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Higher effectiveness of photoinactivation of bacterial spores, UV resistant vegetative bacteria and mold spores with 222 nm compared to 254 nm wavelength

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Eleven selected species of vegetative bacteria, bacteria spores and mold spores were irradiated with different doses of UV radiation of a 222 nm krypton-chloride excimer lamp and a 254 nm mercury lamp under laboratory conditions. Then the inactivation curves were determined. The necessary UV fluences for a respective reduction were higher for the excimer lamp for the tested vegetative bacteria of Bacillus cereus, Arthrobacter nicotinovorans, Staphylococcus aureus and Pseudomonas aeruginosa and slightly higher for the spores of Streptomyces griseus and Clostridium pasteurianum. However, less than 250 J/m2 UV fluence with 222 nm was sufficient for a 4-log reduction, depending on the species. On the other hand, the UV fluences for the 254 nm mercury lamp were much higher for the bacterial spores of Bacillus cereus, Thermoactinomyces griseus and the bacteria of Deinococcus radiodurans and slightly higher for the mold spores of Aspergillus niger and Penicillium expansum. The results show that especially the germs with a higher UV resistance and those with more effective repair mechanisms can be inactivated more efficiently by the 222 nm excimer lamp. This may be due to the fact that low UV fluence mainly affects radiation sensitive microorganisms by DNA damage whereas at higher UV fluence (various) mechanisms of protein damage can presumably be held responsible for inactivation.
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Acta hydrochim. hydrobiol. 2006, 34, 525 – 532 M. Clauß 525
Research Paper
Higher effectiveness of photoinactivation of bacterial spores,
UV resistant vegetative bacteria and mold spores with 222 nm
compared to 254 nm wavelength
Marcus Clauß
Faculty of Biology, University of Bielefeld, Bielefeld, Germany
Eleven selected species of vegetative bacteria, bacteria spores and mold spores were irradiated
with different doses of UV radiation of a 222 nm krypton-chloride excimer lamp and a 254 nm
mercury lamp under laboratory conditions. Then the inactivation curves were determined. The
necessary UV fluences for a respective reduction were higher for the excimer lamp for the tested
vegetative bacteria of Bacillus cereus,Arthrobacter nicotinovorans,Staphylococcus aureus and Pseudo-
monas aeruginosa and slightly higher for the spores of Streptomyces griseus and Clostridium pasteu-
rianum. However, less than 250 J/m2UV fluence with 222 nm was sufficient for a 4-log reduction,
depending on the species. On the other hand, the UV fluences for the 254 nm mercury lamp
were much higher for the bacterial spores of Bacillus cereus,Thermoactinomyces griseus and the
bacteria of Deinococcus radiodurans and slightly higher for the mold spores of Aspergillus niger
and Penicillium expansum.
The results show that especially the germs with a higher UV resistance and those with more
effective repair mechanisms can be inactivated more efficiently by the 222 nm excimer lamp.
This may be due to the fact that low UV fluence mainly affects radiation sensitive microorgan-
isms by DNA damage whereas at higher UV fluence (various) mechanisms of protein damage can
presumably be held responsible for inactivation.
Keywords: Excimer lamp / mercury lamp / disinfection / microorganism / drinking water /
Received: January 17, 2006; accepted: August 7, 2006
DOI 10.1002/aheh.200600650
1 Introduction
Biological effectiveness of UV radiation varies markedly,
depending on the wavelength used for the irradiation [1].
This is caused by varying degrees of absorption of differ-
ent biomolecules such as DNA, membranes or proteins.
According to the kind of chemical bond between the ele-
ments of the molecule, these are able to absorb the
energy of UV light of different wavelengths in a selective
way [2]. In most cases, this energy absorption results in
damage of the molecule. If this damage occurs to a large
extent and cannot be repaired, it harms and in case of
sufficiently high UV fluence even kills the organism.
A wavelength of 254 nm is traditionally used for inacti-
vation of microorganisms. This wavelength corresponds
approximately to the absorption maximum of DNA at
about 260 nm and can easily be obtained with mercury
lamps. At irradiation with UV light at this wavelength
there is a great extent of damage in the DNA such as
strand breaks, photo products and linking-ups, also with
proteins. It is a disadvantage of this wavelength that the
cells have different mechanisms to repair DNA damage
[3]. The light dependent photoreactivation is especially
effective. In a bacteria suspension with E. coli cells of
which 99.99% have been inactivated by irradiation with
254 nm UV light, only 90% killed cells can be found after
a 2-hour-exposition to artificial sunlight. This leads to
Correspondence: Marcus Clauß, Faculty of Biology, University of Biele-
feld, Universittsstr. 25, 33615 Bielefeld, Germany
E-mail: marcus.clauss@uni-bielefeld.de
Fax: +49 521 1066493
i2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
526 M. Clauß Acta hydrochim. hydrobiol. 2006, 34, 525 –532
problems especially for large-scale use of UV inactivation
of microorganisms when processing drinking water and
sewage, i.e. when inactivated cells can get out and are
exposed to sunlight.
This effect can be stopped partially by using wave-
lengths which are absorbed by other molecules than
DNA. Proteins have a strong absorption maximum at 220
nm. UV radiation of approximately this wavelength can
be generated with a 222 nm KrCl-excimer lamp. Examina-
tions comparing the photoreactivation of E. coli after irra-
diation with 222 nm and 254 nm have shown that the
irradiation is considerably less effective at 222 nm than
at 254 nm [4]. Furthermore, additional tests showed that
Bacillus subtilis spores can be inactivated much better
with UV radiation having a wavelength of 222 nm than
with 254 nm [5, 6] whereas the vegetative forms of B. sub-
tilis, Enterococcus faecalis, Candida albicans and E. coli can
be inactivated better with 254 nm.
In this examination, the effectiveness of photoinactiva-
tion with 222 nm wavelength compared to 254 nm wave-
length of further microorganisms is tested. For this, the
inactivation curves for both wavelengths of the microor-
ganisms were taken. Out of the most important groups
different aerobic spore-producing bacteria and mold
spores, the anaerobic and also spore-producing bacter-
ium Clostridium pasteurianum, some important microor-
ganisms relevant for drinking water and sewage and the
highly radiation-resistant Deinococcus radiodurans, which
has extremely effective repair mechanisms, were chosen.
2 Materials and methods
2.1 UV source
A collimated beam device (WEDECO AG Water Technol-
ogy) with interchangeable lamp units was used for irra-
diation as described previously [4]. The UV fluences were
measured with a Bentham Spectrometer DM 150 double
monochromator with a 200450 nm standard sensing
head and verified for the excimer lamp every month with
Bacillus subtilis spores strain ATCC 6051 as biological dosi-
meter after the previously investigated inactivation
curve [6]. For the mercury lamp the irradiance was veri-
fied every week with a Spectroline DRC 100H digital
radiometer with a DIX-254A UV-C sensor (Spectronics
Corporation).
2.2 Organisms and their cultivation
Aspergillus niger ATCC 32625 spores and Penicillium expan-
sum ATCC 36200 spores (American Type Culture Collec-
tion, Manassas, VA): Spores were taken from a slant cul-
ture with a sterile cotton bud and applied evenly on YGC-
Agar (yeast extract (Oxoid) 5.0 g, glucose (Merck) 20.0 g,
chloramphenicol (Selective Supplement OXOID) 0.1 g
and agar (Oxoid) 12.0 g per litre aqua demin). The plates
were incubated at 308C for 5 days. To harvest the spores,
3.0 g sterile quartz sand was dispersed on the plate. Then
the plate was shaken for one minute by hand. The mix-
ture of sand and spores was removed by being washed
with sterile aqua demin with 0.001% Tween 80 and col-
lected in a 300 mL Erlenmeyer flask. To remove the
spores from the sand, the suspension was sonicated for 2
min in an ultrasonic bath (Sonorex RK 102 company Ban-
delin Electronics, Berlin). The supernatant was removed
and centrifuged at 1000 rcf for 10 min at 208C. Then, the
pellet was resuspended in 100 mL sterile aqua demin.
This stock solution was stored in the fridge at 48C.
Bacillus cereus ATCC 11778 spores: 12 colonies were
plated evenly on 5 Nutrient Agar plates (meat extract
(Merck) 3.0 g, tryptone (Oxoid) 5.0 g, agar (Oxoid) 15.0 g,
with 10.0 mg MnSO4NH2O per litre aqua demin). After
72 h at 30 8C the harvest of the spores was done by resus-
pending the colonies on each plate in 10 mL sterile aqua
demin with a cotton bud. The solution was sonicated for
2 min in an ultrasonic bath (Sonorex RK 102 company
Bandelin Electronics, Berlin) and centrifuged (Biofuge 28
RS, company Heraeus) with 3000 rcf for 15 min at 208C.
Afterwards, the pellet was resuspended in 100 mL sterile
aqua demin. This was repeated two times. Then, the solu-
tion was sounded again for 2 min. Vegetative cells were
eliminated by heating the solution for 10 min at 808C.
This stock solution was also stored in the fridge at 48C.
Bacillus cereus ATCC 11778 vegetative bacteria, Staphylo-
coccus aureus ATCC 25923, Pseudomonas aeruginosa ATCC
27853: 12 colonies were plated to Standard-I-Nutrient
medium (Merck) and incubated at 308C for 24 h. Once
again, 12 colonies were inoculated in 100 mL Stan-
dard-I-Nutrient broth (Oxoid) for 1420 h. Bacteria were
harvested by centrifugation (Biofuge 28 RS, company Her-
aeus) with 1000 rcf for 10 min at 208C, the pellet was
resuspended in 100 mL 0.65% NaCl and filtrated through
an 8.0 lm filter (cellulose-nitrate filter, Sartorius).
Arthrobacter nicotinovorans ATCC 49919 and Deinococcus
radiodurans ATCC 13939: The same procedure as
described for B. cereus vegetative bacteria, S. aureus and P.
aeruginosa was done only with Corynebacterium Agar
(tryptone (Oxoid) 10.0 g, yeast extract (Oxoid) 5.0 g, glu-
cose (Merck) 5.0 g, NaCl (Merck) 5.0 g, agar (Oxoid) 15.0 g
per litre aqua demin).
Clostridium pasteurianum ATCC 6013 spores: 12 colo-
nies were plated to 5 GYE-agar plates (glucose (Merck)
20.0 g, yeast extract (Oxoid) 10.0 g, CaCO3(Merck) 10.0 g
and agar (Oxoid) 17.0 g per litre aqua demin) under an-
aerobic conditions in a nitrogen atmosphere. The plates
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Acta hydrochim. hydrobiol. 2006, 34, 525 – 532 More effective photoinactivation with 222 nm 527
were put in an air sealed 2.0 L preserving glass with an
Anaerogen bag (Oxoid atmosphere generation system)
for 2.5 L and incubated for 72 h at 378C. Spores were har-
vested and stored under aerogenic conditions as it was
described for B. cereus spores.
Streptomyces griseus ATCC 10137 spores: 12 colonies
were plated evenly on 5 GYM plates (glucose (Merck) 4.0
g, yeast extract (Oxoid) 4.0 g, malt extract (Merck) 10.0 g,
CaCO3(Merck) 2.0 g, agar (Oxoid) 12.0 g per litre aqua
demin). After 5 days at 288C the harvest of the spores was
done by resuspending the colonies on each plate in
10 mL sterile aqua demin with a cotton bud. The solution
was sonicated for 2 min in an ultrasonic bath (Sonorex
RK 102 company Bandelin Electronics, Berlin) and centri-
fuged (Biofuge 28 RS, company Heraeus) with 5000 rcf for
15 min at 208C. Then, the pellet was resuspended in
100 mL sterile aqua demin. This was repeated two times.
After that, the solution was sounded again for 2 min and
filtrated through an 8.0 mm filter (cellulose-nitrate-filter,
Sartorius). The stock solution was stored in the fridge at
48C.
Thermoactinomyces vulgaris ATCC 43649 spores. The
same procedure as described for S. griseus was done only
with Czapek Peptone Agar for 7 days (sucrose (Merck) 30.0
g, NaNO3(Merck) 3.0 g, K2HPO4(Merck) 1.0 g, MgSO4N7H2O
(Merck) 0.5 g, KCL (Merck) 0.5 g, FeSO4N7H2O (Merck)
0.01 g, yeast extract (Oxoid) 2.0 g, tryptone (Oxoid) 5.0 g,
agar (Oxoid) 15.0 g per litre aqua demin), centrifugation
with 2600 rcf and filtration through a 12.0 lm filter.
2.3 Standardization of bacteria titer
For irradiation the titer of the test suspension was stan-
dardized at 1 N105vegetative bacteria/mL with a photo-
metric method as described previously [4]. The number of
cfu of the spore stock solutions was only one time deter-
mined because the spores will keep in the fridge at 48C
for several months, as well as the spores of the anaerobe
C. pasteurianum. Hence, simply by diluting the stock sus-
pension a concentration for the irradiation of 1 N105
spores/mL is reached (for C. pasteurianum 1N106spores/
mL). The spores were irradiated in sterile aqua demin
and the vegetative bacteria in 0.65% NaCl. The transmis-
sion of each solution for the irradiation was measured
with a photometer (Hitachi U-1100 Spectrometer).
2.4 Irradiation and evaluation of the results
The irradiation was done as described previously [4]. Each
time 25 mL of test suspension were irradiated for differ-
ent periods of times in 85 mm standard polystyrene Petri
dishes (arithmetical thickness of 4.4 mm) without inter-
mixing and shade effects. The measured transmissions
(Hitachi U-1100 Spectrometer, quartz glass cuvette
against 0.65% NaCl) of all bacteria solutions used for irra-
diation were between 96.598.0% for both wavelengths.
In order to determine the exact inactivation kinetic, five
duplicate samples of each of the microorganisms were
irradiated. After irradiation, A. niger and P. expansum
spores were plated in pour-plate method on YGC-Agar
and incubated for 5 days at 308C. B. cereus spores and
vegetative bacteria, S. aureus and P. aeruginosa were pla-
ted in pour-plate method on PC-Agar (tryptone (Oxoid)
5.0 g, yeast extract (Oxoid) 2.5 g, glucose (Oxoid) 1.0 g and
agar (Oxoid) 10.0 g per litre aqua demin) and incubated
for 24 h at 308C. A. nicotinovorans and D. radiodurans were
plated in pour-plate method on Corynebacterium Agar
and incubated at 308C for 24 h for A. nicotinovorans and
72 h for D. radiodurans.C. pasteurianum spores were pla-
ted in pour-plate method on GYE-Agar and incubated in
air sealed 2.0 L preserving glasses each with an Anaero-
gen bag for 2.5 L and incubated for 72 h at 378C. S. griseus
spores were plated on GYM-Agar and incubated for 72 h
at 288C. T. vulgaris spores were plated in pour-plate
method on Czapek Peptone Agar and incubated for 20 h
at 508C. The evaluation of the results was done as
described previously according to the regulations of the
DVGW and the NORM, which lay down the require-
ments and testing of plants for the disinfection of water
using ultraviolet radiation [7, 8]. The dose reduction fac-
tor was determined and plotted logarithmically as func-
tion of the irradiance.
3 Results
The UV fluence/reduction response curves for the tested
mold and bacteria spores are presented in Figure 1, the
curves for the vegetative bacteria in Figure 2. The neces-
sary UV fluences with both wavelengths for reductions of
14-log steps and 9099.99% respectively for all tested
microorganisms are presented in Table 1. The inactiva-
tion curves for both wavelengths are very close for the
two kinds of mold spores, the curves of P. expansum even
cross each other twice (cf. Fig. 1). Furthermore, the curves
do not necessarily show the typical sigmoid gradient of
the other inactivation curves but are rather irregular.
The values of the necessary UV fluences for the corre-
sponding inactivations differ only slightly for both wave-
lengths. However, the inactivation at 222 nm seems to be
more effective. For a 3-log reduction of Aspergillus niger
spores 3250 J/m2at 222 nm are necessary, at 254 nm 3700
J/m2(cf. Table 1). For P. expansum the UV fluence is 420
J/m2at 222 nm and 490 J/m2at 254 nm. For A. niger and
222 nm even 4300 J/m2and for 254 nm 5600 J/m2are
necessary for a reduction of 4-log steps. Therefore, these
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528 M. Clauß Acta hydrochim. hydrobiol. 2006, 34, 525 –532
spores show the highest resistance against UV radiation
among all tested germs. P. expansum fungus spores can be
inactivated correspondingly with only a seventh of the
UV fluence. There is considerably less UV fluence neces-
sary for a corresponding reduction of the spores of B. cer-
eus and T. vulgaris at 222 nm than at 254 nm. The values
i2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www3.interscience.wiley.com/cgi-bin/jhome/5007772
Figure 1. UV fluence/reduction response curves for different mold and bacteria spores. All symbols indicate the
results of five independent series of experiments. Error bars denote the standard deviations.
Acta hydrochim. hydrobiol. 2006, 34, 525 – 532 More effective photoinactivation with 222 nm 529
of the inactivation of B. cereus spores by 3-log steps are for
222 nm 690 J/m2and for 254 nm 1400 J/m2. For T. vulgaris
the values are 550 J/m2and 1400 J/m2for a 4-log reduc-
tion. Consequently, these bacteria are almost 10 times
more resistant against UV radiation of these wavelengths
compared with the spores of C. pasteurianum and S. gri-
i2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www3.interscience.wiley.com/cgi-bin/jhome/5007772
Figure 2. UV fluence/reduction response curves for different vegeta-
tive bacteria. All symbols indicate the results of five independent se-
ries of experiments. Error bars denote the standard deviations.
530 M. Clauß Acta hydrochim. hydrobiol. 2006, 34, 525 –532
seus. For C. pasteurianum the values for a reduction of 4-
log steps at 222 nm are only 96 J/m2and at 254 nm 84
J/m2. For S. griseus the values are 255 J/m2and 182 J/m2.As
a result, these two germs can be inactivated more effi-
ciently with 254 nm. For the vegetative bacteria it can
easily be recognised that the wavelength of 254 nm of
the mercury lamp clearly inactivates almost all germs in
a more efficient way than the 222 nm of the excimer
lamp. The only exception is D. radiodurans which is with
a sufficient UV fluence of 910 J/m2at 222 nm and 1700
J/m2at 254 nm for a 3-log reduction about 10 times more
resistant against UV-radiation of both wavelengths than
the other tested vegetative bacteria. Furthermore, for the
same reduction it needs only half of the UV fluence at
222 nm than at 254 nm. With a 4-log reduction at 179
J/m2at 222 nm and 123 J/m2at 254 nm, the vegetative
bacteria of B. cereus are approx. 10 times more sensitive
against UV-radiation of both wavelengths than the
spores. For A. nicotinovorans, an UV fluence of 198 J/m2at
222 nm and 135 J/m2at 254 nm was sufficient for a 4-log
reduction. For the same reduction, an UV fluence of 178
J/m2and 95 J/m2was necessary for S. aureus. Here, the
wavelength of 254 nm is almost twice as effective as at
222 nm. The most UV-radiation sensitive bacteria is P. aer-
uginosa with a necessary UV fluence of 75 J/m2at 222 nm
and 31 J/m2at 254 nm for a 4-log reduction. For this case,
the wavelength of 254 nm is more than twice effective.
4 Discussion
Contradictory results regarding the inactivation of micro-
organisms can be found in literature because of the differ-
ent experimental conditions and the big variability of the
organisms. For example, the UV fluences for a reduction
of 1- and 3-log steps for S. aureus ATCC 25923 are 45 J/m2
and 67 J/m2[9] and in the present examination 44 J/m2and
73 J/m2showing only a slight difference. In contrast, the
UV fluence for P. aeruginosa is for 1-log step 48 J/m2[10],
but in the present examination it is only 8 J/m2. Another
problem is the lack of literature dealing with the inactiva-
tion of microorganisms with wavelengths less than 254
nm. Here, only few papers can be found [1, 4–6, 11].
The investigated inactivation curves of the tested
germs show approximately the expected typical sigmoid
gradient. The only exceptions are the alternate curve gra-
dients of the mold spores of A. niger and P. expansum for
both wavelengths. These curves are close together, i.e.
the inactivation of the spores each with 222 nm and 254
nm is nearly the same. Nevertheless, the excimer lamp
seems to be better for an inactivation referring to A. niger.
The obtained values of the one curve are in the range of
the standard deviations of the other curve and vice versa.
The data for P. expansum are not clear, too, because of the
high standard deviations. The most UV resistant germ of
all tested microorganisms is A. niger. Together with P.
expansum it belongs to the group of eucaryotic organisms
which are higher developed in contrast to the procaryo-
tic bacteria. But this seems to have no general influence
on the UV resistance, because P. expansum spores are
clearly more sensitive than for example the spores of B.
cereus. Even in previous investigations, cells of the eucar-
yotic yeast C. albicans ATCC 10231 showed a reduction of
4-log steps after an UV fluence of 290 J/m2with a wave-
length of 222 nm and 230 J/m2with 254 nm. This neces-
sary UV fluence is only slightly higher than in the present
investigation for the vegetative bacteria. Also responsible
for the high UV resistance of A. niger is the black spore
pigment aspergillin which is certainly able to absorb
high amounts of the UV radiation to protect the cell [12].
If highly efficient repair mechanisms were responsible
for the resistance, the inactivation curve would show a
higher shoulder [13].
In contrast to the vegetative bacteria of the same
strains, the spores of B. cereus and also the ones of Bacillus
subtilis ATCC 6051, tested in previous investigations [6],
can be inactivated almost twice as effective with 222 nm
than with 254 nm. For the spores of B. cereus and B. subtilis
other authors have investigated similar ratios as well [11]
but no detailed information was given about the irra-
diances of the lamps used and the bacterial strains.
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Table 1. Necessary UV fluences for a 1-4-log reduction of
different microorganisms with 222 nm and 254 nm UV radiation.
UV fluence in J/m2
Tested microorganisms lg(N/N0)1–2–3–4
Aspergillus niger 222 nm 900 2200 3250 4300
spores ATCC 32625 254 nm 1150 2450 3700 5600
Penicillium expansum 222 nm 180 330 420 –
spores ATCC 36200 254 nm 110 380 490 650
Bacillus cereus 222 nm 250 430 690 –
spores ATCC 11778 254 nm 520 930 1400
Clostridium pasteurianum 222 nm 43 61 79 96
spores ATCC 6013 254 nm 34 53 67 84
Thermoactinomyces vulgaris 222 nm 250 380 460 550
spores ATCC 43649 254 nm 550 900 1150 1400
Streptomyces griseus 222 nm 127 168 200 255
spores ATCC 10137 254 nm 85 126 154 182
Bacillus cereus 222 nm 85 111 137 179
veg. bacteria ATCC 11778 254 nm 58 73 85 123
Deinococcus radiodurans 222 nm 440 570 910 –
ATCC 13939 254 nm 1130 1420 1700 2050
Arthrobacter nicotinovorans 222 nm 104 149 177 198
ATCC 49919 254 nm 80 102 120 135
Staphylococcus aureus 222 nm 93 115 138 178
ATCC 25923 254 nm 44 60 73 95
Pseudomonas aeruginosa 222 nm 31 48 59 75
ATCC 27853 254 nm 8 16 23 31
Acta hydrochim. hydrobiol. 2006, 34, 525 – 532 More effective photoinactivation with 222 nm 531
Another investigation shows that the B. subtilis strain HA
101 and two derivates of it which were defective in exci-
sion repair and spore repair of spore photoproduct were
investigated [5]. In addition, one of the strains carried a
mutation which caused a defect in DNA polymerase I. It
was revealed that the inactivation of the wild-type spores
with 222 nm is better than with 254 nm, in contrast to
the two defective strains. These results indicate that one
reason for the better inactivation of spores with 222 nm
is the damage caused to proteins like Polymerase I.
Furthermore, the DNA in dormant spores of Bacillus and
Clostridium species is complexed with a group of unique
proteins, the a/btype SASP (small, acid-soluble proteins)
[14]. These DNA protecting proteins are one reason for
the high resistance of the spores, but they can be
damaged more easily with 222 nm radiation than with
254 nm. Another reason is the presence of an enormous
depot of pyridine-2,6-dicarboxylic acid (dipicolinic acic
(DPA)) [14]. The two –COOH groups absorb the energy of
UV light from about 215225 nm in a selective way [15].
This could lead to a defect of the molecule and thus of
the spores though the absorption of the pyridine is in the
range of 255265 nm. No clear evidence can be given for
the spores of C. pasteurianum referring to the effective-
ness of both wavelengths and the data collected in the
present investigation. The necessary UV fluences for the
excimer lamp are a little bit higher than for the mercury
lamp, but the curves are very close together and the
obtained values of the one curve are in the range of the
standard deviations of the other curve and vice versa.
Remarkable is the UV sensitivity of the Clostridium
spores although they are very similar to the Bacillus
spores. One explanation for this could be the strictly
anaerobic way of living in the soil, the natural habitat of
these bacteria, which means no exposure to oxygen nor
to sunlight. So special protect and repair mechanisms
against UV radiation would be unnecessary. The same
may be presumed for the spores of the aerobe soil bacter-
ium S. griseus. They are also very sensitive to UV radiation,
but they can be inactivated better with 254 nm in con-
trast to the spores of T. vulgaris. This species is also a typi-
cal soil bacterium, but shows an approximately 8 times
higher UV resistance than S. griseus and can clearly be
inactivated better with 222 nm. T. vulgaris is able to pro-
duce a polysaccharide layer which usually protects the
cells against high temperatures. This could be a reason
for the higher UV resistance.
Among the vegetative bacteria D. radiodurans is the
most UV resistant species; even more resistant than the
spores of B. cereus. One reason certainly are the effective
repair mechanisms, clearly shown by the high shoulder
of the 254 nm inactivation curve. At 222 nm this
shoulder is considerably lower and even the necessary
UV fluences for a respective inactivation are only about
50% of those with 254 nm. This indicates that the repair
mechanisms were damaged. This thesis is also supported
by the lower photoreactivation rate, done by the enzyme
photolyase after irradiation with 222 nm [4].
There is another possible reason for the higher effec-
tiveness of 222 nm regarding the inactivation especially
for the UV resistant microorganisms. It could be the
level of the UV fluence itself. If the UV fluence is high
enough, many proteins are damaged to such an extent
that there is no possibility for the cell to maintain the
metabolism. This finally leads to cell death, no matter
how effective the repair mechanisms are. Many procar-
yotic and eucaryotic cells are able to repair proteins to
a certain extent [15]. They can either restore damaged
polypeptides to an active stage or they can remove
them [15]. However, protein synthesis is severely limited
in starved bacteria [16]. Bacterial spores are in fact not
starved but inactive. This also applies for the DNA
repair mechanisms, which do not become active until
germination and outgrowth of the spores. It can there-
fore be assumed that in a case where not only the DNA
structure suffers damage (by irradiation with 254 nm)
but also the proteins (by irradiation with 222 nm)
which are responsible for various repair mechanisms
the caused damage may lead to severe consequences for
the cell which is indicated by the more effective inacti-
vation of UV-resistant species.
The remaining vegetative bacteria are very UV sensi-
tive. They all can be inactivated better with 254 nm. How-
ever, less than 200 J/m2UV fluence with the excimer
lamp is sufficient for a 4-log reduction, depending on the
species. The regulations of the DVGW [7] and the Trink-
wasserverordnung [17] (Drinking Water Ordinance of
Germany) prescribe for UV-devices in Germany a mini-
mum UV fluence of 400 J/m2and a wavelength of
240290 nm. With the demanded irradiation dose suffi-
cient reductions for all tested bacteria are reached with
the 222 nm wavelength of the excimer lamp. With this
dose, there is a reduction of less than 4-log steps for the
especially resistant microorganisms, however, the ratio
still is more favourable for the excimer lamp than for the
mercury lamp (cf. Table 1). Unfortunately, the wave-
length of 222 nm is below the prescribed wavelength
range. Therefore, this type of lamp must not be used for
disinfection of drinking water at least in Germany.
This work was supported by the company WEDECO AG, Her-
ford (Dr A. Kolch). Special thanks go to Dr M. Roth of the
Radium Lampenwerk GmbH for the supply of the KrCl excimer
lamp.
i2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www3.interscience.wiley.com/cgi-bin/jhome/5007772
532 M. Clauß Acta hydrochim. hydrobiol. 2006, 34, 525 –532
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Five types of Bacillus subtilis spores (UVR, UVS, UVP, RCE, and RCF) differing in repair and/or recombinational capabilities were exposed to monochromatic radiations at 13 wavelengths from 50 to 300 nm in vacuum. An improved biological irradiation system connected to a synchrotron radiation source was used to produce monochromatic UV radiation in this extended wavelength range with sufficient fluence to inactivate bacterial spores. From the survival curves obtained, the action spectra for the inactivation of the spores were depicted. Recombination-deficient RCE (recE) and RCF (recF) spores were more sensitive than the wild-type UVR spores in the entire range of wavelengths. This was considered to mean that DNA was the major target for the inactivation of the spores. Vacuum-UV radiations of 125-175 nm were effective in killing the spores, and distinct peaks of the sensitivity were seen with all types of the spores. Insensitivities at 190 and 100 nm were common to all five types of spores, indicating that these wavelengths were particularly impenetrant and absorbed by the outer layer materials. The vacuum-UV peaks centering at 150 nm were prominent in the spores defective in recombinational repair, while the far-UV peaks at around 235 and 270 nm were prominent in the UVS (uvrA ssp) and UVP (uvrA ssp polA) spores deficient in removal mechanisms of spore photoproducts. Thus, the profiles of the action spectra were explained by three factors; the penetration depth of each radiation in a spore, the efficiency of producing DNA damage that could cause inactivation, and the repair capacity of each type of spore.
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