Some previous studies have hypothesized that the interaction between plasma generated RONS and fungal spores is one of the most likely chemical pathways for inactivation. (37,55) When CAP decontamination efficacy is compared against the most frequently used methods for fungi decontamination, conventional UV irradiation or Otreatment, the CAP method is significantly more effective. Recent studies of conventional approaches demonstrated that UVC irradiation achieved a modest 2.8 log reduction ofspores after a 6 min exposure at a light intensity of 3.3 kJ/m (56) Similarly, gaseous Owas found to be even less effective, with a 3 log reduction achieved for various spores types exposed to 35 ppm of Ofor 20 min. (17) Since the low power CAP treatment accomplished a high inactivation level within a short time frame, it is reasonable to conclude that Oalone is not solely responsible. Therefore, the simultaneous exposure to multiple RONS, UV and elevated gas temperatures are likely to result in synergistic effects that accelerate spore inactivation in the presence of plasma.

The biological effects of RONS are well-known. Ois a powerful oxidant; it can oxidize organic matter either directly or through OH produced during the decomposition of O. First, it interacts with double bonds of unsaturated lipids and the sulphydryl groups of membrane enzymes. This damage causes disruption of cell permeability, leading to leakage and oxidation of intercellular contents and biomolecules such as nucleic acids and eventually the lysis of the cell. (48) Another potent oxidant produced by CAP is H, which has inactivation mechanisms similar to those of O. Generally, cytotoxicity caused by Hbegins with its penetration into the cell and then the production of OH. OH is an extremely potent oxidizing agent which can be created from the decomposition of both Oand H. Its primary targets are cell walls and membranes, therefore all the compounds composing of these two structures. The mechanism of OH reaction with lipids refers to its H-abstraction from the unsaturated carbon bonds of the fatty acids, ending in lipid peroxidation. (49−53) Simultaneously, OH also reacts with membrane proteins by H-abstraction from the α-carbon of the peptide bonds between chain peptide-linked amino acids, resulting in peroxidation of proteins. In comparison with the direct activity of O, the mechanism of OH inactivation is less selective. (54) Given that the SEM images in Figures 4 (b and e) demonstrate that spores subjected to CAP treatment exhibit significant structural changes, it is highly probably that ROS species have an etching effect on the spore wall, creating an entry point for RONS to react further with the intracellular contents.

Beyond UV and thermal effects, it is well-known that air plasma generated at atmospheric pressure creates highly reactive short-lived species, for example, O, OH, N, and O, which diffuse away from the plasma region, rapidly reacting to form a variety of longer-lived RONS, including O, H, nitric oxide (NO), and other NO (46) The density of some of these species can be quantified from FTIR absorption measurements obtained during plasma operation, as shown in Figure 5 (b). Under low power conditions, it can be seen that Ois the primary long-lived product of the discharge. Ois primarily produced in the discharge layer through a three-body reaction involving atomic oxygen ( 1 ).Conversely, under high power conditions, the composition of the discharge effluent is dominated by NOincluding nitrous oxide (NO) and nitrogen dioxide (NO) ( Figure 5 (b)). The origins of the transition between Odominated to NOdominated discharge chemistries is 2-fold: first, as discharge power increases, the rate of thermal decomposition of Orapidly increases. (42) Second, an increased power yields more NO which acts to poison Oproduction by simultaneously reducing Othrough direct decomposition to NOand through the consumption of O, a key precursor for Oformation (Reactions 2 and 3 ). (47) Time-resolved FTIR measurements reveal the evolution of O, NO, and NO in the discharge effluent and clearly demonstrate the poisoning effect ( Figures 5 (c–e)). Under high power operation, Oformation is rapidly inhibited as the discharge reactor reaches thermal equilibrium and the nitric oxide concentration increases. As Opoisoning occurs, a significant increase in NOis observed. Finally, it is worth pointing out that several key RONS present in the plasma effluent may not be measured using FTIR, either because they do not actively absorb infrared light, or because they react to form alternative species between the discharge chamber and the FTIR gas cell, nitric oxide being a prime exemplar.

Further analysis of the second positive system makes it possible to obtain an estimate of the gas temperature within the discharge layer. It is well-known that under the atmospheric pressure conditions the rotational temperature of excited nitrogen is in thermodynamic equilibrium with the background gas. By comparing the recorded emission spectra against a synthetic spectra calculated at a known temperature an approximation of the gas temperature was obtained. As shown in the inset of Figure 5 (a), the best-fit temperature at the highest plasma power was found to be 340 K with an error of ±25 K. This value corresponds well with the thermocouple measurements made at the sample location.

To explain the observed decontamination results and aid in identification of the most likely underpinning inactivation pathways involved in gas phase CAP treatment FTIR, OES and temperature measurements were made. Initially, to rule out thermal effects, a thermocouple was placed at a position 5 mm beneath the SBD electrode. In all cases thermal equilibrium was reached within 120 s of CAP exposure and temperatures of 35 °C, 48 °C, and to 52 °C were measured for the low, medium, and high power conditions, respectively. While these temperatures are not insignificant, studies have demonstrated that such temperatures are incapable of achieving a significant level of mold spore inactivation on the time scales considered in this study. (44) Such results provide a strong indication that thermal effects are not the primary inactivation mechanism. To gain an insight into the composition of the plasma, OES was employed to capture the optical signature of the excited species within the discharge layer. The overlaid emission spectra of the discharge operating under low, medium and high power conditions is displayed in Figure 5 (a). The spectra reveal that in all cases the excited nitrogen second positive series was the dominant emission, appearing between 290 and 410 nm. No other significant emission lines were observed within the 280–600 nm wavelength range regardless of operating power. Given the emission data was calibrated for intensity it is possible to conclude that the light intensity produced by the CAP over the UVC, UVB, and UVA ranges is well below that known to have an impact on the growth ofspores, with levels around μW·cm·nm. Therefore, UV effects from the CAP device can also be neglected, a finding consistent with previous studies. (45)

Insight into the mode of inactivation during CAP exposure can be obtained from SEM images of the treated spores. The typical cases of untreated, CAP treated, and Virkon treated spores under two different levels of magnification are presented in Figure 4 . As seen in Figure 4 (b) and 4 (e), a high power plasma treatment resulted in considerable structural damage to thespores. It was observed that a large percentage of the treated spores exhibited similar levels of structural damage and were surrounded by considerable cell debris. In contrast, Virkon treated spores, presented in Figure 4 (c) and (e), showed little difference from the untreated spores.

Figure 3. CAP effluent treatment of A. flavus spores: (a), (b), and (c) log numbers of new grown spore units after low, medium, and high power plasma treatment with a comparison provided against a 1% Virkon solution. The initial concentration of spores for (a), (b), and (c) was 1.3 × 10 6 CFU/mlL. An one way ANOVA was performed between treated samples and control samples (0 s). P -values −0,0332 (*); 0,0021 (**); 0,0002 (***); < 0,0001 (****); (d), (e), (f) metabolic activity of spores exposed to low, medium and high power plasma and a 1% Virkon solution comparison.

spores deposited on glass coverslips were directly exposed to the CAP effluent under the plasma generation conditions outlined in the experimental methods section. The antifungal effect of the treatment is highlighted in Figure 3 . From the figure it is clear that a 480 s CAP treatment resulted in considerable spore inactivation, exceeding a 5 log reduction under both the low and high power conditions ( Figure 3 (a) and (c)). In general, the log reduction increased with exposure time and applied power. The highest power condition gave the most rapid level of inactivation, reaching a 2.19 log reduction after just 120 s of exposure ( Figure 3 (c)). Conversely, the low power condition showed a very little effect until 240 s, at which point a rapid increase in the level of inactivation occurred, ultimately achieving complete inactivation (defined as <1 log surviving CFU) within 480 s ( Figure 3 (a)). To provide a comparison of CAP inactivation efficacy against a widely used liquid decontamination agent, spores were spread on a glass coverslip and exposed to a 1% Virkon solution for a total of 480 s. Under such conditions, no reduction in viable spores was observed ( Figure 3 (a)–(c)). Such results indicate that gas phase plasma treatment is considerably more effective than a standard Virkon solution. These findings were supported by the XTT analysis, which highlights a minor decrease in the metabolic activity of Virkon treated spores; in contrast, plasma treated spores exhibited significantly reduced activity at considerably shorter exposure times ( Figure 3 (d)-(f)).

Antifungal Effect of Plasma Activated Broth

Escherichia coli, Pseudomonas fluorescens, Staphylococcus aureus, Candida albicans, etc. Recently, a considerable research effort has been focused on the use of plasma treated liquids for microbial inactivation applications. Examples include the inactivation ofetc. (42,57) Using a liquid as a carrier for reactive plasma species overcomes some drawbacks associated with a gas plasma treatment including an ability to treat highly porous materials with complex morphologies and enabling the treatment of highly temperature sensitive systems or environments where gaseous plasma cannot be directly used. Another comparative advantage of this treatment modality is the long-lived nature of the PAB (up to 1 week), (58) which can be used when needed, without the need to create a plasma.

A. flavus spores for varying lengths of time. The inactivation results are presented in A. flavus spores were analyzed by SEM after a 6 h incubation in fresh aqueous TSB solution (A. flavus exposed to PAB treated with high power CAP was decreased or arrested ( To assess the potential of PAB for fungal inactivation, a PAB solution was prepared and subsequently used to exposespores for varying lengths of time. The inactivation results are presented in Figure 6 (a–c). Clearly, the inactivation efficacy of PAB is significantly lower than the corresponding gas phase treatment. No significant reduction in spore count was observed with the exception of 24 h exposure to PAB created using high power plasma conditions, yielding a 1.04 log reduction. In contrast, exposure to a 1% Virkon solution led to complete inactivation under all conditions investigated. Interestingly, the metabolic activity of PAB treated spores significantly decreased under all treatment conditions, reaching a value two times lower compared to the control ( Figure 6 (d–f)). Given that previous studies have demonstrated the high antimicrobial potential of PAB, the results from Figure 6 are unexpected since it would be reasonable to assume that similar inactivation pathways are at play. To unravel this, the untreated, PAB treated and Virkon treatedspores were analyzed by SEM after a 6 h incubation in fresh aqueous TSB solution ( Figure 7 ). From the observed morphology, some minor differences were detected between the untreated ( Figure 7 (a) and (d)) and PAB treated spores ( Figure 7 (b) and (e)); however, spores exposed to Virkon exhibit significant structural damage ( Figure 7 (f)). Nevertheless, the growth and the development of hyphae and mycelia ofexposed to PAB treated with high power CAP was decreased or arrested ( Figure 7 (b) and (e)). In comparison, the formation of mycelia can be observed at untreated sample ( Figure 7 (a)).

Figure 6 Figure 6. Exposure of A. flavus spores to plasma activated broth (PAB) generated under low, medium and high power conditions for 480 s with a comparison provided against a 1% Virkon solution: (a), (b), (c) log numbers of new grown spore units after exposure to PAB and Virkon for 3, 6, and 24 h. The initial concentration of spores for (a), (b), and (c) was 2.1 × 106, 3.8 × 106 and 2.5 × 106 CFU/mL, respectively. An one way ANOVA was performed comparing results of treated samples to control (0 s). P-values −0,0332 (*); 0,0021 (**); 0,0002 (***); < 0,0001 (****); (d), (e), (f) metabolic activity of spores exposed to PAB and Virkon for 3, 6, and 24 h.

Figure 7 Figure 7. Characteristic scanning electron microscopy images of A. flavus spores after a 6 h exposure to PAB and a 1% Virkon solution under 5000 x magnification ((a)-(c)) and 25 000× magnification ((d)–(f)): (a) and (c) sample incubated in untreated aqueous TSB broth solution (control) with marked mycelia; (b) and (e) sample treated with PAB; (c) and (f) sample treated with 1% Virkon solution.

2 O 2 , NO 2 –, NO 3 – and pH were measured as a function of CAP treatment time ( In order to understand the results, the nature of PAB must be considered. CAP interaction with liquid induces a wide range of physicochemical changes. The creation of aqueous phase chemical species is strongly influenced by the type of discharge, its underpinning physical processes and the chemical composition of the surrounding environment, such as the type of gas and liquid used. Mass transport from the plasma phase, through the separating gas layer to the liquid phase, leads to the generation of a wide variety of reactive species in the bulk liquid. In this study, the liquid chemistry is mostly affected by long-lived species despite the relatively short distance between the plasma and the liquid surface, as the most reactive chemical species are confined to the visible discharge layer. (46) To follow the kinetics of the aqueous phase reactive species, the concentrations of H, NO, NOand pH were measured as a function of CAP treatment time ( Figure 8 ).

Figure 8 Figure 8. Liquid phase chemistry of PAB. Created using CAP under low, medium and high dissipated powers. A two way ANOVA was performed comparing results of treated samples to control (0 s); (a) pH values of PAB; (b) concentration of hydrogen peroxide. All treated samples were significantly different compared to control (P ≤ 0,001); (c) concentration of nitrite. All treated samples were significantly different (P ≤ 0,001) with exceptions of those treated for 30 s with low power plasma and 480 s with medium and high power plasma; (d) concentration of nitrate. Significant differences (P ≤ 0,001) occurred after 240 s of exposure to low and medium power plasma , and after 120 s of exposure to high power plasma.

2 O 2 in the broth solution. The increase in H 2 O 2 concentration is highly dependent on the discharge power employed. For low and high power treatment conditions, the H 2 O 2 concentration is increased up to an equilibrium or saturation level. Conversely, PAB created under medium power operation CAP operation showed a different trend, with a continual increase. The highest concentration of H 2 O 2 was measured after 480 s in the medium power case, at over 1800 μM. Based on recent plasma modeling studies, 2 O 2 present in the liquid phase was either generated in situ or created in the humid air surrounding the plasma generating electrode, through (4) 2 O 2 at the gas–liquid interface will be minimal ( (5) (6) 2 – was measured using the Griess reagent method. 2 – concentration initially increased with treatment time, reaching a peak, followed by a significant decrease with further treatment. Conversely, the NO 3 – displays an almost linear increase with treatment time under all power conditions ( 2 – and NO 3 – in the CAP treated broth solution are created through numerous reaction pathways. Mass transport of nitrous acid (HNO 2 ) and nitric acid (HNO 3 ) from the gas phase to the liquid provides the primary reactants to form NO 2 – and NO 3 –. Once in the liquid, they are hydrolyzed to form NO 2 –, NO 3 –, and H+ (Reactions (7) (8) 2 generated in the gas phase downstream of the discharge layer (Reactions (9) (10) + rises to a level where hydrolysis reactions are inhibited, contributing to a reduction of pH, 2 – concentration can be explained by considering its reaction with dissolved O 3 and NO 3 to become NO 3 –, 3 – is also generated through the reaction of NO 2 – with H 2 O 2 , forming short-lived ONOO– via (33,60) (11) (12) (13) 2 O 2 , NO 2 – and NO 3 –. Moreover, it is assumed that under acidic conditions ONOO– is generated. Several recent studies have indicated that ONOO– is one of the most potent oxidizing molecules in the field of biology and is a key agent in oxidative stress mechanisms. It is able to oxidize organic molecules directly or through H+ or CO 2 -catalyzed homolysis (yielding NO 2 ·, OH, or carbonate anion radical CO 3 .-). As for direct reactivity, it has affinity with key parts of proteins such as thiols, iron/sulfur centers, and zinc fingers. The lifetime of ONOO– is relatively short, nonetheless, it can still cross cellular membranes and reach deep within the cell, enabling it to interact with most of the important biomolecules. Because of its instability, measuring the amount of ONOO– produced is a considerable challenge. In Figure 8 (b) we suggest that CAP treatment induces the formation of Hin the broth solution. The increase in Hconcentration is highly dependent on the discharge power employed. For low and high power treatment conditions, the Hconcentration is increased up to an equilibrium or saturation level. Conversely, PAB created under medium power operation CAP operation showed a different trend, with a continual increase. The highest concentration of Hwas measured after 480 s in the medium power case, at over 1800 μM. Based on recent plasma modeling studies, (59,60) it is expected that Hpresent in the liquid phase was either generated in situ or created in the humid air surrounding the plasma generating electrode, through Reaction 4 Considering the reasonably large air gap between the plasma layer and liquid surface, it is highly unlikely that OH produced in the discharge will reach the liquid interface in any significant concentration. Consequently, the direct formation of Hat the gas–liquid interface will be minimal ( Figure 9 ). Several studies have used electron spin resonance techniques to measure OH directly in the liquid phase, (60) this has been attributed to in situ generation pathways involving plasma generated RONS that are capable of transport across the dividing gas layer, pertinent reactions include Reactions 5 and 6 The presence of NOwas measured using the Griess reagent method. Figure 8 (c) demonstrates that regardless of the plasma power the NOconcentration initially increased with treatment time, reaching a peak, followed by a significant decrease with further treatment. Conversely, the NOdisplays an almost linear increase with treatment time under all power conditions ( Figure 8 (d)). Both NOand NOin the CAP treated broth solution are created through numerous reaction pathways. Mass transport of nitrous acid (HNO) and nitric acid (HNO) from the gas phase to the liquid provides the primary reactants to form NOand NO. Once in the liquid, they are hydrolyzed to form NO, NO, and H(Reactions 7 and 8 ): (60) Another important pathway involved in the creation of these ionic species involves NO and NOgenerated in the gas phase downstream of the discharge layer (Reactions 9 and 10 ): (60) Eventually, the concentration of Hrises to a level where hydrolysis reactions are inhibited, contributing to a reduction of pH, (33,60) which can also be observed in the presented study. The pH level decreased over time under all examined power conditions, reaching a pH of 2.4 after 480 s of exposure ( Figure 8 (a)) in the highest power case. The results presented in Figure 8 and the observed drop in NOconcentration can be explained by considering its reaction with dissolved Oand NOto become NO Reactions 11 and 12 (61) Under the observed acidic conditions, NOis also generated through the reaction of NOwith H, forming short-lived ONOOvia Reaction 13 Based on the liquid chemistry analysis detailed in Figure 8 , the low rate of fungal spore inactivation observed in Figure 6 remains somewhat surprising. The PAB contained significant amounts of RONS such as H, NOand NO. Moreover, it is assumed that under acidic conditions ONOOis generated. Several recent studies have indicated that ONOOis one of the most potent oxidizing molecules in the field of biology and is a key agent in oxidative stress mechanisms. It is able to oxidize organic molecules directly or through Hor CO-catalyzed homolysis (yielding NO·, OH, or carbonate anion radical CO). As for direct reactivity, it has affinity with key parts of proteins such as thiols, iron/sulfur centers, and zinc fingers. The lifetime of ONOOis relatively short, nonetheless, it can still cross cellular membranes and reach deep within the cell, enabling it to interact with most of the important biomolecules. Because of its instability, measuring the amount of ONOOproduced is a considerable challenge. (32,62,63)

Figure 9 Figure 9. Schematic of main reactions during the CAP treatment of liquid.

Aspergillus spp. spores which have extremely hydrophobic properties due to the presence of proteins known as hydrophobins. These proteins cause spores to clump together and form aggregates in aqueous solution by association of their hydrophobic molecules with each other rather than with other molecules within the liquid. 3 and related ROS are important in terms of fungicidal activity of liquids. 3 reached the aqueous phase due to its low Henry’s law constant, and the number of potential loss pathways arising due to the presence of other plasma generated species, especially at a low pH. In all cases, the pH of the PAB was seen to drop and it is commonly accepted that the fungicidal effect in acidic conditions is less significant since many mold species tolerate low pH. To explain the significant difference in inactivation efficiency between spores exposed to plasma gas phase species and those exposed to PAB, the persistence of the fungal spores must be considered. These difference could be attributed to the surface characteristics ofspp. spores which have extremely hydrophobic properties due to the presence of proteins known as hydrophobins. These proteins cause spores to clump together and form aggregates in aqueous solution by association of their hydrophobic molecules with each other rather than with other molecules within the liquid. (64) Hydrophobic properties of the surface of the spores in relation with their insufficient inactivation in water was already discussed in the study by Ouf et al., where double atmospheric pressure cold plasma was used with intention to inactivate different mold species during water washes of fruit. (65) Conversely, spores spread on glass and allowed to dry are not protected with liquid and are therefore far less likely to form such aggregates, and are therefore more vulnerable to the reactive plasma species. This is in agreement with the SEM images presented in Figure 5 and 7 , where plasma treated spores display considerable damage after gas phase exposure, and little change after PAB exposure. It is well-known that Oand related ROS are important in terms of fungicidal activity of liquids. (66) In the case of this study, it was assumed that only a small amount of Oreached the aqueous phase due to its low Henry’s law constant, and the number of potential loss pathways arising due to the presence of other plasma generated species, especially at a low pH. In all cases, the pH of the PAB was seen to drop and it is commonly accepted that the fungicidal effect in acidic conditions is less significant since many mold species tolerate low pH. (67) This is not the case when PAB is used as an antimicrobial agent, since many bacterial species are significantly less tolerant to a low pH compared to their fungal counterparts. Nevertheless, a low pH can affect the electrostatic properties of the mold spores, resulting in an increase of zeta potential, meaning spores are likely to be more dispersed throughout the liquid volume, which can negatively affect the development of mold hyphae, mycelia and arrest mold growth. (68)

Overall, it is clear that gas phase CAP treatment is an extremely effective and environmentally friendly method for fungal inactivation, which requires no consumables and produced no harmful residues. Based on the results presented, a greater understanding of CAP technology gas been uncovered and thus may accelerate the translation of this innovative green technology for the decontamination of food, living areas or use in other relevant environments. Conversely, the results with plasma treated liquid did not demonstrate promising results. The difference between surface and liquid inactivation efficacy was attributed to the hydrophobic surface properties of spores, which makes them considerably more resilient to RONS when in the liquid phase.