Formation of filamentous fungal biofilms in water and the transformation of resistance to chlor(am)ine disinfection.

Biofilms are composed of complex multi-species in nature, potentially threatening drinking water safety. In this work, the formation of single- and multi-species fungal biofilms formed by Aspergillus niger (A. niger) and Aspergillus flavus (A. flavus), and the inactivation of mature biofilms using chlor(am)ine were firstly investigated. Results revealed that the antagonistic interaction occurred between A. niger and A. flavus. Chloramination at 20 mg/L for 30 min achieved 74.74 % and 76.04 % inactivation of A. flavus and multi-species biofilm, which were 1.69- and 1.84-fold higher than that of chlorine at the same condition. However, no significant difference was observed in the inactivation of A. niger biofilm between chlorine and monochloramine disinfection due to the lower amount of extracellular polymeric substance produced by it (p > 0.05). The inactivation of biofilm by monochloramine fitted the Weibull model well. According to the Weibull model, the monochloramine resistance of biofilm were as follows: A. flavus > multi-species > A. niger biofilm. Besides, an increase in reactive oxygen levels, damage of cell membrane, and leakage of intracellular substances in biofilms were observed after chlor(am)ination. More intracellular polysaccharides and proteins were leaked in chloramination inactivation (p < 0.05). This study provides important implications for controlling fungal biofilm.

The aggregation characteristics of Aspergillus spores under various conditions and the impact on LPUV inactivation: Comparisons with chlorine-based disinfection.

Aggregation is the primary step prior to fungal biofilm development. Understanding the attributes of aggregation is of great significance to better control the emergence of waterborne fungi. In this study, the aggregation of Aspergills spores (A. flavus and A. fumigatus) under various salt, culture medium, and humic acid (HA) conditions was investigated for the first time, and the inactivation via low-pressure ultraviolet (LPUV) upon aggregated Aspergillus spores was also presented. The aggregation efficiency and size of aggregates increased over time and at low salt (NaCl and CaCl2) concentration (10 mM) while decreasing with the continuous increase of salt concentration (100 and 200 mM). Increasing the concentration of culture medium and HA promoted the aggregation of fungal spores. Spores became hydrated, swelled, and secreted more viscous substances during the growth period, which accelerated the aggregation process. Results also suggested that fungal spores aggregated more easily in actual water, posing a high risk of biohazard in real-life scenarios. Inactivation efficiency by LPUV decreased with higher aggregation degrees due to the protection from the damaged spores on the outer layer and the shielding of pigments in the cell wall. Compared to chlorine-based disinfection, the aggregation resulted in the extension of shoulder length yet neglectable change of inactivation rate constant under LPUV treatment. Further investigation of cell membrane integrity and intracellular reactive oxygen species was conducted to elucidate the difference in mechanisms between various techniques. This study provides insight into the understanding and controlling of the aggregation of fungal spores.

The improved resistance of germinated spores to ultraviolet irradiation: Comparison with chlorine.

Fungi outbreaks in water will include a series of processes, including spore aggregation, germination, biofilm, and finally present in a mixed state in the aquatic environment. More attention is paid to the control of dispersed fungal spores, however, there was little knowledge of the control of germinated spores. This study investigated the inactivation kinetics and mechanism of ultraviolet (UV) treatment for fungal spores with different germination percentages compared with dormant spores. The results indicated that the inactivation rate constants (k) of spores with 5%-45% germination were 0.0278-0.0299 cm2/mJ for Aspergillus niger and 0.0588-0.0647 cm2/mJ for Penicillium polonicum, which were lower than those of dormant spores. It suggested that germinated spores were more tolerant to UV irradiation than dormant spores, and it may be due to the defensive barrier (upregulated pigments) and some reductive substance (upregulated enoyl reductase) by absorbing UV or reacting with reactive oxygen species according to transcriptome analysis. Compared to dormant spores, the k-UV of germinated spores decreased by 18.17%-26.56% for Aspergillus niger, which was less than k-chlorine (62.33%-69.74%). A slighter decrease in k-UV showed UV irradiation can efficiently control fungi contamination, especially when dormant spores and germinated spores coexisted in actual water systems. This study indicates that more attention should be paid to germinated spores.

Kinetics and mechanism of sulfate radical-and hydroxyl radical-induced disinfection of bacteria and fungal spores by transition metal ions-activated peroxymonosulfate.

Peroxymonosulfate(PMS)-based advanced oxidation process have been recognized as efficient disinfection processes. This study comprehensively investigated the role of sulfate radical (SO4•-) and hydroxyl radical (•OH)-driven disinfection of bacteria and fungal spores by the PMS/metals ions (Me(II)) systems and modeled the CT value based on the relationship between survival and ∫[Radical]dt, with the aim to provide an accurate and quantitative kinetic data of inactivation processes. The results indicated that •OH played a more central role than SO4•- in the inactivation process, and bacteria were more vulnerable to radical attack than fungal spores due to the differences in antioxidant mechanisms and external structures. The k value of •OH -induced inactivation of E. coli was approximately 3-fold higher than that of A. niger, and the shoulder length of •OH -induced inactivation of E. coli was closely 52-fold shorter than that of A. niger after treated with the PMS/Co(II) system. The morphological and biochemical changes revealed that PMS/Me(II) treatment caused membrane damage, intracellular ROS accumulation and esterase activity loss in microorganisms. This study significantly improved the understanding of the contribution of radicals in the process of microbial inactivation by PMS/Me(II) and would provide important implications for the further development of technologies to cope with the highly resistant fungal spores in drinking water.

Occurrence and control of fungi in water: New challenges in biological risk and safety assurance.

Recently, the contamination of fungi in water has aroused widespread concern, which will pose a threat to water quality and safety, and raise diseases risk in the immunocompromised individuals. In this review, the characteristics and different physiological state of fungi in water are summarized. A comprehensive evaluation of the control efficiency and mechanism of waterborne fungi by the commonly used disinfection methods is provided as well. During the disinfection processes of chlorine, chlorine dioxide, chloramine and advanced disinfection processes (ADPs) such as O3-based ADPs and UV-based ADPs, the fungal spores firstly lost their culturability, followed by membrane integrity, and the intracellular reactive oxygen species level increased at the same time, eventually the fungal spores were completely inactivated. The security strategies of drinking water against the contamination of fungi are also discussed in terms of water sources, water treatment plants and pipe network. Finally, future researches need to be explored are proposed: the rapid detection methods, the production laws and control of mycotoxin, and the outbreak conditions of fungi in water. Specifically, exploring efficient, safe and economical technologies, especially ADPs, is still the main direction in the disinfection of fungi in future studies. This review can offer a comprehensive understanding on the occurrence and control of fungi in water to fill the knowledge gap and provide guidance for the future research.

The protective role and mechanism of melanin for Aspergillus niger and Aspergillus flavus against chlorine-based disinfectants.

Melanin is a critical component of fungal cell wall which protect fungi from adverse environmental tress. However, the role of melanin for fungi during the disinfection with chlorine-based disinfectants has not been elucidated. The results showed that the inactivation rate constants of Aspergillus niger with chlorine and chlorine dioxide decreased from 0.08 to 2.10 min-1 to 0 after addition of 0.32 mg/L melanin. The results indicated addition of extracted fungal melanin inhibited the inactivation efficiency of chlorine and chlorine dioxide. In contrast, the k of Aspergillus niger after inactivation with monochloramine ranged from 1.50 to 1.78 min-1 after addition of melanin which indicated effect of melanin on the inactivation efficiency of monochloramine was negligible. In addition, the extracted fungal melanin exhibited high reactivity with chlorine and chlorine dioxide but very low reactivity with monochloramine. The different inactivation mechanisms of chlorine-based disinfectants and different reactivity of melanin with chlorine-based disinfectants led to the different protective mechanism of melanin for A. niger and A. flavus spores against disinfection with chlorine-based disinfectants. The chlorine and chlorine dioxide appeared to react with functional groups of melanin in cell wall of spores, so sacrificial reactions between melanin and disinfectants decreased the available disinfectants and limited the diffusion of disinfectants to the reactive site on cell membrane, which led to the decrease of the disinfection efficiency for chlorine and chlorine dioxide. The monochloramine could penetrate into cell and damage DNA without the effect of melanin due to its strong penetration and low reactivity with melanin. Our results systematically demonstrate the protective roles of melanin on the fungal spores against chlorine-based disinfectants and the underlying mechanisms in resisting the environmental stress caused by chlorine-based disinfectants, which provides important implications for the control of fungi, especially for fungi producing melanin.

Enhanced solar inactivation of fungal spores by addition of low-dose chlorine: Efficiency and mechanism.

This work demonstrated that the solar inactivation of fungal spores was enhanced by addition of low-dose chlorine. Although the effect of low-dose chlorine alone (2.0 mg/L) on culturability of fungal spores was negligible, the solar/chlorine inactivation on fungal spores performed better than solar alone inactivation, with a lower shoulder length and a higher maximum inactivation rate constant. The enhanced inactivation of Aspergillus niger can be ascribed to the membrane oxidation by chlorine, and the enhanced inactivation of Penicillium polonicum can be ascribed to the membrane oxidation by chlorine and ·OH (·OH plays a major role). The oxidization by chlorine and ·OH led to an increase in membrane permeability of fungal spores, which enhanced the solar inactivation, resulting in an increase in intracellular ROS and more serious morphological damage. Due to the presence of background substances such as dissolved organic matter and metal ions (Fe2+, Mn2+, etc.), the inactivation efficiency in real water matrices was decreased. The main disinfection by-products (DBPs) produced in the inactivation of fungal spores in chlorine alone and solar/chlorine treatments were dichloroacetic acid, trichloroacetic acid, trichloroacetone and trichloromethane. Generally, DBPs formation in solar/chlorine treatment was lower than those in chlorine alone treatment. Moreover, the regrowth potential of the two genera of fungal spores in R2A medium could be inhibited by adding low-dose chlorine.

Inactivation and subsequent reactivation of Aspergillus species by the combination of UV and monochloramine: Comparisons with UV/chlorine.

Ultraviolet (UV)/monochloramine (NH2Cl) as an advanced oxidation process was firstly applied for Aspergillus spores inactivation. This study aims to: i) clarify the inactivation and photoreactivation characteristics of UV/NH2Cl process, ii) compared with UV/Cl2 in inactivation efficiency, photoreactivation and energy consumption. The results illustrated that UV/NH2Cl showed better inactivation efficiency than that of UV alone and UV/Cl2, and could effectively control the photoreactivation. For instance, the inactivation rates for Aspergillus flavus, Aspergillus niger and Aspergillus fumigatus in the processes of UV/NH2Cl (2.0 mg/L) was 0.034, 0.030 and 0.061 cm2/mJ, respectively, which were higher than that of UV alone (0.027, 0.026 and 0.024 cm2/mJ) and UV/Cl2 (0.023, 0.026 and 0.031 cm2/mJ). However, there was no synergistic effect for Aspergillus flavus and Aspergillus fumigatus. As for Aspergillus niger, the best synergistic effect can reach 1.86-log10. This may be due to their different resistance to disinfectants, which were related to the size, an outer layer of rodlets (hydrophobins) and pigments. After UV/NH2Cl inactivation, the degree of cell membrane damage and intracellular reactive oxygen species were higher than that of UV alone. UV/NH2Cl had the advantages of high inactivation efficiency and inhibition of photoreactivation, which provides a new entry point for the disinfection of waterborne fungi.

Differentiation of DNA or membrane damage of the cells in disinfection by flow cytometry.

Recently, the viabilities changes of fungal spores in the water supply system during different disinfection processes have been revealed. SYBR Green I (SG), a nucleic acid stain, its fluorescence intensity is correlated with the amount of double-stranded DNA. This study established a new method through successive SG-SG-PI staining (PI: Propidium Iodide) with flow cytometry (FCM). It could successfully distinguish DNA damage and membrane damage of fungal spores, clearly elucidating the intrinsic disinfection mechanism during the chemical disinfection. This method was briefly described as follows: firstly, (1) the fungal spores were stained with SG and washed by centrifugation; and then, (2) the washed spores were treated with disinfectants and terminated; after that, (3) the disinfected spores were re-stained with SG and analyzed by FCM; finally, (4) the SG re-stained spores were stained with PI and analyzed by FCM. The percentages of spores with DNA damage and membrane damage were determined by the fluorescence intensity obtained from steps (3) and (4), respectively. The repeatability and applicability of this developed method were confirmed. It was further applied to explore the inactivation mechanism during chlorine-based disinfection, and results demonstrated that chloramine attacked the DNA more seriously than the membrane, while chlorine and chlorine dioxide worked in a reverse way.

Efficacy of UV-LED based advanced disinfection processes in the inactivation of waterborne fungal spores: Kinetics, photoreactivation, mechanism and energy requirements.

The contamination of fungi in water supply systems poses great risks to environment and human health. In this work, UV light-emitting diodes (UV-LEDs)-based advanced disinfection processes (ADPs) including UV-LEDs/hydrogen peroxide (H2O2), UV-LEDs/persulfate (PS) and UV-LEDs/peroxymonosulfate (PMS), were adopted for waterborne fungal spores inactivation. Overall comparisons of the UV-LEDs-based ADPs with respect to the control efficiency of photoreactivation and energy consumption were also evaluated. Results showed that culturability reduction of the fungal spores treated by UV-LEDs was not enhanced with the addition H2O2, PMS, and PS according to the results of heterotrophic plate counts and reaction rate constants; A. niger was expected to have higher UV resistance followed by T. harzianum and P. polonicum. However, UV-LEDs-ADPs inactivation, especially at the wavelengths of 280 and 265/280 nm, could accelerate the permeabilization of fungal spores as characterized by flow cytometry. Take P. polonicum for example, the percentage of membrane permeabilized spores was 98.0%, 98.7%, 97.6% and 82.6% after treatment by UV280/H2O2, UV280/PS, UV280/PMS and UV280 alone, respectively at the fluence of 100 mJ/cm2. The direct attack of free radicals in the processes of UV-LEDs-ADPs further enhanced the membrane damage and lowered the photoreactivation level, thus improved the inactivation efficiency. UV-LEDs/H2O2 was considered as an effective process in the disinfection of fungal spores with the advantages of enhancing the damage of membrane, inhibiting photoreactivation and comparable energy consumption compared with UV-LEDs alone.

Sequential use of UV-LEDs irradiation and chlorine to disinfect waterborne fungal spores: Efficiency, mechanism and photoreactivation.

In this work, sequential applications of light-emitting diodes (UV-LEDs) with two wavelengths and chlorine (Cl2) were performed for fungal spores disinfection: UV-Cl2, Cl2-UV, UV/Cl2-UV, UV-UV/Cl2, Cl2-UV/Cl2-Cl2. Overall comparisons of the sequential processes with respect to the inhibitory effect on photoreactivation were also evaluated. According to the evaluation of culturability and membrane permeability, inactivation of fungal spores by UV was not enhanced by prior or post exposure to Cl2, but in the UV/Cl2 process with pre or post UV treatment, the inactivation efficiency was greatly enhanced. Take P. polonicum for example, pre-treatments by UV265 and UV280 (40 mJ/cm2) caused the log count reduction (LCR) of 1.05 log and 0.95 log, then the followed UV265/Cl2 and UV280/Cl2 at the same UV fluence caused additional LCR of 1.80 log and 2.00 log. The permeabilization of P. polonicum was also accelerated in the processes of UV/Cl2-UV and UV-UV/Cl2, especially at the wavelength of 280 nm. In the sequential processes, especially those containing UV/Cl2 or at the wavelength of 280 nm, could promote the formation of intracellular reactive oxygen species (ROS), thus leading to more severe damage to the spores as reflected in the culturability reduction, membrane permeability and inhibition of photoreactivation.

Inactivation of waterborne fungal spores by 1-bromo-3-chloro-5,5-dimethylhydantoin: Kinetics, influencing factors and mechanisms.

Fungal contamination in drinking water source has become a problem worth studying, as waterborne fungi may cause deterioration of water quality and outbreak of diseases. Various disinfection methods have been explored to control fungal spores in drinking water, such as chlor(am)ination, ozonation, chlorine dioxide treatment, but these methods are not appropriate for remote areas, owing to the difficulties in preparation, carriage and storage. In this study, a powdery disinfectant of 1-bromo-3-chloro-5,5-dimethylhydantoin (BCDMH), which facilitated transportation and preservation, was firstly chosen to inactivate opportunistic pathogens of Aspergillus niger (A. niger) and Penicillium polonicum (P. polonicum). The results revealed that the inactivation kinetics of fungal spores by BCDMH fitted to Chick-Watson model well, with the inactivation rate constant of 0.011 and 0.034 L mg-1 min-1 for A. niger and P. polonicum, respectively. Acidic condition and high temperature promoted the inactivation by BCDMH. Compared with chlorine, BCDMH showed relative weaker ability on inactivation of fungal spores. However, it was demonstrated that the inactivation efficiency of BCDMH was obviously enhanced by adding halide ions, with 11 or 36 folds for A. niger and 4 or 15 folds for P. polonicum by adding 40 µM Br- or I-. The inactivation mechanisms were detected by flow cytometry and scanning electron microscope. Fungal spores lost their culturability firstly, then membrane integrity was damaged. Meanwhile, the esterase activity and intracellular reactive oxygen species level changed, and finally intracellular adenosine triphosphate released.

Evaluation of the vital viability and their application in fungal spores' disinfection with flow cytometry.

More attention was focused on fungi contamination in drinking water. Most researches about the inactivation of fungal spores has been conducted on disinfection efficiency and the leakage of intracellular substances. However, the specific structural damage of fungal spores treated by different disinfectants is poorly studied. In this study, the viability assessment methods of esterase activities and intracellular reactive oxygen species (ROS) were optimized, and the effects of chlorine-based disinfectants on fungal spores were evaluated by flow cytometry (FCM) and plating. The optimal staining conditions for esterase activity detection were as follows: fungal spores (106 cells/mL) were stained with 10 µM carboxyfluorescein diacetate and 50 mM ethylene diamine tetraacetic acid at 33 °C for 10 min (in dark). The optimal staining conditions for intracellular ROS detection were as follows: dihydroethidium (the final concentration of 2 µg/mL) was added into fungal suspensions (106 cells/mL), and then samples were incubated at 35 °C for 20 min (in dark). The cell culturability, membrane integrity, esterase activities, and intracellular ROS were examined to reveal the structural damage of fungal spores and underlying inactivation mechanisms. Disinfectants would cause the loss of the cell viability via five main steps: altered the morphology of fungal spores; increased the intracellular ROS levels; decreased the culturability, esterase activities and membrane integrity, thus leading to the irreversible death. It is appropriate to assess the effects of disinfectants on fungal spores and investigate their inactivation mechanisms using FCM.

Simultaneously enhance the inactivation and inhibit the photoreactivation of fungal spores by the combination of UV-LEDs and chlorine: Kinetics and mechanisms.

Waterborne fungi have been recognized as an emerging environmental contaminant in recent years. This work was to investigate the inactivation efficiency and mechanisms of ultraviolet light-emitting diodes (UV-LEDs)/chlorine (Cl2) (265, 280 and 265/280 nm combination) and LPUV/Cl2 (254 nm) treatments for three fungal species compared with individual disinfection processes. Control of photoreactivation for fungal species inactivated by UV-LEDs/Cl2 and LPUV/Cl2 was also evaluated. The results revealed that the combined UV-LEDs/Cl2 and LPUV/Cl2 processes, especially UV-LEDs/Cl2, exhibited better inactivation performance compared to UV alone and Cl2 alone based on the inactivation rate constants, and an evident synergistic effect was observed. For example, the inactivation rates for Penicillium polonicum in the processes of UV265/Cl2, UV280/Cl2, UV265/280/Cl2 and LPUV/Cl2 was 0.142, 0.168, 0.174 and 0.106 cm2/mJ, respectively, which were all approximately 1.5-fold higher than that of UV alone. The synergistic effect of fungal spores inactivation by UV-LEDs/Cl2 and LPUV/Cl2 was due to the high level production of intracellular reactive oxygen species and the reaction of potential extracellular free radicals. Resistance of the tested fungal spores was as follows: Trichoderma harzianum < Penicillium polonicum < Aspergillus niger. In addition, the joint effect of DNA and other cellular damage resulted in the inhibition of photoreactivation of fungal spores inactivated by UV-LEDs/Cl2 and LPUV/Cl2 compared with that of fungal spore inactivated by UV alone. This study may provide reference for controlling the dissemination of waterborne fungi utilizing combined UV-LEDs and free chlorine processes.

Graphene quantum dots as fluorescence probes for turn-off sensing of melamine in the presence of Hg(2+).

A rapid and sensitive fluorescence sensing system for melamine based on charge transfer quenching of the fluorescence of graphene quantum dots (GQDs) in the presence of Hg(2+) is proposed. The synthesized GQDs were strongly luminescent with predominantly aromatic sp(2) domains. Melamine could coordinate with mercury through nitrogen atoms in both its amine and triazine groups and bring more Hg(2+) to the surface of GQDs through π-π stacking, thus leading to quenching of the GQDs' fluorescence. The quenching mechanism was investigated in detail and ascribed to charge transfer from the GQDs to Hg(2+) with melamine acting as the linkage agent. The melamine demonstrated a linear range 0.15-20 µM and a detection limit of 0.12 µM, which was far below the regulatory level, suggesting the promising practical usage of this sensing system. This sensing system also possessed high selectivity for melamine in the presence of possible interferences. Finally, this novel sensor was successfully applied for melamine detection in raw milk and satisfactory recovery was achieved.

Focusing on luminescent graphene quantum dots: current status and future perspectives.

To obtain graphene-based fluorescent materials, one of the effective approaches is to convert one-dimensional (1D) graphene to 0D graphene quantum dots (GQDs), yielding an emerging nanolight with extraordinary properties due to their remarkable quantum confinement and edge effects. In this review, the state-of-the-art knowledge of GQDs is presented. The synthetic methods were summarized, with emphasis on the top-down routes which possess the advantages of abundant raw materials, large scale production and simple operation. Optical properties of GQDs are also systematically discussed ranging from the mechanism, the influencing factors to the optical tunability. The current applications are also reviewed, followed by an outlook on their future and potential development, involving the effective synthetic methods, systematic photoluminescent mechanism, bandgap engineering, in addition to the potential applications in bioimaging, sensors, etc.

Ratiometric fluorescent chemosensor for Hg(2+) based on heptamethine cyanine containing a thymine moiety.

Based on a T-Hg(2+)-T binding mode, a sensitive ratiometric fluorescent chemosensor for aqueous Hg(2+) was developed with a heptamethine cyanine chromophore containing a thymine moiety.