Fungal photoinactivation doses for UV radiation and visible light–a data collection

Nearly two million people die each year from fungal infections. Additionally, fungal crop infections jeopardize the global food supply. The use of 254 nm UVC radiation from mercury vapor lamps is a disinfection technique known to be effective against all microorganisms, and there are surveys of published UVC sensitivities. However, these mainly focus on bacteria and viruses. Therefore, a corresponding overview for fungi will be provided here, including far-UVC, UVB, UVA, and visible light, in addition to the conventional 254 nm UVC inactivation. The available literature was searched for photoinactivation data for fungi in the above-mentioned spectral ranges. To standardize the presentation, the mean log-reduction doses were retrieved and sorted by fungal species, spectral range, wavelength, and medium, among others. Additionally, the median log-reduction dose was determined for fungi in transparent liquid media. Approximately 400 evaluable individual data sets from publications over the last 100 years were compiled. Most studies were performed with 254 nm radiation from mercury vapor lamps on Aspergillus niger, Candida albicans, and Saccharomyces cerevisiae. However, the data found were highly scattered, which could be due to the experimental conditions. Even though the number of individual data sets seems large, many important fungi have not been extensively studied so far. For example, UV irradiation data does not yet exist for half of the fungal species classified as “high priority” or “medium priority” by the World Health Organization (WHO). In addition, researchers should measure the transmission of their fungal suspensions at the irradiation wavelength to avoid the undesirable effects of either absorption or scattering on irradiation results.


Introduction
The importance of bacteria and viruses for human health with hundreds of thousands of infections or even fatalities per year worldwide is undisputed [1][2][3][4].Besides the treatment of infected patients, the disinfection of air, surfaces, or liquid media by chemical or physical measures plays a major role.Among the physical means is the application of UVC radiation at a wavelength of 254 nm, which acts very efficiently by destroying either the DNA or RNA of various pathogens [5][6][7].
However, the required irradiation doses are not the same for all microorganisms.There can be large differences.For example, vegetative bacterial cells such as Bacillus subtilis are much more sensitive than their spores [8].Tabular overviews exist, which list the common irradiation doses required to reduce known pathogens [8,9].However, these tables focus on bacteria and viruses.
The radiation doses necessary for fungi are found only on a much smaller scale, although fungi pose a similar threat to human health as bacteria and viruses.Each year, approximately 150 million fungal infections occur, of which nearly two million are fatal [10][11][12][13].Additionally, the World Health Organization (WHO) has recognized the problem of fungal infections, has called for research and action by researchers, and has even published a list of the most significant fungal pathogens [14] similar to the bacterial ESKAPE pathogens [15,16].Cryptococcus neoformans, Candida auris, Aspergillus fumigatus, and Candida albicans have been identified as particularly important and constitute the "the critical priority group".Seven additional fungi were named in the next most important "high priority group", including three more Candida species.
In addition to the direct impact of fungi on human health, fungi can also cause other very undesirable effects.It is estimated that fungi are largely responsible for food spoilage [17,18] and pose a threat to humans in this regard, as well as the annual amount of spoiled food which would have been sufficient to feed 600 million people.
In principle, UVC radiation can be employed as a universal disinfection measure against all fungi via the DNA-destroying mechanism.In the study presented here, the results of already published UVC inactivation studies are compiled and standardized in their presentation.The existing overviews by Kowalski and Malayeri et al. [8,9] mainly presented data that was obtained with 254 nm UVC radiation from low-pressure mercury vapor lamps.However, other UV spectral ranges also exhibit antimicrobial properties, and the same is true for visible violet or blue light, if the applied dose is high enough [19,20].Therefore, in this study, the relevant irradiation range is extended from Far-UVC-starting at 200 nm to visible blue light of wavelengths up to 480 nm.

Materials and methods
On Pubmed and Google scholar, different combinations of the following terms were searched for: fungi, mold, yeast, inactivation, photoinactivation, reduction, disinfection, antifungal, ultraviolet, UV, UVA, UVB, UVC, Far-UVC, UV-A, UV-B, UV-C, blue light, and violet light.When matching articles were found, the given references were searched for possible further studies.In addition, research was performed to find out which later publications the retrieved paper had cited.
In particular, the mean log-reduction doses were determined for irradiation with either UV or visible light in the spectral range 200-480 nm.If not explicitly stated by the authors themselves, the data were determined from given values or graphs of the respective study as far as possible by determining the mean log-reduction dose from 3 log-reductions.Publications in which either the wavelength or the dose information was either missing or could not be determined were not included.This also applied to experiments within liquid media such as cell culture media or fruit juices, which have very high absorptions, especially in the UV range [21][22][23][24][25], and thus prevent the determination of the irradiation dose or only partially irradiate contaminated samples.
Additionally, studies with shorter wavelengths (below 200 nm), longer wavelengths (above 480 nm), very broadband irradiation (>50 nm), or the combination of radiation with other potentially antimicrobial measures including photosensitizers, heat, or extreme pH values were not included.Here, only experiments in the range between 10 and 40 °C and between pH 5 and 8 were included.
When studies investigated different repair mechanisms after irradiation, the data of cultivation in the dark were selected.Studies on particularly radiation-sensitive or -insensitive fungal mutations were not considered.
Then, a categorization was carried out between fungi in liquids, in the air, and on surfaces.Moreover, a distinction was made between vegetative cells, spores, and hyphae.The results were also sorted by spectral range: Far-UVC (200-230 nm), (residual) UVC (230-280 nm), UVB (280-315 nm), UVA (315-400 nm), violet (400-430 nm), and blue (430-480 nm).For each fungus and spectral range, the medians of the log-reduction dose for the liquid samples were determined.

Results
The literature survey revealed that the study of the disinfecting effect of UV radiation on fungi already started about 100 years ago [26,27]; for example, it was already recognized at that time that dark/pigmented fungi were relatively resistant to radiation [28] and that experiments that were performed in absorbing cell culture media falsified the measurements [29].
In total, over 100 reports on fungi irradiation were found that met the above criteria.The given or determined individual log-reduction doses can be found in Tables 1 and 2 alongside the obtained medians [for transparent liquids] for different fungi in different spectral ranges.Many investigations were performed on human pathogens, though there were also many plant pathogens and environmental species.The most results were found for Saccharomyces cerevisiae, Candida albicans, and Aspergillus niger.
Over 70% of the individual data sets originated from the UVC spectral range 230-280 nm, which is not surprising since mercury vapor lamps, with their 254 nm emission, are efficient, cheap, easy to use, and have been available for more than 100 years [30].
Table 1 provides the log-reduction doses in the spectral ranges Far-UVC (200-230 nm), (residual) UVC (230-280 nm), and UVB (280-315 nm) in mJ/cm 2 .The antifungal impact of UVA, visible violet, and blue light in Table 2 is several orders of magnitude lower; therefore, the log-reduction doses are given in J/cm 2 .Continued on next page median log-reduction dose of the non-pathogenic S. cerevisiae.
With the help of fungi for which the log-reduction dose medians are available for different spectral ranges, a rough comparison of the antifungal effect of radiation from different spectral ranges can be provided.The determined median far-UVC log-reduction doses are mostly slightly lower than the corresponding log-reduction dose observed with conventional UVC irradiation for the same fungus; however, this statement is based on a rather low number of far-UVC results.No major difference in photosensitivity or log-reduction doses can be observed between both ranges.
In contrast, a comparison between UVC and the visible spectral range displays large differences.The violet log-reduction doses are 3 to 4 orders of magnitude higher than those in the UVC range.On the other hand, the differences between violet and UVA are, in most cases, less than a factor of 2, with Cladosporium cladosporiodes (spores) as the only determined exception.

Discussion
Although Tables 1 and 2 may seem rather lengthy, it can be noted that not much has been studied thus far.For example, UVC data are even missing for half of the fungi named in the WHO "high priority group" and the "medium priority group" [14]-even though inexpensive UVC sources (mercury vapor lamps) have been available for more than one hundred years.
In the other spectral ranges, even less fungal inactivation data have been published, although these ranges are also very interesting and allow for disinfection applications without posing a major hazard to humans.This is true for UVA and visible light [151]; however, the radiation has a strong antimicrobial effect, especially for the far-UVC range, and has been considered to be relatively harmless to humans thus far [152,153].Therefore, far-UVC has a great potential to contain the spread of fungi in the future.The individual values in Tables 1 and 2 displayed a large scatter of the log-reduction doses, even within one species and one wavelength range.For A. niger, C. albicans, and S. cerevisiae, there were 1-2 orders of magnitude between each the smallest and the largest UVC log-reduction dose in the liquid samples.
One reason for this is the biological variations or differences between the individual strains and possibly different physiological states.Another reason is probably the differing experimental set-ups and experimental conditions.One important aspect is the culturing condition after antimicrobial irradiation because illumination can lead to photoreactivation [52,60,[154][155][156], which results in higher log-reduction doses compared to dark cultivation.As mentioned above, if results of the different illuminations after the antimicrobial irradiation were published, the dark cultivation results were selected.However, in most cases, no statements on the illumination conditions were provided.
Besides this, even for standard irradiation with low-pressure mercury vapor lamps, which all mainly emit at 254 nm, different temperatures, irradiances, and durations have been mentioned.The latter does not lead to major effects due to the Roscoe-Bunsen law; however, there is another very critical point, which, by itself, can lead to variations in the determined log-reduction doses by a factor of 10.As already observed by Coblentz in 1924 [29], and as already mentioned above, absorption [and scattering] in the irradiated medium can lead to lower disinfection success.This would manifest itself, for example, in larger log-reduction doses and a stronger non-mono-exponential behavior.Some authors seem to be aware of the problem [39,43,45,53,73,100,109,150,157], though most published studies did not comment on transmission at the irradiation wavelength.This does not only concern the pure medium, but also fungal suspensions.A double-digit number of authors provided cell or spore concentrations of  10 7 CFU/mL.In our own (unpublished) measurements on 10 7 S. cerevisiae per mL, we observed an optical density at 600 nm of OD600 = 0.3.For 254 nm, the optical density under these conditions was OD254 = 1.7.For a path length of 10 mm, this resulted in an irradiance decrease by almost 2 orders of magnitude to about 2% of the initial value.Many authors applied thinner layers of fungal suspensions; however, even behind a 2 mm thin layer, the irradiance would have dropped by about 50%.

Conclusions
Up to now, the topic of radiation disinfection of fungi did not seem to be of great importance.Even the photoinactivation properties of many health-endangering fungi have been insufficiently studied thus far.Hopefully, this may now somewhat change with the WHO report on the most dangerous fungi [14].These should be preferentially examined in detail, and for all fungi-or even all pathogens-the far-UVC range seems particularly promising.
Regarding the implementation of the required irradiation experiments, we would recommend always measuring at least the transmission of the fungal suspension to be irradiated at the respective wavelength and, if possible, to achieve a high transmission of more than 50% better 90%.

Figure 1 .
Figure 1.Box-Plots of published fungal UVC log-reduction doses for the WHO "critical priotity group" together with the number of reported single log-reduction doses in brackets.For comparison, the corresponding data for S. cerevisiae and A. niger (spores) are added.(Two outliers for A. niger (spores) are above 250 mJ/cm 2 and not displayed here.)

Table 1 .
Log-reduction doses in mJ/cm 2 for Far-UVC, UVC and UVB for different fungi and various sample media.Besides the exact wavelength, additional information on strain, medium, temperature, and pH is given, if available.

Table 2 .
Log-reduction doses in J/cm 2 for UVA and visible violet and blue light for different fungi and various sample media.Besides the exact wavelength, additional information on strain, medium, temperature, and pH is given, if available.