AHCBAU 33 (6) 573-644
`(2005)
`ISSN 0323-4320 · Vol. 33
`No. 6 · December 2005
`I
`l
`Official JoumJ1 of t_lle...,Water
`Chemical Sodie'\y· - a Division
`of the Gennan Chemical Society
`
`Acta
`[h)W©lIT'@chimica et
`[h)W©lU'@biolog ica
`
`Editorial Board
`
`Advisory Board
`
`Edltorlal Office
`
`Publisher
`
`F. H. Frimmel, Karlsruhe (Editor-in-Chief)
`S. H. Eberle, Karlsruhe (Deputy Editor)
`U. Forstner, Hamburg
`U. Obst, Karlsruhe
`W. V, T0mpllng, Erfurt
`P. Wilderer, Garchlng
`
`G. Becher, Oslo, N
`P. G. Hatcher, Columbus OH, USA
`I. Holoubek, Brno, CZ
`H. Horth, Medmenham, GB
`K. L. E. Kaiser, Burlington, CON
`H. Klapper, Magdeburg, D
`P. Lischke, Magdeburg, D
`N. Matsche, Wien, A
`E. Salameh, Amman, JOA
`Y. I. Skurlatov, Moscow, AUS
`C. E. W. Steinberg, Berlin, D
`S. C. Whalen, Chapel Hill NC, USA
`A. J. B. Zehnder, Zurich, CH
`L. Zilllox, Strasbourg, F
`
`B. C. Gordalla, Karlsruhe
`
`WILEY-VCH Verlag GmbH & Co. KGaA, Postfach 101 161 , 69451 Weinheim,
`Germany, Phone +49(0)6201/606-0, Fax +49(0)6201/606-328
`
`Manuscripts and Correspondence
`
`address to: Prof. Dr. Fritz H. Frimmel, Editor-in-Chief, Universitat Karlsruhe, Engler-Bunte(cid:173)
`lnstitut, Engler-Bunte-Ring 1, 76131 Karlsruhe, Germany, Phone +49(0)721/608-2959,
`Fax +49(0)721/608-7051 , E-mall acta@ebi-wasser.uni-karlsruhe.de
`
`Instructions to Authors
`
`see first issue of the volume, or request from editorial office, or see
`http://www3.interscience.wiley.com/homepages/5007772l2047 _forauthors.pdl
`
`Cover Illustration: Pool water activities - a challenge for treatment and hygiene (see also this issue, pp. 585 - 594).
`
`With kind permission of E. Karle, Karlsruhe.
`
`1
`
`EXHIBIT 1005
`
`

`

`Aims and Scope
`
`Acta hydrochimica et hydrobiologica is an international journal that
`publishes peer-reviewed scientific articles in the current field of water
`research. Papers on the following topics will be weloome for publication
`In the sections REVIEWS, RESEARCH PAPERS, and SHORT CoMMUNICATIONS:
`• chemistry and biochemistry of waters, In particular surface waters
`such as streams, rivers, lakes, tidal and coastal waters, deposits,
`groundwater, seepage water, leachates of soil, sediments and
`waste, municipal and industrial wastewaters:
`• analysis and monitoring of water by physical, chemical, biochemical,
`and biological methods, including analysis of adjacent environmental
`compartments with regard to their effect on water quality:
`
`• waters, sediments, and their interaction;
`• fate and transport of pollutants in aquatic systems;
`• water conditioning ar:id wastewater treatment together with their
`chemical, biological, and technological fspects;
`• limnological and hydrobiological investigations with regard to water
`quality.
`In the column FORUM, specialists have the opportunity to briefly discuss
`topical themes and controversial questions arising in their research
`fields. In the section THESES IN WATER Sc1ENCES, young scientists may
`present short abstracts of their recently finished PhD theses.
`
`Editorial Office:
`Prof. Or. F. H. Frlmmel, Dr. B. C. Gordalla
`Unlversltat Karlsruhe, Engler-Bunte-lns1ttut,
`Engler-Bunte-Ring 1, 76131 Karlsruhe, Gennany
`Productl011: Sandra MOiier
`Ma:ketlng: Veronique Bluteau, CMstlan Waldbusser
`Copy~ght perm~lons: Claudia Jer1<e, Benlna Loyd<e
`(~ghts@wltey•v<:h.de)
`
`Advertising:
`Marion Schulz
`Advertising, WILEY-VCH Ve~ag GmbH & Co. KGaA
`Boschslrasse 12, 69469 Welnhelm, Germany
`Tel. (+49) 6201 606 557. Fax (+49) 6201 606 551
`e-mall: adsalesOwlley-v<:h.de
`
`Published blmootNy by WILEY-VCH Verlag GmbH & Co. KGaA,
`69469 Welnhelm, Germany.
`e 2005 WILEY•VCH Venag GmbH & Co. KGaA, Welnlleim,
`Germany
`Typeset by paglna media GmbH, Hemsbach, Germany
`Pnnted on acid-free paper by Betz-Druck GmbH, Darmstadt,
`Germany
`
`Annual subscription rates 2D05
`Institutional•
`€
`907 .00/824.00
`Gennany/Europe
`SFr 1402.00/1274,00
`Swltzenand
`Outside Europe
`US$ 1050.00/954.00
`pnnt and eleclronlc/prlnt or,ly or electronic only
`Postage and handling charges lnciUded. All Wiley-VCH
`prices are exclusive cl VAT.
`Pr1oes are subject to cllange.
`
`Order lhrough your bookseller or directly at the publisher.
`WILEY-VCH Vertag GmbH & Co. KGaA, P.O. Box 101161,
`69451 Welnhelm, Germany. Fax (+49) 6201 606 172
`e-man: subservlceOwiley-v<:h.da,
`
`Bank accounts (bank. acc. no., son code):
`Dresdner Sank, Welnhelm, Germany, 75111880, 670 800 SO;
`Postglro Frankfun, Germany, 145314-600, 500 100 60
`
`For the USA and <:anada: "Acta hydrochlmlca et hydro(cid:173)
`blotogica" (ISSN 0323-4320) Is published bimonthly by
`Wilay-VCH PO Box 191161, D 69451 Welnheim, Germany.
`Air frel!,hl and mafllng In the USA by Publ!caUons Expediting
`Inc. 200 Meacham Ave .. Elmont, NY 11003. PeJlodlcals
`postage paid at Jamaica NY 11431. US POSTMASTER:
`send adctres,; changes to Advanced functional Matertals,
`c/o Wilay-VCH, 111 River Stree~ 1-tob<?k8n, NJ 07030.
`
`All rtghts reserved (Including those of rranslaUon Into foreign
`languages). No part ol this Issue may be reprO<luced In any
`lorm - by photoprtnt, mlcrofllm, or any other means - nor
`transmitted or lranslated lnlo a maclllne language without
`wrtnen perml:;slon from the publishers, Only single copies of
`conllibutlons, or parts thereol, may be made for personal use.
`This J011rnal was carefully produced In all Its parts. Neverthe•
`less, authors, editors and publisher do not guarantee the In·
`formation eontatned therein to be lree or errors. Registered
`names, lrademarks, etc. used In this !oumal, even wnen not
`marked as such, are not to be considered unprotected by law.
`
`V1l1d lor uters In the USA: Toe copyrtght owner agrees
`that copies of Iha artlde may be made for personal or Internal
`use, or !or tlle personal or Internal use of specific clients. This
`coosent Is given on the condition. howeve,, that the copier pay
`the stated per~ fee through the Copyl'lght Clearance Cen(cid:173)
`ter, Inc. (CCC) tor copying beyond that permitted by Sectioris
`107 or 108 of the U.S. Copyright Law. This consent does not
`extend to olher kinds of copy!ng, such as copying tor general
`dlslrlbU!lon, for advertising or promotional purposes, lor crea(cid:173)
`dng new collectlve wo<1<s, or for resale. For copying from back
`volumes of lhls Joumal see "Permissions to Photo Copy: Pul>(cid:173)
`llsher's Fee Usr ol lhe CCC.
`
`© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
`
`www.wiley-vch.de/home/actahydro
`
`2
`
`

`

`Acta
`[ruw@·u@chimica et
`[ruw@u@biologica
`
`AHCBAU 33 (6) 573-644
`(2005)
`ISSN 0323-4320 · Vol. 33
`No. 6 • December 2005
`
`Contents
`
`M. ClauB, R. Mannesmann,
`A. Kolch
`
`T. Glauner, F. Kunz,
`C. Zwiener, F. H. Frimmel
`
`K. Michel, B. Ludwig
`
`A. Ostojic, s. Curclc,
`L. Comic, M. Topuzovic
`
`J. Flores-Burgos,
`S. S. S. Sarma, S. Nandlni
`
`Research Papers
`
`579 Photoreactivation of Escherichia coll and Yersinia enterolytica after
`Irradiation with a 222 nm Excimer Lamp Compared to a 254 nm Low(cid:173)
`pressure Mercury Lamp
`Photoreaktivierung von Escherichia coli und Yersinla enterolytica nach
`Bestrahlung mit einem 222 nm-Exclmerstrahler im Verglelch mit einem
`254 nm-Nlederdruck-Quecksilberstrahler
`
`585 Elimination of Swimming Pool Water Disinfection By-products with
`Advanced Oxidation Processes (AOPs)
`Verringerung von Deslnfektionsnebenprodukten bei der Schwimm(cid:173)
`beckenwasseraufbereitung
`durch erweiterte Oxldationsverfahren
`(AOP - Advanced Oxidation Processes)
`
`595 Modelling of Seepage Water Composition from Experiments with an Acid
`Soil and a Calcareous Sediment
`Modelllerung der Slckerwasserzusammensetzung ausgehend von
`Experlmenten mit einem sauren Boden und elnem kalkhaltigen Sedi(cid:173)
`ment
`
`605 Estimate of the Eutrophication Process In the Gruza Reservoir (Serbia
`and Montenegro)
`Einschatzung des Eutrophlerungsprozesses im Stausee von Gruza
`(Serblen und Montenegro)
`
`614 Effect of Single Species or Mixed Algal (Chlorella vulgaris and Scene(cid:173)
`desmus acutus) Diets on the Life Table Demography of Brachionus
`calyciflorus and Brach/onus patulus (Rotlfera; Brachionldae)
`Ole Wirkung einer Dlat von einzelnen oder gemischten Algen ( Ch/ore/la
`vutgaris und Scenedesmus acutus) auf die Lebenstafel-Demographle
`von Brach/onus catyclflorus und Brachionus patulus (Rotlfera: Brachlo(cid:173)
`nidae)
`
`© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
`
`3
`
`

`

`A. Riethmuller, E. Langer
`
`622 Salsonales Vorkommen von Arlen der Saprolegnlales und Leptomitales
`im Auesee und in der Fulda in Kassel (Hessen) unter Beri.icksichtigung
`fischpathogener Arlen
`Seasonal Occurrence of Species of Saprolegniales and Leptomitales in
`Lake Aue and the River Fulda in Kassel (Hesse) with Special Considera(cid:173)
`tion of Fish Pathogenic Species
`
`635 Theses in Water Sciences
`
`637 Meetings
`
`638 New Publications
`
`640 Recent Contents
`
`© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
`
`www.wiley-vch.de/home/actahydro
`
`4
`
`

`

`Acta hydrochim. hydrobiol. 33 (2005) 6, 579-584
`
`DOI 10.1002/aheh.200400600
`
`579
`
`Marcus ClauB•,
`Rolf Mannesmann•,
`Andreas Kolchb
`
`a Faculty of Biology,
`University of Bielefeld,
`33615 Bielefeld, Germany
`b WEDECO AG Water
`Technologies,
`Boschstr. 6, 32051 Herford,
`Germany
`
`Photoreactivation of Escherichia coli and
`Yersinia enterolytica after Irradiation with a
`222 nm Excimer Lamp Compared to a 254 nm
`Low-pressure Mercury Lamp
`
`Photoreactivation of Escherichia coli ATCC 11229 and Yersinia enterolytica ATCC 4780
`after irradiation with a 222 nm krypton-chloride excimer lamp compared to a 254 nm mer(cid:173)
`cury lamp was investigated under laboratory conditions. The bacteria samples were
`irradiated each with different doses of both wavelengths. After irradiation one sample of
`the bacteria was illuminated with fluorescent light, the other sample was stored in darkness
`to prevent photoreactivation. The inactivation curves were determined. Without photoreac(cid:173)
`tivation, an irradiation of 69 J/m2 at 254 nm was sufficient for a 4 log reduction for E. coli,
`and only 59 J/m 2 for Y. enterolytica. To get a 4 log reduction with following photoreacti(cid:173)
`vation, 182 J/m 2 were necessary for E. coli and 180 J/m2 for Y. enterolytica. After irradiation
`with the 222 nm excimer lamp the ratios were different. Without photoreactivation, an
`irradiation of 106 J/m2 at 222 nm was sufficient for a 4 log reduction for E. coli and
`88 J/m 2 for Y. enterolytica. With photoreaclivation 161 J/m2 were necessary for E. coli to
`get a 4 log red,uction and 117 J/m2 for Y. enterolytica.
`When the photoreactivation after irradiation is excluded, the mercury lamp with 254 nm
`clearly shows better results regarding inactivation. Whereas, when included, the excimer
`lamp with 222 nm wavelength obviously shows better results.
`
`Photoreaktlvlerung von Escherichia coli und Yersinia enterolytica nach Bestrahlung
`mlt einem 222 nm-Exclmerstrahler im Verglelch mit einem 254 nm•Niederdruck(cid:173)
`Quecksllberstrahler
`
`Die Photoreaktivierung van Escherichia coli ATCC 11229 und Yersinia enterolytica ATCC
`4780 nach Bestrahlung mil einem 222 nm-Krypton-Chlorld-Excimerstrahler im Vergleich
`zu einem 254 nm-Niederdruck-Quecksilberstrahler wurde unter Laborbedingungen unter(cid:173)
`sucht. Proben beider Bakterienarten wurden mit verschiedenen Dasen beider Wellen(cid:173)
`langen bestrahlt. Danach wurde zur Photoreaktivierung eine Probe Fluoreszenzlicht aus(cid:173)
`gesetzt, die andere dunkel gehalten, um diese zu verhindern. Dann wurden die lnaktivie(cid:173)
`rungskurven ermittelt. Bei der Bestrahlung mil dem 254 nm-Quecksilber-Niederdruck(cid:173)
`strahler waren ohne anschlieBende Photoreaktivierung fur eine lnaktivierung van 4 log(cid:173)
`Stufen 69 J/m2 fur E. coli und 59 J/m2 fur Y. enterolytica ni:itig. Mil anschlieBender Photo(cid:173)
`reaktivierung waren es dagegen 182 J/m2 tor E. coli und 180 J/m2 fur Y. enterolytica. Bei
`Bestrahlung mil dem 222 nm-Excimerstrahler zeigen sich deutliche Unterschiede bei den
`Verhaltnissen. Ohne anschlieBende Photoreaktivierung war hier fur eine Reduktion von
`4 log-Stufen eine Bestrahlung von 106 J/m2 fur E. coll und 88 J/m2 fur Y. enterolytica
`notig. Mit Photoreaktivierung waren es 161 J/m2 fur E. co/iund 117 J/m2 fur Y. enterolytica.
`Wird die Photoreaktivierung ausgeschlossen, zeigt der Quecksilberstrahler bessere
`Ergebnisse bei der lnaktivierung, mil anschlieBender Photoreaktivierung jedoch der
`Excimerstrahler.
`
`Keywords: Ultraviolet Radiation, Water Disinfection, Photolyase, Proteins
`
`Schlagworter: Ultraviolette Strahlung, Wasserdesinfektion, Photolyase, Proteine
`
`Correspondence: M. ClauB, E-mail: marcus.clauss@uni-bie1efeld.de
`
`© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinhelm
`
`J:
`(.)
`"(cid:173)«s
`
`i Q) ,~
`
`I°'
`L -·
`
`5
`
`

`

`580 M. ClauB et al.
`
`1 Introduction
`
`Low-pressure mercury lamps are traditionally used for water
`disinfection. Their nearly monochromatic emission of
`254 nm almost corresponds with the maximum of DNA ab(cid:173)
`sorption at approx. 260 nm. This absorption causes damage
`to DNA by altering nucleotide base paring, especially 6-4
`photoproducts and thymine dimers formation [1 , 2]. If the
`damage remains unrepaired, DNA transcription and repli(cid:173)
`cation is blocked. This finally leads to cell death.
`
`There are also other targets in the cell which are damaged
`by UV radiation by different wavelengths. Damage of mem(cid:173)
`branes has been reported to occur in cells of Escherichia
`coli only after irradiation with UV radiation above 305 nm (3].
`In contrast to this, membranes of Saccharomyces cerevisiae
`yeast cells were damaged by radiation at wavelengths less
`than 200 nm only [4]. This membrane damage is predomi(cid:173)
`nantly caused by UV radiation formed radicals which react
`to escharotic lipoperoxides in the unsaturated fatty acids of
`the membrane [5]. Much more important seems to be the
`damage done to amino acids, and thus also in proteins com(cid:173)
`posed out of it. Out of the 20 most common amino acids
`only phenylalanin, tyrosin, tryptophan, cystein, cystin [6] and
`histidin [7] show a peak UV absorption in the area of
`280 nm and a higher one at 220 nm. At wavelengths ex(cid:173)
`ceeding 200 nm the absorption spectra of proteins and those
`of the composition of their constituents are comparable (6,
`8]. Thus, proteins also show absorption maxima at 220 nm
`and 280 nm.
`
`Both prokaryotic and eukaryotic cells have special mecha(cid:173)
`nisms to remove DNA defects (1 ). Among the nucleotide ex(cid:173)
`cision repair (NER), also known as dark repair, one of the
`most important repair mechanisms is the photoreactivation
`[9]. This process has been well researched and uses a
`single enzyme called photolyase to repair UV-induced dam(cid:173)
`age in the DNA {10- 12]. The photolyase of E. coli is basi(cid:173)
`cally specific for repair of pyrimidine dimer. It catalyses the
`reaction from cis-syn pyrimidin dimers to the original pyrimi(cid:173)
`din monomers in DNA. It is a light-dependent process which
`requires specific wavelengths ranging from 300 ... 500 nm
`and is much more effective and faster than the NER (9]. This
`leads to problems when treated water is exposed to light
`and microorganisms which obviously have already been in(cid:173)
`activated begin to reactivate, for example in UV-treated
`wastewater after its discharge to runoff ditches. One
`possible solution being applied in practice is to increase the
`irradiance to such a high value that the DNA is extensively
`damaged and photoreactivation is no longer possible. How(cid:173)
`ever, the higher power consumption is still a disadvantage.
`Due to the fact that the photoreactivation only repairs DNA
`damage !t would be interesting to investigate the photoreac(cid:173)
`tivation of bacteria after irradiation with wavelengths in the
`range of the absorption maxima of proteins.
`
`Acta hydrochim. hydrobio/. 33 (2005) 6, 579-584
`
`Several authors investigated the use of other types of UV
`lamps for water disinfection like medium-pressure mercury
`lamps with a broader emission spectrum from far UV to infra(cid:173)
`red [13- 15] excimer lamps [16, 17] and excimer laser [18].
`But special reactivation studies in the past only focused on
`DNA repair of microorganisms following UV-exposure from
`low-pressure [19, 20] and medium-pressure lamps [13- 15].
`The broad emission spectrum of a medium-pressure lamp
`indeed contains wavelengths also in the range of the absorp(cid:173)
`tion maxima of proteins, but it is rather unspecific. In contrast
`to this a krypton-chloride excimer lamp shows a relatively
`sharp emission spectrum with a peak at 222 nm (Fig. 1 ).
`
`The intention of the following experiment was to compare
`the photoreactivation of Escherichia coli and Yersinia enter(cid:173)
`o/ytica after irradiation with a 222 nm (near protein absorp(cid:173)
`tion max.) excimer Jamp with a 254 nm (near DNA absorp(cid:173)
`tion max.) low-pressure mercury lamp under laboratory con(cid:173)
`ditions. The bacteria were irradiated with the UV radiation
`from the two lamps. Afterwards the suspension was illumi(cid:173)
`nated with fluorescent light. Then the reduction of the colony
`forming units was investigated after different irradiation
`times with and without photoreactivation.
`
`2 Materials and methods
`
`UV source. A collimated beam device (WEDECO AG Water
`Technology) was used for irradiation corresponding to the
`details of the DVGW-guideline W 294 [21]. This device con(cid:173)
`tains interchangeable lamp units. One lamp unit is equipped
`with four low-pressure mercury lamps, type NLR 2036; the
`other unit is equipped with two KrCl-excimer lamps. The dis(cid:173)
`tance between the probes and the UV lamps was 51 cm. For
`both units the emission spectrum and the irradiance were
`measured with a Bentham Spectrometer DM 150 Double
`Monochromator with a 200 .. .450 nm standard sensing head.
`The real power consumption taken from the main supply
`was 185 W for the mercury lamp and 86 W for the excimer
`lamp.
`
`Figure 1 shows the measured irradiance of both lamps
`plotted logarithmically against the wavelength. The ir(cid:173)
`radiance of the mercury lamp in the whole UV region
`(200 ... 380 nm) is 22. 10 W/m2 and therefore much higher
`than that of the excimer lamp with 3.55 W/m2• In the UV-C
`region (200 ... 280 nm) the irradiances are 20.95 W/m2 and
`3.38 W/m2 •
`
`Photoreactivation. The inactivation was followed by expo(cid:173)
`sure to fluorescent lamps. For the photoreactivation the
`probes were illuminated with four (7 cm horizontally apart)
`fluorescent tube lamps (Osram Biolux 18 W, 600 mm length;
`daylight spectrum from 360 ... 700 nm). To ensure that the
`light intensity in the Petri dishes was even, the illuminations
`
`© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
`
`www.wiley-vch.de/home/actahydro
`
`6
`
`

`

`Acta hydrochim. hydrobiol. 33 (2005) 6, 579- 584
`
`Photoreaclivation of Bacteria at 222 nm 581
`
`100
`
`10
`
`0,1
`
`E
`~
`,.;
`B
`
`C:
`
`"' E
`
`0,01
`
`0,001
`200
`
`- - low -pressure
`mercury lallfJ
`- - krypton chloride
`excimer lallfJ
`
`>-
`
`I
`i ~ J
`
`I
`L J
`
`250
`
`- -
`
`A.,
`400
`
`I ~ ~
`
`r----...
`
`300
`Wavelength in nm
`
`350
`
`cording to the regulations of the DVGW [21] and the
`ONORM [22), which lay down the requirements and testing
`of plants for the disinfection of water using ultraviolet radi(cid:173)
`ation. Each lime 25 ml of test suspension were irradiated
`for different periods of times (depending on the lamps and
`the microorganisms to be irradiated) in 85 mm standard
`polystyrene Petri dishes (arithmetical thickness of 4.4 mm)
`without intermixing. In order to determine the exact inacti(cid:173)
`vation kinetic, five duplicate samples of each of the microor(cid:173)
`ganisms were irradiated. After irradiation one sample of the
`bacteria was illuminated with fluorescent light, the other
`sample was stored in darkness to prevent photoreactivation.
`To set a decimal dilution series after irradiation 1 ml of the
`test suspension was taken from the centre of the Petri dish
`each time. 100 µL of the dilutions were plated 3 x for the
`dilution steps 10- 2 to 1 o-5 in pour-plate method with PC(cid:173)
`Agar: Tryptone (Oxoid LP0042) 5.0 g, yeast extract (Oxoid
`lP0021) 2.5 g, glucose (Oxoid lP0071) 1.0 g and agar
`(Oxoid lP0011) 10.0 gin 1 l distilled water. For the dilution
`steps 1 o0 and 10- 1, 1 ml and 100 µL of the test suspension
`were taken from the centre of the Petri dish and directly
`plated. The following incubation of the microbes was done
`at 37°C in a darkened incubator for 24 hours. For the arith(cid:173)
`metical evaluation of the results three agar plates each of
`this dilution step with 10 ... 300 colonies were used. These
`were counted and the results arithmetically averaged. The
`mean was then divided by the corresponding dilution step
`and the common logarithm was calculated. From these five
`lg concentrations the average value was calculated. The cor(cid:173)
`responding reduction for the respective irradiation lime is
`calculated by lg(N/N0). This dose reduction factor was plot(cid:173)
`ted logarithmically as function of the irradiance.
`
`3 Results
`
`The UV inactivation results for E. coli and Y. enterolytica are
`presented in Figures 2 and 3. They show only slight differ(cid:173)
`ences in the UV sensitivity of the two bacteria species. The
`UV irradiation/reduction response curves of E. coli and
`Y. entero/ytica without photoreactivation developed in this
`study differ from the curves with photoreactivation.
`
`Without photoreactivation, an irradiation of 69 J/m2 at
`254 nm was sufficient for a 4 log reduction for E. coli, and
`only 59 J/m2 for Y. enterolytica. In -contrast, the same ir(cid:173)
`radiation with following photoreactivation showed an approx.
`0.5 log reduction only for E. coli and 0.7 for Y. enterolytica.
`To get a 4 log reduction after irradiation and following
`photoreactivation, 182 J/m2 were necessary for E. coli and
`180 J/m2 for Y. enterolytica.
`
`After irradiation with the 222 nm excimer lamp the ratios
`were different compared to the 254 nm mercury lamp. With(cid:173)
`out photoreactivation, an irradiation of 106 J/m2 at 222 nm
`
`Fig. 1: Emission spectra of the 254 nm mercury lamp and
`the 222 nm KrCl-excimer lamp from 200 .. .400 nm wave(cid:173)
`length.
`
`Emissionsspektren des 254 nm-Quecksilber-Niederdruck(cid:173)
`strahlers und des 2.22 nm-Krypton-Chlorid-Excimerstrahlers
`im Wellenlangenbereich 200 ... 400 nm.
`
`were done in a box (62 cm I x 50 cm h x 32 cm w) lined
`with aluminium foil to reflect scattered light and to exclude
`light from outside.
`
`Organisms and their cultivation. Culture: Escherichia coli
`ATCC 11229 and Yersinia enterolytica ATCC 4780 (Amer(cid:173)
`ican Type Culture Collection, Manassas, VA). Incubation of
`E. co/ifor 14 h in End o's broth: Meat extract (Merck 103979)
`3.0 g and tryptone (Oxoid LP0042) 2.5 g per litre aqua de(cid:173)
`min. Incubation of Y. enterolytica for 14 h in Case's broth:
`Tryptone (Oxoid lP0042) 15.0 g, peptone from soymeal
`(Oxoid LP0044) 5.0 g, NaCl (Oxoid lP0005) 5.0 g per litre
`aqua demin. Bacteria were harvested by centrifugation (Bio(cid:173)
`fuge 28 AS, company Heraeus) with 2600 upm for 10 min
`at 20°C, the pellet was resuspended in 100 ml 0.65% NaCl
`and filtrated through a 5.0 µm filter (Cellulose-Nitrate-Filter,
`Sartorius).
`
`Standardization of bacteria titer. For irradiation the titer of
`the test suspension was standardized at 1 • 106 bacteria/ml
`as follows: The extinctions of the respective bacteria sus(cid:173)
`pension were measured at 510 nm in a 5 cm quartz glass
`cuvette against a 0.65 % NaCl solution in a photometer
`(Hitachi U-1100 Spectrometer) and then further diluted with
`the solution until an extinction of 0.100 was reached. To this
`extinction the respective titer was determined. Then, the
`number of organisms/ml to 1 . 106 could be adjusted by
`diluting the corresponding stock suspension.
`
`Irradiation and evaluation of the results. The irradiation
`and the consequent evaluation of the results were done ac-
`
`© 2005 WlLEY-VCH Verlag !}mbH & Co. KGaA, Weinheim
`
`www.wiley-vch.de/home/actahydro
`
`7
`
`

`

`582 M. Clauf3 et a!.
`
`Acta hydrochim. hydrobiol. 33 (2005) 6, 579-584
`
`Escherichia coli
`
`Yersinia enterolytica
`
`0
`
`--0
`
`z z -1
`:§i
`2
`0 .,
`u.
`C:
`0 = 0 :,
`al er
`a,
`8
`Q
`
`-2
`
`-3
`
`-4
`
`-5
`
`-6
`
`6. 222 nm
`... .. ........ ......... A 222nm+pr
`C 254nm
`
`0
`~ z
`:§i
`'o
`tS
`tf
`C:
`.Q
`tl
`:,
`"O a,
`O'.
`a,
`"' 0
`
`Q
`
`-0
`
`-1
`
`-2
`
`-3
`
`-4
`
`-5
`
`-6
`
`0
`
`40
`
`80
`
`120
`
`160
`
`200
`
`0
`
`40
`
`Irradiation in J/m'
`
`80
`
`120
`Irradiation in Jtm2
`
`160
`
`200
`
`Fig. 2: Inactivation curves for E. coli ATCC 11229 after irradi(cid:173)
`ation with a 222 nm KrCI excimer lamp and a 254 nm low(cid:173)
`pressure mercury lamp with and without photoreactivation
`{pr) afterwards. All symbols indicate the mean of five inde(cid:173)
`pendent series of experiments. Error bars denote the highest
`and lowest value.
`
`Fig. 3: Inactivation curves for Y. entero/ytica ATCC 4780
`after irradiation with a 222 nm KrCI excimer larT)p and a
`254 nm low-pressure mercury lamp with and without photo(cid:173)
`reactivation {pr) afterwards. All symbols indicate the results
`of five independent series of experiments. Error bars denote
`the highest and lowest value.
`
`lnaktivierungskurven fur E. coli ATCC 11229 nach Bestrah(cid:173)
`lung mit einem 222 nm-Krypton-Chlorid-Excimerstrahler und
`einem 254 nm-Quecksilber-Niederdruckstrahler mit und
`ohne anschlieBende Photoreaktivierung {pr). Die Symbole
`stehen fur den Mittelwert aus funf unabhangigen Versuchs(cid:173)
`reihen. Die Fehlerbalken zeigen den hochsten und den nied(cid:173)
`rigsten Wert an.
`
`lnaktivierungskurven fur Y. enterolytica ATCC 4780 nach Be•
`strahlung mil einem 222 nm-Krypton-Chlorid-Excimerstrah(cid:173)
`ler und einem 254 nm-Quecksilber-Niederdruckstrahler mil
`und ohne anschlief3ende Photoreaktivierung (pr). Die Sym(cid:173)
`bo!e stehen fur den Mittelwert aus fiinf unabh~ngigen Ver(cid:173)
`suchsreihen. Die Fehlerbalken zeigen den hochste'1' und
`den niedrigsten Wert an.
`
`was sufficient for E. coli for a 4 log reduction. However, the
`same irradiation with following photoreactivation still showed
`a 1.4 Jog reduction . In this case only 161 J/m2 were neces(cid:173)
`sary for E. coli to get a 4 log reduction. Also Y. enterolytica
`showed similar results. Without photoreactivation, an ir(cid:173)
`radiation of 88 J/m2 at 222 nm was sufficient for a 4 log
`reduction. In contrast, the same irradiation with photoreacti(cid:173)
`vation showed an approx. 2.3 log redudion. In this case
`117 J/m2 were necessary to get a 4 log reduction.
`
`When the photoreactivation after irradiation is excluded, the
`mercury lamp with 254 nm clearly shows better results re(cid:173)
`garding inactivation. Whereas, on the other hand with photo(cid:173)
`reactivation afterwards the excimer lamp with 222 nm wave(cid:173)
`length obviously shows better results.
`
`4 Discussion
`
`Photolyase activity and the resulting photoreactivation were
`found in many prokariotic and eukaryotic organisms [9].
`E. coli was selected for this study because it is commonly
`used as a biological indicator of disinfection efficiency in
`water systems. Its dark- and photorepalr processes following
`exposure to UV radiation are well known and have been ex-
`
`tensively studied. This strain was specifically chosen be(cid:173)
`cause it is known to undergo photorepair folldWing low(cid:173)
`pressure UV exposure up to a dose of 280 J/m2 (20].
`Y. enterolytica is not a common test organism but similar to
`E. coli, it has a highly-efficient photorepair mechanism for
`UV radiation induced damage at 254 nm and is actually able
`to undergo photorepair up to 320 J/m2 [20].
`
`The bacterial low-pressure UV irradiation/survival response
`curves developed in this study are similar to other published
`curves for these bacteria [12, 17, 23, 24]. Also values for 3
`or 4 log reductions are similar. After a 4 log reduction of the
`colony count {106/ml- 102/mL) trough UV rays, !He re(cid:173)
`duction rate could be decreased to only 1 log (105/ml) after
`UV disinfection and photoreactivation (19]. The same ratio
`was investigated in this study. But sometimes the values dif(cid:173)
`fer from some authors; however, the ratios are similar. For
`example, an irradiation for a 4 log reduction without photore(cid:173)
`activation was given for E.coli ATCC 11229 and Y. enteroly(cid:173)
`tica {no strain was given), each with 100 J/m2 [20] (in this
`study 69 J/m2 for E. coli and 59 J/m2 for Y. enterolytica). For
`the same reduction with photoreactivation 280 • J/m2 for
`E. coli and 320 J/m2 for Y. enterolytica were necessary
`{182 J/m2 and 180 J/m2 in this study). It is commonly known
`that results regarding the inactivation of bacteria which can
`
`© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
`
`www.wiley-vch.de/home/actahydro
`
`8
`
`

`

`Acta hydrochim. hydrobiol. 33 (2005) 6, 579-584
`
`Photoreactivalion of Bacteria at 222 nm 583
`
`be found in literature are contradictory, due to the different
`experimental conditions and the large variability of the bac(cid:173)
`teria.
`
`No bacterial UV irradiation/survival response curve for the
`irradiation with a 222 nm excimer lamp could be found for
`these bacteria except for E. coli in preliminary investigations
`[17J and only some data in the literature [16). In preliminary
`investigations it was found that under laboratory conditions
`the required irradiation for a 4 log reduction of E. coli ATCC
`25922 is 60 J/m2 with 254 nm UV radiation and 86 J/m2 with
`222 nm UV radiation [17). At least the ratio is very similar to
`69 J/m2 for 254 nm and 106 J/m2 for 222 nm found in this
`investigation. A 0.4 log reduction of E. coli as found in litera(cid:173)
`ture, irradiated with a 222 nm KrCI excimer lamp in a flowing
`system compared to a more than 4 log reduction under the
`same conditions with a 254 nm low-pressure UV lamp (16).
`This reduction correlates with the one investigated in this
`study.
`
`Also no data for photoreactivation at 222 nm could be found
`in literature. Nevertheless, clues are given in some papers
`that deal with the photoreactivation after irradiation with low(cid:173)
`and medium-pressure UV sources [14, 15, 25]. The general
`conclusion in these papers is that the survival ratio of the
`bacteria after photoreactivation following medium-pressure
`lamps is smaller than that of low-pressure lamps. The emis(cid:173)
`sion spectrum of medium-pressure mercury lamps contains
`also wavelengths of around 222 nm. It could thus be as(cid:173)
`sumed that this effect also occurs here.
`
`In general, it is recognizable that without photoreactivation
`the inactivation with UV radiation with 254 nm wavelength
`near the absorption maxima of ONA is most effective, and
`DNA has always been regarded as the most important target
`molecule for UV radiation. To get the same inactivation re(cid:173)
`sults with 222 nm wavelength the necessary irradiation has
`to be 50% higher. This is according to preliminary results for
`irradiated E. coli, Enterococcus faecalis and Candida al(cid:173)
`bicans, which were inactivated 1.5 times better with 254 nm
`than with 222 nm [17]. But when the bacteria get the chance
`to photoreactivate, the ratios change. With photoreactivation
`and irradiation with 254 nm the bacteria has to be irradiated
`300% more to obtain the same reduction as without photore(cid:173)
`activation. At 222 nm a higher irradiation of only 25% for
`E. coli and 50% for Y. enterolytica are necessary to get the
`same inactivation as without photoreactivation.
`
`In summary, the photoreaclivation has a lower level after ir(cid:173)
`radiation wlth 222 nm wavelength compared to 254 nm.
`These results indicate the damage of other molecules at
`222 nm among the DNA, because photoreactivalion is a
`ONA repair process only and not able to remove damage in
`other cell compartments like membranes or proteins. It is
`
`assumed that protein damage is most probable [6-8],
`whereas other authors suppose that this is due to the fact
`that repair of photodimers and 6-4 photoproducts is better
`than the one of damage induced due to photoionizalion at
`lower wavelength (18). Another possibility is, that wave(cid:173)
`length of 220 ... 300 nm reduced the subsequent photorepair,
`possibly by causing a disorder in endogenous photolyase,
`the enzyme specific for photoreact!vation [25).
`
`With 3.38 W/m2 measured in the UV-C region the excimer
`lamp has a much lower irradiance than the mercury lamp
`with 20.95 W/m2 (Fig. 1 ), but the real power, actually taken
`from the main supply, is with 185 W for the mercury lamp
`2.1 times higher than for the excimer lamp with 86.6 W. With
`regard