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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
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/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 microorgan-
isms by DNA damage whereas at higher UV fluence (various) mechanisms of protein damage can
presumably be held responsible for inactivation.
DOI 10.1002/aheh.200600650
problems especially for large-scale use of UV inactivation Agar (yeast extract (Oxoid) 5.0 g, glucose (Merck) 20.0 g,
of microorganisms when processing drinking water and chloramphenicol (Selective Supplement OXOID) 0.1 g
sewage, i. e. when inactivated cells can get out and are and agar (Oxoid) 12.0 g per litre aqua demin). The plates
exposed to sunlight. were incubated at 308C for 5 days. To harvest the spores,
This effect can be stopped partially by using wave- 3.0 g sterile quartz sand was dispersed on the plate. Then
lengths which are absorbed by other molecules than the plate was shaken for one minute by hand. The mix-
DNA. Proteins have a strong absorption maximum at 220 ture of sand and spores was removed by being washed
nm. UV radiation of approximately this wavelength can with sterile aqua demin with 0.001% Tween 80 and col-
be generated with a 222 nm KrCl-excimer lamp. Examina- lected in a 300 mL Erlenmeyer flask. To remove the
tions comparing the photoreactivation of E. coli after irra- spores from the sand, the suspension was sonicated for 2
diation with 222 nm and 254 nm have shown that the min in an ultrasonic bath (Sonorex RK 102 company Ban-
irradiation is considerably less effective at 222 nm than delin Electronics, Berlin). The supernatant was removed
at 254 nm [4]. Furthermore, additional tests showed that and centrifuged at 1000 rcf for 10 min at 208C. Then, the
Bacillus subtilis spores can be inactivated much better pellet was resuspended in 100 mL sterile aqua demin.
with UV radiation having a wavelength of 222 nm than This stock solution was stored in the fridge at 48C.
with 254 nm [5, 6] whereas the vegetative forms of B. sub- Bacillus cereus ATCC 11778 spores: 1…2 colonies were
tilis, Enterococcus faecalis, Candida albicans and E. coli can plated evenly on 5 Nutrient Agar plates (meat extract
be inactivated better with 254 nm. (Merck) 3.0 g, tryptone (Oxoid) 5.0 g, agar (Oxoid) 15.0 g,
In this examination, the effectiveness of photoinactiva- with 10.0 mg MnSO4 N H2O per litre aqua demin). After
tion with 222 nm wavelength compared to 254 nm wave- 72 h at 30 8C the harvest of the spores was done by resus-
length of further microorganisms is tested. For this, the pending the colonies on each plate in 10 mL sterile aqua
inactivation curves for both wavelengths of the microor- demin with a cotton bud. The solution was sonicated for
ganisms were taken. Out of the most important groups 2 min in an ultrasonic bath (Sonorex RK 102 company
different aerobic spore-producing bacteria and mold Bandelin Electronics, Berlin) and centrifuged (Biofuge 28
spores, the anaerobic and also spore-producing bacter- RS, company Heraeus) with 3000 rcf for 15 min at 208C.
ium Clostridium pasteurianum, some important microor- Afterwards, the pellet was resuspended in 100 mL sterile
ganisms relevant for drinking water and sewage and the aqua demin. This was repeated two times. Then, the solu-
highly radiation-resistant Deinococcus radiodurans, which tion was sounded again for 2 min. Vegetative cells were
has extremely effective repair mechanisms, were chosen. 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-
2 Materials and methods coccus aureus ATCC 25923, Pseudomonas aeruginosa ATCC
27853: 1…2 colonies were plated to Standard-I-Nutrient
2.1 UV source medium (Merck) and incubated at 308C for 24 h. Once
A collimated beam device (WEDECO AG Water Technol- again, 1…2 colonies were inoculated in 100 mL Stan-
ogy) with interchangeable lamp units was used for irra- dard-I-Nutrient broth (Oxoid) for 14…20 h. Bacteria were
diation as described previously [4]. The UV fluences were harvested by centrifugation (Biofuge 28 RS, company Her-
measured with a Bentham Spectrometer DM 150 double aeus) with 1000 rcf for 10 min at 208C, the pellet was
monochromator with a 200…450 nm standard sensing resuspended in 100 mL 0.65% NaCl and filtrated through
head and verified for the excimer lamp every month with an 8.0 lm filter (cellulose-nitrate filter, Sartorius).
Bacillus subtilis spores strain ATCC 6051 as biological dosi- Arthrobacter nicotinovorans ATCC 49919 and Deinococcus
meter after the previously investigated inactivation radiodurans ATCC 13939: The same procedure as
curve [6]. For the mercury lamp the irradiance was veri- described for B. cereus vegetative bacteria, S. aureus and P.
fied every week with a Spectroline DRC 100H digital aeruginosa was done only with Corynebacterium Agar
radiometer with a DIX-254A UV-C sensor (Spectronics (tryptone (Oxoid) 10.0 g, yeast extract (Oxoid) 5.0 g, glu-
Corporation). cose (Merck) 5.0 g, NaCl (Merck) 5.0 g, agar (Oxoid) 15.0 g
per litre aqua demin).
2.2 Organisms and their cultivation Clostridium pasteurianum ATCC 6013 spores: 1…2 colo-
Aspergillus niger ATCC 32625 spores and Penicillium expan- nies were plated to 5 GYE-agar plates (glucose (Merck)
sum ATCC 36200 spores (American Type Culture Collec- 20.0 g, yeast extract (Oxoid) 10.0 g, CaCO3 (Merck) 10.0 g
tion, Manassas, VA): Spores were taken from a slant cul- and agar (Oxoid) 17.0 g per litre aqua demin) under an-
ture with a sterile cotton bud and applied evenly on YGC- aerobic conditions in a nitrogen atmosphere. The plates
were put in an air sealed 2.0 L preserving glass with an against 0.65% NaCl) of all bacteria solutions used for irra-
Anaerogen bag (Oxoid atmosphere generation system) diation were between 96.5…98.0% for both wavelengths.
for 2.5 L and incubated for 72 h at 378C. Spores were har- In order to determine the exact inactivation kinetic, five
vested and stored under aerogenic conditions as it was duplicate samples of each of the microorganisms were
described for B. cereus spores. irradiated. After irradiation, A. niger and P. expansum
Streptomyces griseus ATCC 10137 spores: 1…2 colonies spores were plated in pour-plate method on YGC-Agar
were plated evenly on 5 GYM plates (glucose (Merck) 4.0 and incubated for 5 days at 308C. B. cereus spores and
g, yeast extract (Oxoid) 4.0 g, malt extract (Merck) 10.0 g, vegetative bacteria, S. aureus and P. aeruginosa were pla-
CaCO3 (Merck) 2.0 g, agar (Oxoid) 12.0 g per litre aqua ted in pour-plate method on PC-Agar (tryptone (Oxoid)
demin). After 5 days at 288C the harvest of the spores was 5.0 g, yeast extract (Oxoid) 2.5 g, glucose (Oxoid) 1.0 g and
done by resuspending the colonies on each plate in agar (Oxoid) 10.0 g per litre aqua demin) and incubated
10 mL sterile aqua demin with a cotton bud. The solution for 24 h at 308C. A. nicotinovorans and D. radiodurans were
was sonicated for 2 min in an ultrasonic bath (Sonorex plated in pour-plate method on Corynebacterium Agar
RK 102 company Bandelin Electronics, Berlin) and centri- and incubated at 308C for 24 h for A. nicotinovorans and
fuged (Biofuge 28 RS, company Heraeus) with 5000 rcf for 72 h for D. radiodurans. C. pasteurianum spores were pla-
15 min at 208C. Then, the pellet was resuspended in ted in pour-plate method on GYE-Agar and incubated in
100 mL sterile aqua demin. This was repeated two times. air sealed 2.0 L preserving glasses each with an Anaero-
After that, the solution was sounded again for 2 min and gen bag for 2.5 L and incubated for 72 h at 378C. S. griseus
filtrated through an 8.0 mm filter (cellulose-nitrate-filter, spores were plated on GYM-Agar and incubated for 72 h
Sartorius). The stock solution was stored in the fridge at at 288C. T. vulgaris spores were plated in pour-plate
48C. method on Czapek Peptone Agar and incubated for 20 h
Thermoactinomyces vulgaris ATCC 43649 spores. The at 508C. The evaluation of the results was done as
same procedure as described for S. griseus was done only described previously according to the regulations of the
with Czapek Peptone Agar for 7 days (sucrose (Merck) 30.0 DVGW and the NORM, which lay down the require-
g, NaNO3 (Merck) 3.0 g, K2HPO4 (Merck) 1.0 g, MgSO4 N 7H2O ments and testing of plants for the disinfection of water
(Merck) 0.5 g, KCL (Merck) 0.5 g, FeSO4 N 7H2O (Merck) using ultraviolet radiation [7, 8]. The dose reduction fac-
0.01 g, yeast extract (Oxoid) 2.0 g, tryptone (Oxoid) 5.0 g, tor was determined and plotted logarithmically as func-
agar (Oxoid) 15.0 g per litre aqua demin), centrifugation tion of the irradiance.
with 2600 rcf and filtration through a 12.0 lm filter.
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.
spores show the highest resistance against UV radiation UV fluence. There is considerably less UV fluence neces-
among all tested germs. P. expansum fungus spores can be sary for a corresponding reduction of the spores of B. cer-
inactivated correspondingly with only a seventh of the eus and T. vulgaris at 222 nm than at 254 nm. The values
of the inactivation of B. cereus spores by 3-log steps are for tion. Consequently, these bacteria are almost 10 times
222 nm 690 J/m2 and for 254 nm 1400 J/m2. For T. vulgaris more resistant against UV radiation of these wavelengths
the values are 550 J/m2 and 1400 J/m2 for a 4-log reduc- compared with the spores of C. pasteurianum and S. gri-
Another investigation shows that the B. subtilis strain HA shoulder is considerably lower and even the necessary
101 and two derivates of it which were defective in exci- UV fluences for a respective inactivation are only about
sion repair and spore repair of spore photoproduct were 50% of those with 254 nm. This indicates that the repair
investigated [5]. In addition, one of the strains carried a mechanisms were damaged. This thesis is also supported
mutation which caused a defect in DNA polymerase I. It by the lower photoreactivation rate, done by the enzyme
was revealed that the inactivation of the wild-type spores photolyase after irradiation with 222 nm [4].
with 222 nm is better than with 254 nm, in contrast to There is another possible reason for the higher effec-
the two defective strains. These results indicate that one tiveness of 222 nm regarding the inactivation especially
reason for the better inactivation of spores with 222 nm for the UV resistant microorganisms. It could be the
is the damage caused to proteins like Polymerase I. level of the UV fluence itself. If the UV fluence is high
Furthermore, the DNA in dormant spores of Bacillus and enough, many proteins are damaged to such an extent
Clostridium species is complexed with a group of unique that there is no possibility for the cell to maintain the
proteins, the a/b type SASP (small, acid-soluble proteins) metabolism. This finally leads to cell death, no matter
[14]. These DNA protecting proteins are one reason for how effective the repair mechanisms are. Many procar-
the high resistance of the spores, but they can be yotic and eucaryotic cells are able to repair proteins to
damaged more easily with 222 nm radiation than with a certain extent [15]. They can either restore damaged
254 nm. Another reason is the presence of an enormous polypeptides to an active stage or they can remove
depot of pyridine-2,6-dicarboxylic acid (dipicolinic acic them [15]. However, protein synthesis is severely limited
(DPA)) [14]. The two –COOH groups absorb the energy of in starved bacteria [16]. Bacterial spores are in fact not
UV light from about 215…225 nm in a selective way [15]. starved but inactive. This also applies for the DNA
This could lead to a defect of the molecule and thus of repair mechanisms, which do not become active until
the spores though the absorption of the pyridine is in the germination and outgrowth of the spores. It can there-
range of 255…265 nm. No clear evidence can be given for fore be assumed that in a case where not only the DNA
the spores of C. pasteurianum referring to the effective- structure suffers damage (by irradiation with 254 nm)
ness of both wavelengths and the data collected in the but also the proteins (by irradiation with 222 nm)
present investigation. The necessary UV fluences for the which are responsible for various repair mechanisms
excimer lamp are a little bit higher than for the mercury the caused damage may lead to severe consequences for
lamp, but the curves are very close together and the the cell which is indicated by the more effective inacti-
obtained values of the one curve are in the range of the vation of UV-resistant species.
standard deviations of the other curve and vice versa. The remaining vegetative bacteria are very UV sensi-
Remarkable is the UV sensitivity of the Clostridium tive. They all can be inactivated better with 254 nm. How-
spores although they are very similar to the Bacillus ever, less than 200 J/m2 UV fluence with the excimer
spores. One explanation for this could be the strictly lamp is sufficient for a 4-log reduction, depending on the
anaerobic way of living in the soil, the natural habitat of species. The regulations of the DVGW [7] and the Trink-
these bacteria, which means no exposure to oxygen nor wasserverordnung [17] (Drinking Water Ordinance of
to sunlight. So special protect and repair mechanisms Germany) prescribe for UV-devices in Germany a mini-
against UV radiation would be unnecessary. The same mum UV fluence of 400 J/m2 and a wavelength of
may be presumed for the spores of the aerobe soil bacter- 240…290 nm. With the demanded irradiation dose suffi-
ium S. griseus. They are also very sensitive to UV radiation, cient reductions for all tested bacteria are reached with
but they can be inactivated better with 254 nm in con- the 222 nm wavelength of the excimer lamp. With this
trast to the spores of T. vulgaris. This species is also a typi- dose, there is a reduction of less than 4-log steps for the
cal soil bacterium, but shows an approximately 8 times especially resistant microorganisms, however, the ratio
higher UV resistance than S. griseus and can clearly be still is more favourable for the excimer lamp than for the
inactivated better with 222 nm. T. vulgaris is able to pro- mercury lamp (cf. Table 1). Unfortunately, the wave-
duce a polysaccharide layer which usually protects the length of 222 nm is below the prescribed wavelength
cells against high temperatures. This could be a reason range. Therefore, this type of lamp must not be used for
for the higher UV resistance. disinfection of drinking water at least in Germany.
Among the vegetative bacteria D. radiodurans is the
most UV resistant species; even more resistant than the This work was supported by the company WEDECO AG, Her-
spores of B. cereus. One reason certainly are the effective ford (Dr A. Kolch). Special thanks go to Dr M. Roth of the
repair mechanisms, clearly shown by the high shoulder Radium Lampenwerk GmbH for the supply of the KrCl excimer
of the 254 nm inactivation curve. At 222 nm this lamp.
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