It is well-known that bacteria and other microorganisms are capable of repairing their DNA following damage by ultraviolet (UV) radiation. Known as ‘reactivation’, it is a natural defense mechanism that has evolved over millions of years. Some microorganisms need visible light in order to repair their DNA (known as ‘photoreactivation’), while others can repair their DNA without light (‘dark repair’). This self-repair ability poses obvious problems when UV disinfection technology is used to treat potable water, swimming pool water, effluent or other liquids.
There are two main types of UV disinfection technology currently in use: low pressure and medium pressure. Low pressure UV lamps contain mercury gas at a low pressure (<10 torr) which, when excited by an electrical charge, emit UV light at 254nm. Medium pressure lamps contain mercury gas at much higher pressures (~1000 torr). These lamps produce UV of a higher intensity and over a broader range of wavelengths than low pressure lamps.
Recent research comparing microbial DNA photoreactivation after exposure to UV from low and medium lamps has shown that the DNA of E. coli was repaired following low pressure irradiation, but not after exposure to medium pressure UV. These results are highly significant and could have an important effect on the decisions specifiers have to make when choosing UV plant.
Effects of UV light on DNA and other biomolecules
The UV section of the electromagnetic spectrum is divided into three main wavelength ranges which have differing effects on DNA, RNA and other molecules, such as enzymes, within the cell. The main UV spectra that have a damaging effect are UV-C (200 – 280nm), UV-B (280 – 315nm) and UV-A (315-400nm). Low pressure UV lamps have a peak output at 254nm, while medium pressure lamps have a broader output between about 185-400nm.
According to Von Sonntag (1986), DNA has its maximum absorption at both 200nm and 265nm. Maximum absorption does not occur at 254nm, the wavelength produced by low pressure lamps and often wrongly assumed to be optimum wavelength for killing microorganisms. At 200nm most absorption occurs in the ‘backbone’ DNA molecules of ribose and phosphate. At 265nm, UV absorption mainly occurs in the nucleotide bases: adenine, guanine, cytosine and thymine (and uracil in the case of RNA). The most common products resulting from damage by UV radiation are thymine dimers, which are formed when two adjacent thymine molecules become fused. The formation of these dimers and other photoproducts prevents the DNA from being able to replicate, effectively killing the cell.
In some cases UV is effective above 265nm. It has been shown, for example, that the optimum wavelength for destroying Cryptosporidium parvum oocysts is 271nm (15% more effective than 254nm) (Linden, 2001), while the optimum wavelength for Bacillus subtillis is 270nm (40% more effective than 254nm) (Waites, 1988).
In addition to DNA and RNA, UV also causes photochemical reactions in proteins, enzymes and other molecules within the cell. Absorption in proteins peaks around 280nm, and there is some absorption in the peptide bond (-CONH-) within proteins at wavelengths below 240nm. Other biological molecules with unsaturated bonds may also be susceptible to destruction by UV – examples include coenzymes, hormones and electron carriers. The ability of UV to affect molecules other than DNA and RNA is particularly interesting in the case of larger microorganisms such as fungi, protozoa and algae. In these microorganisms, although UV may be unable to penetrate as far as the DNA, it could still have a lethal effect by damaging other molecules.
Recovery from UV damage
The need to recover from or repair UV damage is common to virtually all microorganisms that are regularly exposed to UV light in nature. Known as reactivation, the process can take place in both light and dark conditions and is called, respectively, photoreactivation and dark repair. The ability to reactivate varies significantly depending on the type of UV damage inflicted and by the level of biological organization of the microorganism. The repair mechanism is not universal and there are no clearly defined characteristics determining which species can repair themselves and those which cannot.
The part of cells most vulnerable to UV damage is the DNA and RNA. This is due partly to its unique function as the depository of the cell’s genetic code, and also because of its highly complex structure and large size. It is hardly surprising therefore that all known molecular repair mechanisms have evolved to act upon the macromolecular nucleic acids, particularly DNA. In photoreactivation repair is carried out by an enzyme called photolyase which reverses the UV-induced damage, while in the case of dark repair it is carried out by a complex combination of more than a dozen enzymes. To begin reactivation (both light and dark), these enzymes must first be activated by an energy source – in photoreactivation this energy is supplied by visible light (300-500nm), and in dark repair it is provided by nutrients within the cell. In both cases, reactivation is achieved by the enzymes repairing the damaged DNA, allowing replication to take place again.
Common strains of E. coli contain about 20 photolyase enzymes, each of which can repair up to five thymine dimers per minute – this means that, in a single cell, up to 100 such dimers can be repaired per minute. 1mJ/cm2 of UV produces approximately 3000-4000 dimers (Oguma, 2002) so, theoretically, damage induced by 1mJ/cm2 of UV can be repaired in just 30 minutes.
Repair after exposure to low and medium pressure UV lamps
Low pressure UV lamps have traditionally been used in water treatment plants because their UV output at 254nm closely matches the absorption peak of DNA bases at 265nm. A number of studies, however, have shown that microbial DNA is capable of photoreactivation after exposure to low pressure UV (Hoyer, 1998; Sommer et al, 2000).
Because of these findings, and because of the increased use of medium pressure UV lamps in water and effluent treatment, recent research has begun to look at whether medium pressure UV can permanently inactivate the DNA of microorganisms. It has been suggested that the broader wavelengths emitted by medium pressure lamps not only damage DNA but also cause damage to other molecules, making it more difficult for cells to repair their DNA. UV-A is known to affect membranes and membrane functions, while UV-B and UV-C have been shown to be absorbed by proteins (Jagger, 1985). All these wavelengths are produced in abundance by medium pressure UV lamps.
Zimmer et al (2002) and Oguma et al (2001) compared the effects of low pressure and medium pressure UV on the ability of microorganisms to repair their DNA. In their tests they compared the ability of E. coli to recover in photoreactivating light after being exposed to different amounts of low and medium pressure UV. E.coli was used in the study as it is a useful ‘biological indicator’ of disinfection efficiency in water systems. The results of both studies showed a significant difference in photoreactivation following low and medium pressure radiation. While high levels of photorepair were observed after low pressure irradiation, with maximum repair occurring after 2 – 3 hours, there was virtually no photorepair after medium pressure treatment. This was particularly the case at higher log reductions (log 3 and above). (See Table 1).
Zimmer et al proposed a number of reasons why medium pressure UV causes irreparable damage, while low pressure UV does not. One hypothesis is that there is a synergistic effect between the various wavelengths emitted by medium pressure lamps that causes irreparable damage to the DNA. Another possible explanation is that the repair enzymes themselves are damaged. Harm (1980), for example, has shown that absorption by proteins in the UV-B and UV-C regions is equal to absorption by DNA at 265nm. According to Zimmer et al, while absorption of UV by proteins is considered of little importance to cells, any damage to repair enzymes would be critical due to that fact that there are so few of them present in the cell.
All these studies concluded that polychromatic medium pressure UV radiation was more effective than monochromatic low pressure UV at causing permanent, irreparable damage to the DNA of E. coli.
Implications of the findings
The implications of these findings are far-reaching. For any industry where UV is used to disinfect water or effluent, the operator needs to be sure that the treatment is permanent. This is especially the case when the treated liquid will subsequently be exposed to light. Zimmer at al suggest that medium pressure UV could therefore provide better protection against photoreactivation if UV treatment occurs prior to any process units in which water is exposed to light for even a short time (30 – 180 minutes). According to their study, “Using low pressure UV in this type of situation should be avoided, since repair occurs rapidly following exposure to light.”
The applications affected by these findings include any where the treated water or effluent is subsequently exposed to light. Examples include wastewater, bottled water, fisheries and swimming pools. Also important, due to the possibility of dark repair, are drinking water and process water applications.
Zimmer at al recommended that further research be carried out with medium pressure to determine precisely which wavelengths cause the additional damage and where the damage occurs. They also recommend further research involving real water treatment plants.
Many microorganisms can repair UV-damaged DNA with enzymes in both light and dark conditions. Research comparing photoreactivation of E. coli DNA following exposure to low and medium pressure UV has shown that the DNA underwent extensive photorepair after exposure to low pressure UV, but virtually none following exposure to medium pressure UV.
It is still not clear exactly what wavelengths, or combination of wavelengths, causes this permanent deactivation of the DNA. However, as Zimmer et al suggest, it is the very fact that medium pressure lamps produce such a broad output right across the spectrum of UV light that produces this desirable effect.
The initial results of these studies suggest that medium pressure UV offers better protection against photoreactivation than low pressure UV. If there is any chance that UV-treated treated water or effluent will be exposed to light – even for as little as half an hour – it may be advisable for operators to use medium pressure rather than low pressure UV lamp systems.
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Zimmer, J. L. & Slawson, R. M. (2002). Potential repair of Escherichia coli DNA following exposure to UV radiation from both medium- and low-pressure UV sources used in drinking water treatment. Applied & Environmental Microbiology, Vol. 68, No. 7, 3293-3299.