By Dr. Sara E. Beck and Dr. Paul Onkundi Nyangaresi, University of British Columbia; Dr. Sandra Probst-Rüd, City of Winterthur (Switzerland)
As UV LEDs continue to be incorporated into water systems at household and municipal levels, researchers, manufacturers and utility operators increasingly are considering taking advantage of the synergistic effect of UV-A and UV-C irradiation in the photoinactivation of some bacteria by adding LEDs that emit in the UV-A range. Such a change could not only reduce costs but also lead to improved disinfection. There has been a justifiable need to understand the basis of this synergistic effect when combining the two wavelength ranges with different inactivation mechanisms. The authors’ recent paper in Water Research, from the doctoral work of Dr. Sandra Probst-Rüd under Dr. Martin Ackermann and Dr. Kris McNeill at ETH-Zurich, with the support of Dr. Sara Beck, Dr. Paul Nyangaresi and Adefolawe Adeyeye at the University of British Columbia, answered this question. 1
Using advanced molecular biology methods, including time-lapse microscopy, microfluidics and an engineered strain of E. coli, the authors found a basis why UV-A (315-400 nm) damage is more effective when followed shortly afterward by UV-C (200-280 nm).
With time-lapse microscopy and microfluidics, the authors showed that sub-lethal UV-A caused a transient and reversible growth arrest in E. coli. In other words, the E. coli cells lined up in a microfluidics channel, feeding on a buffet of nutrient broth, and it was possible to time how long it took for the cells to divide. Interestingly, when the cells were exposed to UV-A light, it slowed their division time, as though the bacteria had been stunned or tased, but then they resumed their normal division a few hours later. Once restarted, the cell division time resembled that of unirradiated cells (see Figure 1).
The UV doses the bacteria were exposed to (240-1,200 mJ/cm2) are considered sublethal in that they can cause a growth delay but have a negligible effect on cell viability alone. However, when the sublethal UV-A doses were combined with UV-C (at 268 nm, doses of 10-20 mJ/cm2), it enhanced the UV-C inactivation by several orders of magnitude. The enhanced inactivation, which the authors have demonstrated in bacteria but not viruses, indicated a specific cellular response mechanism to UV-A irradiation. 2
This response was traced to UV-induced damage of the transfer RNA (or tRNA), a component of the cell that provides a key link in the translation of DNA into proteins. When this critical link is damaged by UV-A light, proteins that are vital for cellular processes, such as DNA repair, cannot be synthesized. Exposing the tRNA to UV-A light changes their structure and prevents the amino acids from being loaded, halting the whole assembly process and stopping the production of vital proteins. Importantly, while tRNA normally is transparent to UV-A, E. coli and other bacteria have thiouridine-containing tRNA that absorbs UV-A, making them sensitive to this wavelength range.
The UV-A damage was traced to the tRNA by working with a mutant strain of E. coli that had evolved a resistance to combined UV-A and UV-B light. 3 In the Ackermann Lab at Eawag, Dr. Probst-Rüd exposed a wild-type strain of E. coli to a combination of UV-A and UV-B wavelengths for eight hours at a time. The cells then were grown to exponential phase and the process was repeated for eight days in a row. Over time, the surviving exposed cells evolved a resistance to the synergistic effects of UV-A and UV-B light. This resistance was traced to a genomic mutation, which then was identified through whole genome sequencing: Each E. coli strain that evolved the resistance to combined UV-A and UV-B light had a mutation in the ThiI gene, which prevented the tRNA from absorbing UV-A.
When exposing this mutated strain of E. coli to UV-A, it did not show the same growth-delay and did not have the same synergistic effect from UV-A followed by UV-C as in the wild type strain (exposure to UV-B showed no difference, however). In other words, the strain that evolved a resistance to UV was unable to absorb the UV-A light. In contrast, the wild-type E. coli, with tRNA genes that had not mutated, had a specific component (a photosensitive uridine) that induced a halt in protein synthesis upon exposure to the UV-A light. This halt left the cells less able to respond to stress or manage damage induced by subsequent UV-C (or also UV-B) irradiation. 1,3
The synergistic effect was greatest when UV-A was applied immediately before UV-C; whereas, when there was a time lag of 16-24 hours between exposures, the cells had more time to recover from the UV-A-induced damage. Recovery from the UV-A induced state was prolonged with higher UV-A doses. Cells exposed to UV-A immediately followed by UV-C remained damaged 24 hours later.
What’s exciting about this study is that the authors were able to demonstrate that UV-A wavelengths can cause direct photochemical damage to components of bacterial cells. In general, when it comes to UV damage of microorganisms, there is a tendency to see direct damage in the UV-C range and indirect damage in the UV-A range, where damage is induced by free radicals and other high-energy species produced by the action of UV-A on the system. The authors’ paper showed that UV-A wavelengths also can cause direct photochemical damage to components of the cell that are fundamental for cellular processes. E. coli’s + RNAs absorb strongly below 380 nm, making them susceptible to damage induced by UV-A.3 Although the study was conducted with E. coli, it is expected that the same direct photolysis mechanism would be seen in other bacteria, such as Bacillus spores where we have seen similar synergistic effects. 2
This study answers a question that has long since puzzled engineers and scientists. The knowledge acquired will inform and enhance water treatment in both household and municipal UV LED systems.
This research was presented at IUVA Americas in Orlando, Florida, in May 2024 and the American Chemical Society in San Diego, California, in March of 2025. Dr. Sara Beck is an assistant professor in the Department of Civil Engineering at the University of British Columbia (UBC), Vancouver Campus, in Vancouver, British Columbia, Canada. Paul Onkundi Nyangaresi is a research associate with UBC. Sandra Probst-Rüd is the project manager for Environmental and Health Protection for the City of Winterthur, Switzerland. For more information, contact sara.beck@ubc.ca.
References
- Probst-Rüd, S. et al. Synergistic effect of UV-A and UV-C light is traced to UV-induced damage of the transfer RNA. Water Research 252, 121189 (2024).
- Nyangaresi, P. O., Rathnayake, T. & Beck, S. E. Evaluation of disinfection efficacy of single UV-C, and UV-A followed by UV-C LED irradiation on Escherichia coli, spizizenii and MS2 bacteriophage, in water. Science of The Total Environment 859, 160256 (2023).
- Probst-Rüd, S., McNeill, K. & Ackermann, M. Thiouridine residues in tRNAs are responsible for a synergistic effect of UVA and UVB light in photoinactivation of Escherichia coli. Environmental Microbiology 19, 434–442 (2017).
