secondary effluent by u.v. radiation from a medium pressure (HPK) lamp after passage through 4cra Super-Q (irradiation apparatus version 2); (O) Z2=28.0, (/%) Z2 = 38.3. , Predicted inactivation curve.
Modelling polychromatic u.v.-disinfection the sets of inactivation data exactly followed this model. In fact all measured curves had negative y-intercepts. Several explanations for this phenomenon can be offered. The phage suspension used was a relatively crude preparation and may have contained virions with different degrees of protection from radiation by association with organic matter or cell debris. Alternatively, the upward concave shape of the inactivation curves may have been the result of inhomogeneous distribution of irradiance or from insufficient stirring of the irradiated solution (Budowsky et al., 1981). The practical significance of this observation is relatively small, because most inactivation curves could be forced to fit a firstorder model passing though the origin with high statistical validity. Most experiments could be fitted with a model using k =0.0106m2/L This value is very close to data originally reported by Mattern et aL (1965), who used the same experimental conditions. Using original laboratory notes (personal communication), a value of k = 0.0098 m2/j could be calculated. It should also be noted that in these experiments a significantly negative y=intercept of 0.09 log~-units was found. Severin et al. (1983) reported a value of k =0.0072m2/j for the closely related phage f2 in experiments carried out at 20°C. Hence, the repeatability of inactivation experiments with RNA=bacteriophages appears to be good, which is an advantage when proposing these organisms as biological standards for calibration of u.v.=lamps and disinfection units. Acknowledgements--Calibration of the irradiation units was performed at the Quality Department of Philips Lighting B.V. We thank Mrs I. E. Mattern (TNO, Rijswijk, The Netherlands) for making available original data. The work at the National Institute of Public Health and Environmental Protection was carried out as a part of project 148506 on behalf of the Department-General for Environmental
1393
Protection (Ministry of Public Housing, Physical Planning and Environmental Protection). REFERENCES
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