UV-c Inactivation of Microorganisms
Prof Clive Beggs, Medical Biophysics Group, University of Bradford
(e-mail: c.b.beggs@bradford.ac.uk)
The lethal effect of ultraviolet (UV) light on microorganisms has been known for many years.
In the 1920's and 1930's a number of researchers demonstrated the susceptibility of a variety
of microorganisms to UV-C light (i.e. 100 - 280 nm) [1-4] and by the 1940’s UV-C light was
routinely used to disinfect air in tuberculosis (TB) wards in the USA [5, 6]. However, the use of
UV lamps in hospital wards fell out of favour with the introduction of improved drug therapies.
Notwithstanding this, with the world-wide resurgence of TB in the 1990’s [7-9] and the
emergence of multiple-drug resistant strains of TB [10-12], interest has been renewed in UV
air disinfection [13-21]. Although many researchers have studied the effects of UV induced
damage in microorganisms, most of this work has been experimental in nature; by
comparison relatively little theoretical work has been undertaken to analyse the kinetics of the
UV inactivation process.
When photons of UV light strike biological cells, the energy in the photons is absorbed by
chromophores at discrete wavelengths. The biological impact of UV radiation is primarily due
to the absorption of photons by nucleic acids. DNA has an absorption spectra which has a
maximum in the 260 – 265 nm region and which rapidly declines towards longer wavelengths
[22]. Low pressure UV lamps have a strong UV-C spectral emission at 253.7 nm [23] which is
absorbed by nucleic acids with the result that pyrimidine lesions are formed in DNA. Although
both purine and pyrimidine bases in DNA are strong absorbers of UV-C photons, the
photodecomposition of pyrimidines is much greater than that of purines, with the result that
the UV quantum yield required to create dimers in pyrimidines is of the order of 10 -3, while
that for purines is in the region of 10-4 [24]. It is therefore the photoproducts derided from
pyrimidines that are of most biological importance. These include cyclobutane pyrimidine
dimers, 6-4 pyrimidine-pyrimidone photoproduct and 5-thyminyl-5, 6-dihydrothymine often
termed spore photoproduct [18, 22, 25].
Cyclobutane pyrimidine dimers are generally considered to be the most important class of
DNA photoproduct. They are formed in double stranded DNA when two adjacent pyrimidine
bases fuse together to form a dimer. There are three types of pyrimidine dimers, thyminethymine (T<>T), thymine-cytosine (T<>C), and cytosine-cytosine (C<>C). The frequency with
which these dimer types occur varies with the DNA base composition [22], as can be seen in
Table 1.
DNA source
Haemophilus
influenzae
Escherichia coli
Micrococcus luteus
(A + T) : (G + C)
ratio
Breakdown of
dimers formed
T<>T
C<>T
C<>C
1.63
71%
24%
5%
1.00
0.43
59%
19%
34%
55%
7%
26%
Table 1 Breakdown of pyrimidine dimers formed by 265 nm irradiation at a fluence (i.e. UV
dose) of 200 J/m2 [22]
Like cyclobutane dimers, 6-4 pyrimidine-pyrimidone adducts are formed between adjacent
pyrimidine bases in DNA. The frequency with which 6-4 pyrimidine-pyrimidone photoproducts
are formed depends on the base composition of the DNA. In the Escherichia coli (E. coli) lacI
and lacZ genes cyclobutane pyrimidine dimers and 6-4 pyrimidine-pyrimidone photoproducts
form in a 2:1 ratio during UV irradiation [25]. Spore photoproduct is a thymine lesion which is
the predominant photoproduct formed when bacterial spores are irradiated with UV light [22,
26]. Spore photoproduct has also been reported in isolated vegetative cell DNA at low relative
humidity (RH) levels [27, 28] and in aerosolised bacteria at humidities below 65% RH [18].
When investigating the kinetics of UV inactivation it is helpful to consider a UV field as a
series of photon ‘bullets’, each containing a specific package of energy, which are fired at
a randomly moving series of target microorganisms (or stationary targets if the
microorganisms are on solid media). The energy contained in each photon of light can be
determined using equation 1.
Q
Where;
Q

h
c
=
=
=
=
hc
(1)

Energy of a single photon (J)
Wavelength of the light (m)
Planck’s constant (i.e. 6.623  10-34 Js)
Speed of light (i.e. 2.998  108 m/s)
In many instances a log-linear plot of the number of microorganisms surviving against UV
fluence (i.e. UV dose in J/m 2) reveals a straight line which passes through unity on the y axis.
This pattern of inactivation is often referred to as a one-hit model and is characterised by the
simple exponential equation:
Nt
 kt
e
No
Where:
Nt
No
k
t
=
=
=
=
(2)
Number of living microorganisms at time t seconds (cfu)
Number of living microorganisms at time 0 seconds (cfu)
Inactivation rate constant (s-1)
Duration of exposure to UV irradiation (s)
Equation 2 can be modified to incorporate the UV fluence rate.
Nt
 ZEt
e
No
where:
E
Z
=
=
(3)
UV fluence rate (W/m 2)
UV susceptibility constant of particular microorganism (m 2/J)
In the one-hit model it is assumed that; (i) a single hit is sufficient to inactivate a cell, (ii) the
number of hits achieved is directly proportional to the UV fluence applied, and that (iii) the
microorganism population is uniformly susceptible to the UV irradiation [29].
Although the equations 2 and 3 can be successfully applied to many UV inactivation systems,
the one-hit model does not fully explain the inactivation process. In reality, a great many
photon hits are required before a microorganism finally becomes inactivated. In addition, the
various DNA repair mechanisms play an important role in repairing damage sustained by UV
photons. The number of photons absorbed by a target microorganism can be determined
using equation 4 and the quantum yield using equation 5.
P
Where;
Pabs

abs

=
=
Et
Number of photons absorbed by the target microorganism
Inactivation cross-sectional area (m 2)

Where;

M
(4)
hc
=
=
M
Pa bs
(5)
Quantum yield
Number of molecules altered
Jagger [24] estimates that at a UV fluence of 0.1 J/m 2 at 260 nm one thymine dimer is
produced per 200 microns of E. coli DNA. This equates to approximately 5 dimers per E.coli
chromosome. Similarly, Harm [30] states that for E. coli a fluence of 1.0 J/m 2, at 254 nm, will
produce 65 pyrimidine dimers in a chromosome. By applying equations 4 and 5 to a 2.5 m 
1 m E. coli bacterium it is possible to analyse and compare Jagger and Harm’s assertions
(see Table 2).
Researcher
Jagger (24)
Harm (30)
UV
fluence
Wavelength
(J/m2)
(nm)
0.1
1.0
260
254
Number of photons
absorbed by a
single E.coli target
Average number of
photon hits required to
create a dimer
Quantum
Yield
327361
3198065
65472
49201
1.527  10-5
2.032  10-5
Table 2 Analysis of Jagger and Harm’s data
From Table 2 it can be seen that the quantum yields derived from Jagger and Harm’s data
are of a similar order of magnitude and that they are both well within the expected range of
10-3 to 10-6 [31]. This implies that many thousands of photon hits occur per bacterium before a
cyclobutane pyrimidine dimer is formed. Given that the quantum yield of a pyrimidine dimer is
about 1 [32], it can be concluded that most hits cause no photochemical reaction at all. This is
not at all surprising given that the target area presented by a bacterium is much greater than
that of a single dimer.
Given the discussion above it is clear that the one-hit model does not satisfactorily describe
the UV inactivation process and that a superior model is required. This is especially the case
for UV inactivation curves which exhibit a shoulder, such as the survival curve shown in
Figure 1. It is important to note that in many experiments, microorganisms do not exhibit a
shoulder simply because the UV fluence rates used are too high and the microorganisms are
very quickly overwhelmed by the photons. If lower fluence rates are used then in many
instances a shoulder will be observed. Shoulders are often observed in microorganisms which
have efficient DNA repair mechanisms.
83.8
79.8
75.8
71.8
67.8
63.8
59.9
55.9
51.9
47.9
43.9
39.9
35.9
31.9
27.9
20
16
12
7.98
3.99
0
23.9
tc
1
0.1
Survival fraction
0.01
0.001
0.0001
0.00001
0.000001
0.0000001
UV fluence (J/m2)
Figure 1
Typical shouldered UV Survival Curve
From Figure 1 it can be seen that at low UV doses there is a shoulder in the inactivation curve
and that after a certain threshold dose the curve becomes a straight line which is similar to
that for the one-hit model [29]. This type of curve is often referred to as a multi-target model
[29]. The shoulder in the curve represents the period in which the microorganisms receive a
sub-lethal dose and little or no inactivation occurs.
The UV field can be considered as a series of photon ‘bullets’ which are fired at a randomly
moving series of target microorganisms. Each target microorganism will receive many photon
hits before one final hit eventually inactivates it. So if the nth photon to hit a target
microorganism is lethal, it follows that all the photon hits up to and including n – 1, will
produce a sub-lethal effect. If it is assumed that all the target microorganisms are equally
susceptible to UV damage and that the photons strike all the targets at roughly the same rate,
then they will all accumulate photon hits until each has received n – 1 hits. Thereafter, the first
microorganism to be struck by the nth photon will become inactivated. Similarly, the second
microorganism to receive a nth hit will be inactivated, and so on, until all the microorganisms
present are inactivated. However, as more and more microorganisms are removed, it
becomes increasingly difficult for the photons to hit targets. The first tranche of photons will
easily inactivate microorganisms, but as the process continues successive tranches of
photons will be aiming at fewer and fewer targets. Consequently, more and more photons
must be fired in order to maintain the same kill rate. So although only one photon eventually
kills any particular microorganism, there is a cumulative component to the inactivation
process, since many non-lethal photon hits are required before the first lethal hits occur.
Equations 6 and 7 are expressions derived by Kowalski et al. [33] to describe the multi-target
inactivation curve. These equations utilise a quasi-threshold term, tc, (see Figure 1) which
represents the time at the point where the extended straight line decay curve intersects the
unity line.
t  2tc
If;
Then;
Nt
( zE / 4tc )t 2
e
No
where:
tc
And if;
t  2tc
=
(6)
Quasi-threshold time (s)
Then;
Nt
 zE (t tc )
e
No
(7)
REFERENCES
1. F.L. Gates, A study of the bacterial action of ultraviolet light, J. Gen. Physiol. 13
(1929) 231 - 260
2. D.G. Sharp, A quantitative method of determining the lethal effect of ultraviolet light
on bacteria suspended in air, (1937) 589 – 599
3. D.G. Sharp, The lethal action of short ultraviolet rays on several common pathogenic
bacteria, (1938) 447 – 460
4. H.C. Rentschler, R. Nagy, Bacterial action of ultraviolet radiation on air-borne
organisms, (1941) 85 - 91
5. L.J. Buttolph, H. Haynes, Ultraviolet air sanitation. General Electric Lamp Division,
Cleveland, Ohio, USA, 1953
6. W.W. Stead, et al. Probable role of ultraviolet irradiation in preventing transmission of
tuberculosis: A case study. Infection Control & Hospital Epidemiology, 17 (1996) 11 –
13
7. J.M. Grange, A. Zumia, Paradox of the global emergency of tuberculosis. Lancet, 353
(1999) 996
8. WHO. TB - a global emergency: WHO report on the TB epidemic. WHO, Geneva,
1994
9. WHO. Global Tuberculosis Programme. Global tuberculosis control. WHO report,
WHO, Geneva. WHO/TB/98 – 237, 1998
10. V. Gleissberg, The threat of multidrug resistance: is tuberculosis ever untreatable or
uncontrollable? Lancet, 353 (1999) 998 – 999
11. T.R. Freiden, et al. A multi-institutional outbreak of highly drug resistant tuberculosis:
Epidemiology and Clinical Outcomes, Journal of the American Medical Association.
276 (1996) 1229 - 1235.
12. A.S. Breathnach, A. de Ruiter, G.M. Holdsworth, N.T. Bateman, D.G. O’Sullivan, P.J.
Rees, D. Snashall, H.J. Milburn, B.S. Peters, J. Watson, F.A. Drobniewski, G.L.
French, An outbreak of multi-drug-resistant tuberculosis in a London teaching
hospital. Journal of Hospital Infection. 39 (1998) 111 - 117.
13. J.M. Macher, L.E. Alvantis, Y.L. Chang, K.S. Liu, Effect of ultraviolet germicidal lamps
on airborne microorganisms in an outpatient waiting room, Applied. Occupational
and Environmental Hygiene. 7 (1992) 505 – 513
14. S.L. Miller, J.M. Macher, Evaluation of a methodology for quantifying the effect of
room air ultraviolet germicidal irradiation on airborne bacteria, Aerosol Science and
Technology. 33 (2000) 274 – 295
15. C.B. Beggs, K.G. Kerr, JK. Donnelly, P.A. Sleigh, D.D. Mara, G. Cairns, An
engineering approach to the control of Mycobacterium tuberculosis and other
airborne pathogens: a UK hospital based pilot study, Transactions of the Royal
Society of Tropical Medicine and Hygiene. 94 (2000) 141 – 146
16. G. Ko, MW First, HA Burge, Influence of relative humidity on particle size and UV
sensitivity of Serratia marcescens and Mycobacterium bovis BCG aerosols. Tubercle
and Lung Disease. 80 (2000) 217 – 228
17. J.L. Peccia, H.M. Werth, M.T. Hernandez, Effects of relative humidity on the UVinduced inactivation of bacterial bioaerosols. Journal of Aerosol Science. 31 (2000)
Suppl. 1, S959 – S960
18. J.L. Peccia, H.M. Werth, S.M. Miller, M.T. Hernandez, Effects of relative humidity on
the ultraviolet induced inactivation of airborne bacteria. Aerosol Science and
Technology. 35 (2001) 728 – 740
19. C.B. Beggs, P.A. Sleigh, A quantitative method for evaluating the germicidal effect of
upper room UV fields. Journal of Aerosol Science. 23 (2002) 1681-1699
20. C.B. Beggs, C.J. Noakes, P.A. Sleigh, L.A. Fletcher, K.G. Kerr, Methodology for
determining the susceptibility of airborne microorganisms to irradiation by an upperroom UVGI system. Journal of Aerosol Science. 37 (2006) 885-902
21. C.J. Noakes, P.A. Sleigh, A.R. Escombe, C.B. Beggs, Use of CFD analysis in
modifying a TB Ward in Lima, Peru. Indoor and Built Environment. 15 (2006) 41-47
22. W. Harm, Biological effects of ultraviolet radiation. Cambridge University Press,
Cambridge, Chapter 3, 1980
23. Philips Lighting. Disinfection by UV-radiation: Germicidal lamps, 1993
24. J. Jagger, Introduction to research in ultraviolet photobiology. Prentice-Hall, New
Jersey, Chapter 4, 1967
25. D. Chandrasekhar, B. Van Houten. In vivo formation and repair of cyclobutane
pyrimidine dimers and 6-4 photoproducts measured at the gene and nucleotide level
in Escherichia coli. Mutation Research, 450 (2000) 19 – 40.
26. W.L Nicholson, T.A. Slieman, DNA in dormant Bacillus subtilis spores exposed to
solar radiation accumulates strand breaks and cyclobutane pyrimidine dimers in
addition to spore photoproduct.
27. M.H. Patrick, D.M. Grey, Independence of photoproduct formation on DNA
conformation. Photochem. Photobiol. 24 (1976) 507 – 513.
28. R.O. Rahn, L.C. Landry, Pyrimidine dimer formation in poly (d-dT) and apurinic acid.
Biochim. Biophys. Acta. 247 (1971) 197 – 206.
29. D.L. David, W.D.J. Jones, C.M. Newman, Ultraviolet light inactivation and
photoreactivation in the mycobacteria. Infect. Immun. 4 (1971) 319 – 329
30. W. Harm W, Biological effects of ultraviolet radiation. Cambridge University Press,
Cambridge, Chapter 7, 1980
31. W. Harm, Biological effects of ultraviolet radiation. Cambridge University Press,
Cambridge, Chapter 6, 1980
32. J. Jagger, Introduction to research in ultraviolet photobiology. Prentice-Hall, New
Jersey, Chapter 3, 1967
33. W.J. Kowalski, Design and optimisation of UVGI air disinfection systems. PhD Thesis,
Pennsylvania State University, (2001)
CB Beggs
24th October 2006