Use of ultraviolet radiation for inactivation of bacteria and coliphages in pretreated wastewater

Wat. Res. Vol. 27, No. 3, pp. 397--403, 1993 Printed in Great Britain

0043-1354/93 $6.00 + 0.00 Pergamon Press Ltd

USE OF ULTRAVIOLET R A D I A T I O N F O R INACTIVATION OF BACTERIA A N D COLIPHAGES IN P R E T R E A T E D WASTEWATER HALIM DIZER, WOLFRAM BARTOCHA, HARTMUT BARTEL, KARSTEN SEIDEL, JUAN MANUEL LOPEz=PILA and ANDREAS GROHMANN Institute for Water, Soil and Air Hygiene, Federal Health Office, Corrensplatz 1, D-1000 Berlin 33, Germany

(First received April 1992; accepted in revised form August 1992) Abstract--The inactivation of bacteria and coliphages by u.v. radiation was tested in a full-scale pilot plant with a flow rate of 180 m3/h. The investigated water contained about 70% secondary effluent from sewage treatment plants and 30% surface water. The minimal rated radiation density was 13.3 mW/cm 2 (60% of u.v. transmission in water), and the radiation exposure lasted for 3.54 s resulting in a u.v. radiation dose of 47 roWs/sin2. This type of u.v. radiation chamber decreased the concentration of total coliform organisms, E. coli, fecal streptococci, Salmonella sp. and coliphages in the influent by 1-2 logs. Strains of bacteria, Streptococcus faecalis and Salmonella enteritidis, seeded artificially into the influent showed a reduction of about 2-4 logs after u.v. radiation. The coliphage f2 was more resistant than the tested bacteria and reduced by less than 2 logs through u.v. radiation. The inactivating effect of u.v. radiation was counteracted by the binding of the coliphage f2 to suspended turbid particles. It can be recommended to use u.v. treatment of effluents of wastewater plants after a flocculation and filtration step to improve the efficiency of the u.v. radiation.

Key words--bacteria, total coliform organisms, E. coli, fecal streptococci, Streptococcus faecalis, Salmonella enteritidis, coliphage f2, wastewater, u.v. radiation, wastewater treatment

INTRODUCTION Ultraviolet radiation as a means of reducing microorganisms is gaining more importance both in the treatment of certain drinking waters and in the advanced treatment of wastewater. Recent studies have demonstrated its effectiveness for secondary and tertiary effluents (Whitby et al., 1986; Zukovs et aL, 1986; Gelzhauser, 1989). In much of those scale pilot plants the disinfection of microorganisms varied greatly depending on the properties of sewage and the u.v. radiation density (Quails et aL, 1985; Whitby et al., 1986; Schleypen et aL, 1989). " F i l m " formation on the u.v. lamps by running for a long period reduces the disinfecting effect (Anonymous, 1991). Therefore, the running conditions of large-scale u.v. plants should be empirically determined especially in consideration of the influent quality. After regulation of technical properties in those plants, a high disinfection effectivity can be achieved (Anonymous, 1991). The inactivating effect o f u.v. radiation on bacteria and coliphages tested in a pilot plant which was connected to the outlet o f the phosphate elimination plant in Berlin-Tegel (PEP-Tegel) is described in this report.

MATERIALS AND METHODS

Ultraviolet pilot plant The surface water of a channel in Berlin which is up to 70% polluted by secondary effluent is treated at PEP-Tegel by flocculation and filtration in order to reduce eutrophication in the following lake Tegel (Heinzn~un et aL, 1991). The u.v. pilot system on the outlet of PEP-Tegel consists of the following units: a conveyor pump for a maximum output of 400 m3/h, an inductive stream meter and a basin of 0.75 x 0.51 x 2.25 m. Six frames are mounted longitudinally in the basin. Each of the frames carries four u.v. lamps, one above the other, at a distance of 85 mm (Fa. Wedeco, Type 078, 60 W). Each of the u.v. emitters has a 1.8 m radiation length (Fig. 1). The lamps are 105 mm vertically and 85 mm horizontally apart from one another to ensure the calculated radiation density of 13.3 mW/cm 2 (calculated at 60% of the transmission in water). In order to minimize short-circuit flow in the basin, an unperforated metal sheet was installed at the inlet and a perforated one at the inlet and outlet. The experiments were carried out at a flow rate of 180m3/h. Initially, tests were performed with uranine to further verify uniform flow in the tank. From these experiments we calculated that the water stayed under radiation, on average, for 3.54 s and that the average radiation dose was approx. 47 nd/cm 2.

Bacteria Streptococcus faecalis ATCC 19433 and Salmonella enteriddis were used as test organisms for the model experiments. The bacterial strains were cultivated in 101. 397

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casein-peptone-soy bean peptone broth (Merck No. 5459) for 16 h at 36°C. The cell suspensions reached a density of about 108 cfu per mi. For detection of fecal streptococci in the native water, samples were enriched in azide-D-glucose broth (Merck No. 1590) by incubation for 24-48 h at 36°C. The positive samples (cloudy broth) were placed on Slanetz-Bartley agar (Merck No. 5262) and incubated at 36°C. The appearance of colonies with Gram-positive diplococci was chosen as the criterion for a positive test result. For detection of Salmonella, I000 ml samples were filtered through a membrane filter (Sartorius, 0.45 #m) and the filter incubated for 24 h at 36°C in 50 ml tetrathionate broth (Oxoid No. CM 29). The positive samples (turbid broth) were plated on brilliant green-phenol red-lactose agar (Difco No. 0100-01). Suspected colonies of Salmonellae were picked and identified using biochemical and serological methods. E. coil, other coliform organisms and aerobic bacteria were identified according to the German Standard Methods for Water, Wa.~tewaterand Sludge Examinations (Anonymous, 1971).

Coliphages f 2 Determination was carded out on E. coil KI3 indicator strain according to Havelaar (1986). Naturally occurring coliphages were concentrated according to the following procedure: 2mi of a 10% A12(SO4)3 solution were added to 1 litre of the sample, the pH was adjusted to 5.5 with HCI and the sample left overnight at 6°C. The AI(OH)3 flocs which had formed together with the adsorbed phages were centrifuged at 3000g for 5rain. The sediment was then resuspended in 10ml of 0.1 M citrate buffer with a pH of 4.7. I mi concentrate was mixed with 0.5 mi of an E. coil suspension containing 10s cfu/ml and with 5 ml soft afar (0.7 g in 1 0 0im) . The tube containing the mixture was thoroughly mixed and poured into a Petri dish containing 20 mi of trypsine yeast extract agar. The plates were counted after incubation for 24 h at 36°C. The titre of coliphages f2 in the stock suspension taken for seeding the incoming efliuent was about 109 plaque forming units (pfu)/mi.

Ultraviolet treatment process The suspensions of test bacteria and coliphages were introduced into the inlet of the u.v.-pilot plant using a peristaltic pump delivering 2.4 l/rain so as to reach a final concentration of c. 105cfu/ml bacteria and 10+pfu/ml phages. Samples were collected at three points of the perforated sheet before the outlet of the irradiation chamber (Fig. 1). In order to reach stability of the emitted energy we started innoculating the bacteria and phages after the u.v. lamps had been in operation for at least I h. After taking 4 or 5 samples, the u.v. lamps were turned off and a further 3-5 control samples were collected without u.v. radiation. A further experiment was designed to see the influence of suspended fine sandy soil particles in the water on the inactivation of phages. A suspension containing c. 10s phages/ ml was mixed with a fine sand soil sediment (grain size >0.2ram) to form a slurry of c. 0.1 g/ml. The slurry was shaken for 30 rain to allow adsorption of the phages to the particles and subsequently pumped into the inlet as described above. Samples were collected during irradiation and after turning off the lamps. The three samples taken at the perforation sheet were combined and the phages enumerated using the following procedure: The coliphages f2 adsorbed to colloidal particles were separated from the free coliphages f2 by centrifuging at 3000 g for 5 min and the supernatant tested for phages as described. The sediment was then resuspended in I ml of a 1.6% nutrient broth solution (Difco No. 0003-01-6) at pH 8 and the phages eluted by gently shaking for 15 rain. The amount of coliphages f2 in the eluate was then determined as above. RESULTS

Inactivation o f the bacteria and coliphages present in flocculated effluent o f the PEP-Tegel by u.v. radiation N a t u r a l l y occurring E. coil a n d o t h e r coliform organisms, fecal streptococci, Salmonella sp., aerobic

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Fig. 2. Occurrence of several bacteria in the effluent of the phosphate elimination plant Berlin-Tegel with or without u.v. irradiation. bacteria on standard plate as well as coliphages were determined in the effluent of PEP-Tegel, and the results are shown in Fig. 2. The titre of E. coli/coliform organisms in untreated water was between 10 -L and 10 -2 (e.g. 0.01-0.1 ml was the smallest volume found positive), and for fecal streptococci between l0 t and 10-z. After passage through the u.v. reactor,

both parameters were reduced by a factor of 10-100. The colony count of bacteria in the inlet was about 3 x 103 cfu/ml and in the outlet, after u.v. treatment, it was about 102 cfu/ml. Figure 3 shows the results with the coliphages; the maximum elimination of coliphages by the u.v. radiation was 2 logs.

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Inactivation of artificially added bacteria by u.v. radiation Because the concentrations of the indicator bacteria and coliphages naturally present in the outlet of PEPTegel were relatively low, a suspension of laboratory cultures was introduced into the u.v. reactor as described in the methods. The results with S. faecalis are presented in Fig. 4. The figure shows the titres found with and without radiation. The minimal elimination of S. faecalis in experiment 1 was 3 logs (the difference between smallest titre estimated without u.v. radiation and maximal titre during u.v. radiation); and in experiment 2 it was only 1 log. The results also show that the titres from the three different sample collection sites varied considerably in both experiments. In several cases, this variability was as much as 2 logs. Salmonella enteritidis was no longer found after u.v. treatment and therefore showed a reduction of at least 4 logs (detection limit). Inactivation of seeded coliphages by u.v. radiation The result of the coliphage t"2 tests showed a titre reduction of 1-2 logs (Fig. 5). Small variations between the different collection sites were observed: samples from the middle spigot had a maximum reduction of 97%; samples from the bottom spigot had a maximum reduction of 89%.

Inactivation o f adaorbed coliphages by u.v. radiation In this experiment we investigated the influence of suspended particles associated with coliphages on the disinfecting effect of u.v. radiation. Figure 6 shows the concentration of fre¢ and adsorbed coliphages f2 before and during u.v. treatment. The results show that the u.v. radiation caused an activation of free cofiphages f2 of less than 2 logs (max. 94%). The coliphges f2 which were adsorbed to suspended particles were reduced by 54% only. DISCUSSION

The reduction of various bacteria and of coliphages f2 was tested at PEP-Tegel with a minimum radiation flow of 13.3 mW/cm 2 and an exposure period of 3.5 s. The results described stress the importance of the following topics in evaluating disinfection by u.v. radiation: Differences in u.v. sensitivity between sewage born bacteria and laboratory cultures, as well as between bacteria and viruses The bacteria in the outlet of the PEP-Tegel (colony counts, fecal streptococci, E. coli, other coliform organisms and Salmonella sp.) experienced an elimination of 1-3 logs while passing through the u.v. reactor. The reduction of coliphages f2 in the same

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reduced by 3-4 logs; coliphages f2 up to less than 2 logs only. Numerous laboratory experiments have recently provided similar information about the high sensitivity of various laboratory species as compared with naturally occurring bacteria (Martiny et ai., 1988a, b,

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1989; Sommer et al., 1989). The reduction of the naturally present bacteria and viruses should therefore have priority for evaluating the efficiency of u.v. devices. In comparison to bacteria, viruses show a much higher resistance to u.v. radiation (Rodgers et al., 1982; Quails et al., 1984). Chang et al. (1985) were able to establish the following sequence of various microorganisms according to the level of sensitivity to u.v. radiation resulting from equal doses of exposure: E. coil > coliform organisms > poliovirus type 1 > spores of B. subtilis. According to Sommer et al. (1989), the high sensitivity of E. eoli is caused by a very extensive enzyme system which provides a wide spectrum for attack by u.v. rays and which poliovirus lacks. Therefore, the evaluation of disinfection by u.v. devices should include tests with viruses too. Because of the relatively short time needed and low material costs of the test methods, it is suggested to use coliphages as an indicator, at least to obtain an approximate picture of the viral reduction (Havelaar, 1986). Influence o f suspended particles on the inactivation o f bacteria and viruses by u.v. Heavy microbial contamination of water is usually accompanied by higher turbidity. It is known that suspended particles can influence the efficiency of u.v. radiation due to adsorption or scatter (Leuker and Hingst, 1990). In our experiments, the suspended particles of a fine sandy soil influenced the disinfecting function of u.v. radiation on coliphages f2 considerably. The reduction was lowered from 97% to an unsatisfactory 54%. Rodgers et al. (1982) estimated that the inactivating action of u.v. radiation on poliovirus type 1 was considerably lowered in a 15% fecal suspension. Poliovirus type 1 survived even high doses of u.v. radiation in seawater contaminated with wastewater (Hill et al., 1967, 1979). Because of the negative effect of suspended particles on the inactivation of bacteria and viruses, only prefiltered surface water should undergo u.v. treatment. This is suggested for the functioning of the u.v. disinfection systems (Anonymous, 1983; FIGAWA, 1987). In sand-filtered river water, Bewig (1985) achieved a reduction of bacteria of about 3 logs. The colony counts of bacteria in water from the River Maas were reduced from 102-103cfu/ml to about 10cfu/ml as a result of u.v. treatment with u.v. radiation doses of 20.6 and 12 mWs/cm2 (Kruithof et al., 1986). In relatively turbid pond water from a fish-hatchery treated with u.v. radiation, Weigend (1986) was able to record a colony count of c. 103 cfu/ml, while the untreated water in the control basin contained about 10' cfu/ml. Acknowledgements--The authors would like to express their thanks to Mr H. Wedecamp for the installation of u.v.

radiation pilot plant and to Mrs P. Kirschner for technical assistance. REFERENCES

Anonymous (1971) Deutsche Einheitaverfahren zur Wasser-, Abwasser- und Schlammuntersuchung. Nachweis yon E. coli und Coliformen Bakterien (K 6). Chemic, Weinheim. Anonymous (1983) Hinweise fiir den Einsatz yon UVAnlagen zur Desinfektion von Trinkwasser und yon Brauchwasser ffir Lebensmittelbetriebe. Bayerisches Landesamt ffir Wasserwirtschaft, Merkblatt Nr. I. 7-3 vom 29.11.1983. Anonymous (1991) Untersuchungen zur Keimreduktion im gereinigten Abwasser durch UV-Bestrahlung. Bayerisches Landesamt ffir Wasserwirtschaft, Informatiosberichte, p. 151, Mfinchen. Bewig F. (1985) Praktische Erfahrungen mit UV-Strahlen. In Desinfektion yon Trinkwasser durch UV-Bestrahlung (Edited by Bartz W. J. and Wippler E), pp. 75-85. Expert, Sindelfingen. Chang J. C. H., Ossoff S. F., Lobe D. C., Dorfman M. H., Dumais C. M., Quails R. G. and Johnson J. D. 0985) UV inactivation of pathogenic and indicator microorganisms. Appl. envir. Microbiol. 49, 1361-1365. FIGAWA (1987) Technische Mitteilung der Bundesvereinigungen der Firmen im Gas-und Wasserfach e.V.: UV-Bestrahlung in der Wasseraufbereitung, Teil 3: UV-Bestrahlung in der Trinkwasseraufbereitung. Brunnennbau-bbr 38, pp. 190-193. Gelzh.~user P. (1989) Keimreduktion im Abwasser dutch UV-Bestrahlung. Korrespond. Abwass. 36, 68-75. Havelaar A. H. (1986) F-specific RNA bacteriophages as model viruses in water treatment processes. Proefschrift,in bet Rijksinstituut voor Volksgesundheiden Mifieuhygiene, Bilthoven, Holland. Heinzmann B., Sarfert F. and Stengel A. (1991) Die Phosphateliminationsanlage Tegel in Berlin. gwf Wass.Abwass. 132, 674-684. Hill W. F., Hamblett F. E. and Akin E. W. (1967) Survival of poliovirus in flowing turbid seawater treated with UV light. Appl. Microbiol. 15, 533-566. Hill W. F., Hamblett F. E., Benton W. and Akin E. W. (1979) Ultraviolet devitalization of eight selected enteric viruses in estuarine water. Appl. Microbiol. 19, 805812. Kruithof J. C., van der Leer R. C. and Hijnen W. A. M. (1986) Experiences with UV-disinfection of drinking water in the Netherlands. In Ozone-Ultra-violet Water Treatment, p.4. l-p.4.15. IOAC, Amsterdam. Leuker G. and Hingst V. (1990) Mikrobiologiscbe Untersuchungen zur Bedeutung natiirlicher und simutierter Transmission des Wassers bei der Wertbestimmung yon UV-Anlagen zur Wasserdesinfektion. Zbl. Hyg. 190, 365-379. Martiny H., Wlodavezzyk K. and Riiden H. (1988a) Anwendung yon UV-Strahlen zur Desinfektion yon Wasser. II. Mitteilung: MikrobiologischeUntersuchungen in Oberfl,~ichenwasser.Zbl. Bakt. Hyg. B 186, 344-359. Martiny H., Wlodavezzyk K., Harms G. and Rfden H. (1988b) Anwendung yon UV-Strahlen zur Desinfektion yon Wasser. I. Mitteilung: Mikrobiologische Untersuchungen in Trinkwasser. Zbl. Bakt. Hyg. B 185, 350-367. Martiny H., Seidel K. and Rfiden H. (1989) Anwendung yon UV-Strahlen zur Desinfektion von Wasser. III. Mitteilung: UV-Sensibilit/it von Legionella pneumophila unterschiedlichen Alters in kaltem und warmem Trinkwasser. Zbl, Hyg. 188, 35-46. Qualls R. G., Chang J. C. H., Ossoff $. F. and Johnson J. D. (1984) Comparison of methods of enumerating coliforms after UV disinfection. Appl. envir. Microbiol. 48, 699-701.

Ultraviolet radiation Quails R. G., Ossof S. F., Chang J. C. H., Dorfman M. H., Dumais C. M. and Johnson J. D. (1985) Factors controlling sensitivity in ultraviolet disinfection of secondary effluents. J. Wat. Pollut. Control Fed. 57, 1006-1011. Rodgers F. G., Gurrie R. and Bulmore M. (1982) Effect of faecal material on the inactivation of poliovirus type 1 by ultraviolet irradiation. In Viruses and Disinfection of Water and Wastewater (Edited by Butler M., Medlen A. R. and Morris R.), pp. 378-384. University of Surrey Print Unit, Guildford. Schleypen P., Lesel T. and Popp W. (1989) UV-Bestrahlung yon Abwasser unter Betriebsbedingungen. Korrespond. Abwass. 36, 458-461. Sommer R., Weber G., Cabaj A., Wekerle J., Keck G.

403

and Schauberg G. (1989) UV-Inaktivierung yon Mikroorganismen in Wasser. Zbl. Hyg. 189, 214-224. Weigand F. (1986) The effect of the operation of UV-water disinfection the quality of fish and water in fishrearing systems. In Ozone-Ultra -violet Water Treatment, D.5.l-D.5.9. IOAC, Amsterdam. Whitby G. E., Palmateer G., Cook W. G., Maarschalkerweerd J., Huber D. and Flood K. 0984) Ultraviolet disinfection of secondary effluent. J. Wat. Pollut. Control Fed. 56, 844-850. Zukovs G., Kollar J., Monteith H. D., Ho K. W. A. and Ross S. A. (1986) Disinfection of low quality wastewaters by ultraviolet light irradiation. J. War. Pollut. Control Fed. 58, 199-206.