3.1 Chlorine-based disinfectant disinfection
3.1.1 (Free) chlorination
Chlorine began to be used for disinfection of drinking water in the early 20th century (Metzger et al, 1976). Due to its good disinfection effect, convenient use, low price and ability to maintain a certain amount of residual chlorine, chlorine is still the most widely used disinfection method in the world. When chlorine is used to disinfect drinking water, the free chlorine concentration and reaction time should be controlled reasonably so as to meet the requirements of disinfection and reduce the generation of by-products. The effectiveness of disinfectants is usually expressed by Ct (concentration×reaction time) values required to inactivate a certain proportion of bacteria. The Ct value required to reach 0.7 log removal was 60 min·mg/L with the addition of 3 mg/L of chlorine to the actual aqueous substrate (pH7, 21℃) represented by Cladosporium, Penicillium, and Trichoderma (Pereira et al., 2013). The experimental results under another condition (pH 8, 25℃) were as follows: when the Ct value was 40 min·mg/L, the fungi inactivation rate was 1.2-log, when the Ct value increased to 60 min·mg/L, the fungi inactivation rate reached to 1.7-log (Huang et al, 2016). In practical application, in addition to controlling chlorine concentration and reaction time, water temperature and pH should also be considered.
Chlorine has a good inactivation effect on fungi. Nevertheless, different fungi have different resistance to chlorine disinfection (Ma and Bibby, 2017, Wen et al, 2016, Liu, 2015, Pereira et al., 2013). The nature of the bacteria itself affects the inactivation rate. The larger the size of the fungal spores, the stronger the hydrophilicity, and the better the inactivation effect (Wen et al., 2016).
Water quality indexes have a significant impact on the efficacy of chlorine inactivation. The inactivation rate of chlorine disinfection increases with the increase of chlorine concentration, with the extension of reaction time (Liu, 2015, Huang et al., 2016), and with the increase of temperature. Additionally, the inactivation rate under acidic conditions is higher than that under alkaline conditions (Liu, 2015, Zhao et al., 2017). There was a "lag" effect in the initial inactivation, probably because chlorine reacted with reducing substances in water first, which slowed down the inactivation rate of fungi and affected the inactivation effect (Ma and Bibby, 2017, Zhao, 2016, Huang et al., 2016).
By collecting the SEM images of fungal spores after inactivation by chlorine, it was found that the surface of fungal spores was depressed, wrinkled and damaged to varying degrees. Nitrogenous organic matter leaked from cells. Extracellular ATP, DNA and protein concentrations increased significantly but no organelle leakage was found (Wen et al., 2016, Zhao, 2016). It is speculated that chlorine first acts on the cell wall and membrane, increasing the permeability of cells, further leading to the leakage of intracellular organic matter, so as to inactivate cells and die.
Fungi show stronger resistance to chlorine than bacteria (Ma and Bibby, 2017, Liu, 2015, Pereira et al., 2013). The Ct99% required to inactivate Penicillium sp., Trichoderma sp. and Cladosporium sp. is 71~1404min·mg·L-1, and only 0.6min·mg·L-1 is required to inactivate E. coli. This is arisen from the different cell composition. Fungi have intact nuclei, which makes it more difficult for chlorine to destroy genetic material. In addition, the main component of the cell wall of fungi is chitin, while the main component of the cell wall of bacteria is peptidoglycan. Chitin has a stronger protective effect on cells than peptidoglycan. Moreover, fungal cells contain more organelles than bacteria, and fungal cells are larger than bacteria, requiring more chlorine consumption (Wen et al., 2016).
3.1.2 Chlorine dioxide disinfection
Chlorine dioxide is a more powerful disinfectant than chlorine. The inactivation of fungal spores with ClO2 at 2 mg/L reached to a 2.48-log removal for Penicillium spp., a 1.5-log removal for Trichoderma sp. and a 0.5-log removal for Cladosporium sp. in 1min. Meanwhile, the inactivation of E. coli by ClO2 under the same conditions has achieved 2.42-log removal at 30s inactivation time (Xu, 2018). These results indicate that bacteria are much less resistant to ClO2 than fungal spores.
Within a certain range, the increase of ClO2 concentration and the increase of temperature will enhance the inactivation effect of ClO2 on fungi (Zhao, et al., 2017, Wen, et al., 2017a). However humic acids and substrates in water can adversely affect the inactivation of fungal spores. Different microorganisms have different sensitivity to ClO2 inactivation, that is, different resistance, which may be caused by the different size of microbial cells (Zhao et al., 2017, Wen et al., 2017a).
Three fungi were inactivated with 2mg/L ClO2 solution. The content of extracellular DNA, protein and ATP in the fungal spore suspension after inactivation increased to varying degrees compared with that before inactivation. Additionally, SEM images of the spores after inactivation showed that the spore cell surface was depressed and folded to varying degrees (Wen et al., 2017a). This shows that ClO2 destroys the cell wall and cell membrane of the fungal spores and enters the cell to destroy the nucleus, thus causing cell death.
The inactivation efficiency of ClO2 is better than that of chlorine. The CT value of ClO2 required for fungi to reach 2.0-log removal is 1.845 min•mg/L (pH 7, 27℃) (Xu, 2018), while the CT value required for fungi to reach 0.7-log removal rate by chlorine is 60min•mg/L (pH 7, 21℃) (Huang et al., 2016). ClO2 is more effective than chlorine inactivation probably because: ClO2 (1.51V) has a higher redox potential than chlorine (1.36V), and reacts faster with proteins or nucleic acids. Otherwise, ClO2 is a neutral molecule and easy to diffuse. Although hypochlorous acid, the main substance in chlorine disinfection, is also a neutral molecule, hypochlorous acid is easily decomposed into hypochlorite (OCl-) depend on pH, which inactivation ability is weakened (Xu, 2018, Wen et al., 2017a). However, the cost of ClO2 is at least 3 times higher than Cl2 (Peeters et al., 1989).
3.2 Ozone disinfection
In 1893, ozone was first used in disinfection and sterilization of drinking water plants (Liu, 2015). As ozone molecules are neutral molecules, they can easily penetrate the cell membrane and enter the interior of cells. At the same time, ozone have strong oxidation ability, which makes ozone inactivate fungi at a fast rate (Straub et al., 1995). When ozone with an initial concentration of 1mg/L was applied to groundwater samples for 1 minute (pH 8, 25℃), the log removal of fungi reached to 0.3-log (Huang, 2016). Further, the log removal reached about 0.5-log after exposure for 15 minutes (pH 8.2, 20℃) (Liu, 2015). When CT=9min•mg/L, the log removal of fungi was 1-log in underground water. When CT=24min•mg/L, the fungus log removal was 1.2-log (Huang, 2016). When 2.0-log inactivation was achieved, the Ct value of A. niger was 5.65 min·mg/L, Bacillus subtilis was 2.5 min·mg/L (Choi et al., 2007), and the virus only needed 1.9~8.0×10−3 min·mg/L (Wolf et al., 2018). Fungi are more resistant to ozone inactivation than bacteria and viruses due to their more complex cellular structure. Different from fungi, the genetic material of bacteria exists in cytoplasm rather than nucleus, which makes bacteria more likely to be inactivated. Viruses without cell structure are always inactivated faster (Wen et al., 2020).
In a certain range, the fungal inactivation rate increased with the increase of ozone contact time. In high concentration (1.5~2.5 mg/L) ozone solution, the fungal inactivation was close to 1-log in 10min. The higher the concentration, the higher the inactivation rate. However, the inactivation rate was 0.35-log for 3 min of exposure and 0.7-log for 30 min of exposure at low concentrations (1 mg/L). For low concentrations of ozone, extending the time can increase the inactivation rate to some extent. When the inactivation tends to equilibrate, the inactivation rate will not change again even if the time is increased (Liu, 2015). The increase in temperature promotes the inactivation of the fungus because the increase in temperature accelerates ozone decomposition to produce hydroxyl radicals (Wen et al., 2020). The log reduction of A. niger, T. harzianum and P. polonicum at 4 mg/L ozone concentration was 2.48, 2.27 and 1.76 times higher than that at 1 mg/L ozone concentration at an exposure time of 10 min. An increase in temperature from 5℃ to 33℃ increased the k values of A. niger, T. harzianum and P. polonicum by 55.8%, 127.2% and 136%, respectively. T. harzianum and P. polonicum were much more sensitive to temperature than A. niger.
3. 3 Ultraviolet disinfection
It is generally believed that ultraviolet light with wavelength between 240nm~280nm penetrates cell wall and cell membrane and is absorbed by nucleic acid (Liu, 2004, Liu, 2015, Zhu, 2017), generating photochemical products cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine dimers, blocking nucleic acid replication and transcription, leading to cell death (Wen et al., 2017b, Sinha and Häder, 2003).
When the UV flunce was 70 mJ/cm2, the inactivation of fungi in underground water could reach 1-log. When the UV flunce was 100mJ/cm2, the inactivation was 1.6-log (Zhao et al.,2016). When the fungal concentration was 1000 cfu/ml, the UV flunce was 12.45mJ/cm2, 16.6mJ/cm2 and 20.75mJ/cm2 for Aspergillus fumigatus, Aspergillus niger and Aspergillus flavus to achieve the inactivation of 4.0-log, respectively. Obviously, the inactivation efficiency of A. fumigatus > A. niger > A. fumigatus (Nourmoradi et al., 2012). Comparing the UV doses required to achieve 100% inactivation rates of several fungi, Trichoderma only needs 50mJ/cm2, Cladosporium needs 60mJ/cm2, and Penicillium needs 80mJ/cm2. As for Cladosporium, even when the UV dose reaches 100mJ/cm2, the log removal is only 1.1-log (Zhu, 2017). This indicates that different fungi have different resistance to UV inactivation. more UV is absorbed by the cytoplasm before reaching the accounting position for the larger size fungal spore, which reduces the inactivation efficiency (Nascimento et al., 2010).
After inactivation, DNA, protein and ATP in the suspension of fungal spores did not increase compared with those before inactivation (Zhu, 2017, Wen et al., 2017b). The morphology of fungal spores has different degrees of shrinkage and depression, but the cells have not broken, no content flow out. It indicates that in addition to the direct destruction of nucleic acid, UV can also denaturate proteins on the cell membrane, resulting in changes in cell permeability and the imbalance of osmotic pressure inside and outside the cell body, resulting in folds (Zhu, 2017, Oliveira et al.,2020).
However, some studies (Hijnen et al., 2006, Pescheck et al., 2019, Wang et al., 2019a) have shown that microorganisms in water may revive after UV disinfection, which will greatly reduce the efficiency of UV disinfection (Guo et al., 2012, Li et al., 2017). Photoactivation is one of the ways in which microorganisms reactivate by specifically binding CPDs to photoenzymes and using light energy (310~480nm) to reverse damage (Lehmann, 1979, Sinha and Häder, 2002). Studies have shown that different fungal spores exhibit different degrees of photoactivation under the same conditions (Wen et al., 2019a, Sousa et al., 2017), which may be due to differences in cell structure and repair genes. The more thoroughly UV flunce is used, the less favorable it is to photoactivation (Wen et al., 2019a). The photoreactivation percentages of spores of T. harzianum, A. niger, and P. polonicum increased from 33.26%, 22.1%, and 1.35% in PBS solution to 52.8%, 34.07%, and 3.86% in actual groundwater. In addition, the photoreactivation percentages of spores of T. harzianum, A. niger, and P. polonicum increased from 18.52%, 4.87%, and 0.98% at 5℃ to 91.4%, 75.28%, and 2.47% at 35℃. Moreover, after 24 h of dark delay, the photoreactivation percentages of spores of T. harzianum and A. niger decreased from 50.17% and 15.76% to 38.09% and 13.11%. Therefore, in real water substrate, fungal spores exhibited higher levels of photoactivation. Low temperature and dark delay could effectively inhibit the fungal light response (Wen et al., 2019b). Another method of microbial resurrection is dark repair, in which damaged DNA is replaced by new, undamaged nucleotides unaffected by light (Sinha and Häder, 2002). Dark repair is not obvious at different temperatures for fungal spores (Wen et al., 2019b).
3. 4 Comparison of inactivation effects
Comparing the above four methods of fungal inactivation, the results are shown in Table 3.