Plasma-activated Liquids Research Articles

Effects of combined treatment of plasma activated liquid and ultrasound for degradation of chlorothalonil fungicide residues in tomato

The effects of combined treatment (PAL-U) of plasma-activated liquid (PAL) including plasma-activated water (PAW) and plasma-activated buffer solution (PABS) and ultrasound (U) for the degradation of chlorothalonil fungicide on tomato fruit was investigated. Distilled water and buffer solution were activated by radiofrequency plasma jet for durations of 1, 3, 5, and 10 min to obtain PAL1 to PAL10. Fruits were immersed in PAL for 15 min and also in distilled water with sonication for 15 min for individual treatments, and in PAL with sonication for 15 min for combined treatments. The maximum chlorothalonil fungicide residues were reduced by 89.28 and 80.23% for PAW10-U and PABS10-U, respectively. HPLC-MS characterization revealed chlorothalonil degradation pathway and formation of 2,4,5-trichloroisophthalonitrile, 2,4-dichloroisophthalonitrile, 4-chloroisophthalonitrile, isophthalonitrile and phenylacetonitrile as degradation products. Treatments also showed no negative effects on tomato quality. Therefore, PAL and PAL-U treatments could serve as effective methods for degrading pesticides on tomatoes.

Systematic Safety Evaluation of Cold Plasma-Activated Liquid in Rabbits

Plasma-activated liquid (PAL) can effectively and selectively kill various types of cancer cells both in superficial and deeper tumors. As a promising novel approach to oncotherapy, the safety of PAL is essential in the clinic but has not been thoroughly assessed. In myeloma and blood tumors, the pathogenesis is in the bone marrow cavity. We have therefore evaluated the safety of PAL in New Zealand rabbits by intra-bone marrow injection, and provide a basis of further clinical research and application of PALs. In this study, both a plasma jet and plasma surface were used to treat saline solution, phosphate-buffered solution, and cell culture medium, to produce PAL. Then, oscillograms and optical emission spectra were evaluated to characterize the plasma discharge. Acute toxicity tests and safety evaluation studies were conducted by intra-bone marrow injection of PAL into New Zealand rabbits, while control rabbits received saline only. Body weight, vital organ coefficient, organ appearance, organ histopathology, blood cell and hemoglobin parameters, and blood biochemical indicators were tested on the 30th day after injection. We found that there was no mortality or loss of mobility throughout the experimental period. Acute toxicity tests showed that there were no PAL-related side effects in rabbits receiving the maximum dose of 700 μL PAL. PAL treatments did not affect body weight, organ coefficient, organ appearance, organ histopathology, or blood biochemical indicators. However, the percentage of lymphocytes decreased while the percentage of neutrophil granulocytes increased compared with the control group. In summary, our results indicate that PAL can be safely injected into bone marrow of New Zealand rabbits without significant toxicity.

Selective Apoptotic Effect of Plasma Activated Liquids on Human Cancer Cell Lines.

Plasma medicine is a new field focusing on biomedical and clinical applications of cold gas plasmas, including their anticancer effects. Cold plasmas can be applied directly or indirectly as plasma-activated liquids (PAL). The effects of plasma-activated cell growth medium (PAM) and plasma-activated phosphate buffered saline (PAPBS) were tested, using a plasma pen generating streamer corona discharge in ambient air, on different cancer cell lines (melanoma A375, glioblastoma LN229 and pancreatic cancer MiaPaCa-2) and normal cells (human dermal fibroblasts HDFa). The viability reduction and apoptosis induction were detected in all cancer cells after incubation in PAL. In melanoma cells we focused on detailed insights to the apoptotic pathways. The anticancer effects depend on the plasma treatment time or PAL concentration. The first 30 min of incubation in PAL were enough to start processes leading to cell death. In fibroblasts, no apoptosis induction was observed, and only PAPBS, activated for a longer time, slightly decreased their viability. Effects of PAM and PAPBS on cancer cells showed selectivity compared to normal fibroblasts, depending on correctly chosen activation time and PAL concentration, which is very promising for potential clinical applications. This selectivity effect of PAL is conceivably induced by plasma-generated hydrogen peroxide.

The Antimicrobial Property of Plasma Activated Liquids (PALs) against Oral Bacteria Streptococcus mutans

The Antimicrobial Property of Plasma Activated Liquids (PALs) against Oral Bacteria Streptococcus mutans

Multivariate Optimization of the FLC-dc-APGD-Based Reaction-Discharge System for Continuous Production of a Plasma-Activated Liquid of Defined Physicochemical and Anti-Phytopathogenic Properties

To the present day, no efficient plant protection method against economically important bacterial phytopathogens from the Pectobacteriaceae family has been implemented into agricultural practice. In this view, we have performed a multivariate optimization of the operating parameters of the reaction-discharge system, employing direct current atmospheric pressure glow discharge, generated in contact with a flowing liquid cathode (FLC-dc-APGD), for the production of a plasma-activated liquid (PAL) of defined physicochemical and anti-phytopathogenic properties. As a result, the effect of the operating parameters on the conductivity of PAL acquired under these conditions was assessed. The revealed optimal operating conditions, under which the PAL of the highest conductivity was obtained, were as follows: flow rate of the solution equaled 2.0 mL min−1, the discharge current was 30 mA, and the inorganic salt concentration (ammonium nitrate, NH4NO3) in the solution turned out to be 0.50% (m/w). The developed PAL exhibited bacteriostatic and bactericidal properties toward Dickeya solani IFB0099 and Pectobacterium atrosepticum IFB5103 strains, with minimal inhibitory and minimal bactericidal concentrations equaling 25%. After 24 h exposure to 25% PAL, 100% (1−2 × 106) of D. solani and P. atrosepticum cells lost viability. We attributed the antibacterial properties of PAL to the presence of deeply penetrating, reactive oxygen and nitrogen species (RONS), which were, in this case, OH, O, O3, H2O2, HO2, NH, N2, N2+, NO2−, NO3−, and NH4+. Putatively, the generated low-cost, eco-friendly, easy-to-store, and transport PAL, exhibiting the required antibacterial and physicochemical properties, may find numerous applications in the plant protection sector.

Recent progress in the application of plasma-activated water (PAW) for food decontamination

Plasma-activated water (PAW) is a novel and promising alternative to traditional food sanitizers. Recently, the inactivation efficacy of PAW has been demonstrated on a wide range of food products against foodborne pathogens , spoilage microorganisms, and harmful chemicals. The effectiveness of PAW relies on various factors related to the plasma generation mechanisms, the target microorganisms, and the food matrix. The inactivation mechanisms of PAW are attributed to the damage of cell integrity and intracellular components by various reactive oxygen and nitrogen species (RONS). Utilization of plasma-activated liquids and hurdle technologies can enhance the inactivation efficacy and diversify the application of this technology. Scaling-up of PAW is still at the very beginning stage and needs further studies before industrial application.

Controlling: the composition of plasma-activated water by Cu ions

It is demonstrated that Cu ions can be used to control the ageing of plasma-activated liquids through the Fenton-type reaction, which acts as a competitor to the + H2O2 + H+ → products reaction, and thus moderates the recombination of ions. It is shown that the ageing dynamics is influenced by the type of Cu-ions source used, due to the different ion dissolution dynamics. Using CuNP-colloidal and copper acetate salt solutions, respectively, similar ageing dynamics can be achieved: the lifetime of can be increased to 104 min comparing to 102 min obtained in plasma-activated water, while the H2O2 concentration decreases to about half of its initial value. While the treated salt solutions contain the degradation products of the salt compounds, the CuNPs can be filtered out from the plasma-activated colloid (PAC). It is shown that filtration does not influence the concentration of active species, while the ageing of the filtrated PAC depends on the moment of filtration, which determines the concentration of dissolved Cu ions. Using copper foil, the dissolved Cu-ion concentration can be controlled by removing the foil during ageing, and in this way the lifetime of species and the H2O2 concentration can be adjusted. The effect has been validated in the case of two different plasma sources: a surface-wave microwave discharge and a kHz plasma jet, which produce PALs with different concentration ratio, which is the main governor of the ageing process.

Effect of plasma activated water and buffer solution on fungicide degradation from tomato (Solanum lycopersicum) fruit

This study investigated the effect of plasma-activated liquid (PAL) including plasma-activated water (PAW) and plasma-activated buffer solution (PABS) for the reduction of chlorothalonil (CTL) and thiram (THM) pesticide residues on tomato fruit. Results revealed that the PAL obtained by using atmospheric air as the feed gas, CTL residues were decreased to 85.3% and 74.2% and THM residues decreased to 79.47 and 72.21% after treatments with PAW10 and PABS10, respectively, and increasing the activation time caused a significant reduction in fungicide residues. In addition, CTL and THM residues were also decreased while increasing the activation time of PAL using Ar/O2 as the feed gas, the concentrations of the CTL residues were decreased to 75.07 and 69.89% for PAW10 and PABS10, respectively and THM residues decreased to 65.89 and 61.91% for PAW10 and PABS10, respectively. Oxidation-reduction potential (ORP) and electrical conductivity (EC) were increased significantly after plasma treatment, while pH values of both solutions were decreased significantly with activation time. NO3− and NO2− concentrations of PAW increased significantly, while for PABS, NO3− concentration decreased but NO2−, with increasing the plasma activation time. Additionally, washing with PAW and PABS caused no notable negative impact on tomato fruit. Results confirmed that PAL treatments showed a significant reduction of CTL and THM fungicide residues (p < 0.05) in tomato without affecting the quality.

Role of Short- and Long-Lived Reactive Species on the Selectivity and Anti-Cancer Action of Plasma Treatment In Vitro.

Simple SummaryOne fundamental feature that has emerged from in vitro application of cold plasmas in cancer treatment is the key role of the liquid phase covering the cells. In the present work, we investigated the effect of direct and indirect plasma treatments on two cancer and three normal cell lines to assess the benefits of one treatment over the other in terms of death of tumor versus healthy cells. Our results demonstrate that indirect plasma treatment is as efficient at killing tumor cells as an appropriate combination of H2O2, NO2− and acidic pH in ad hoc solutions, while sparing normal cells. However, direct plasma treatment is far more efficient at killing normal than tumor cells, and we provide evidence that short- and long-lived reactive species contribute synergistically to kill normal cells, while having an additive effect regarding tumor cell death. Collectively, our results call the use of plasma-activated liquid in cancer treatment into question.(1) Plasma-activated liquids (PAL) have been extensively studied for their anti-cancer properties. Two treatment modalities can be applied to the cells, direct and indirect plasma treatments, which differ by the environment to which the cells are exposed. For direct plasma treatment, the cells covered by a liquid are present during the plasma treatment time (phase I, plasma ON) and the incubation time (phase II, plasma OFF), while for indirect plasma treatment, phase I is cell-free and cells are only exposed to PAL during phase II. The scope of this work was to study these two treatment modalities to bring new insights into the potential use of PAL for cancer treatment. (2) We used two models of head and neck cancer cells, CAL27 and FaDu, and three models of normal cells (1Br3, NHK, and RPE-hTERT). PBS was used as the liquid of interest, and the concentration of plasma-induced H2O2, NO2− and NO3−, as well as pH change, were measured. Cells were exposed to direct plasma treatment, indirect plasma treatment or reconstituted buffer (PBS adjusted with plasma-induced concentrations of H2O2, NO2−, NO3− and pH). Metabolic cell activity, cell viability, lipid peroxidation, intracellular ROS production and caspase 3/7 induction were quantified. (3) If we showed that direct plasma treatment is slightly more efficient than indirect plasma treatment and reconstituted buffer at inducing lipid peroxidation, intracellular increase of ROS and cancer cell death in tumor cells, our data also revealed that reconstituted buffer is equivalent to indirect plasma treatment. In contrast, normal cells are quite insensitive to these two last treatment modalities. However, they are extremely sensitive to direct plasma treatment. Indeed, we found that phase I and phase II act in synergy to trigger cell death in normal cells and are additive concerning tumor cell death. Our data also highlight the presence in plasma-treated PBS of yet unidentified short-lived reactive species that contribute to cell death. (4) In this study, we provide strong evidence that, in vitro, the concentration of RONS (H2O2, NO2− and NO3−) in combination with the acidic pH are the main drivers of plasma-induced PBS toxicity in tumor cells but not in normal cells, which makes ad hoc reconstituted solutions powerful anti-tumor treatments. In marked contrast, direct plasma treatment is deleterious for normal cells in vitro and should be avoided. Based on our results, we discuss the limitations to the use of PAL for cancer treatments.

Distinct Chemistries Define the Diverse Biological Effects of Plasma Activated Water Generated with Spark and Glow Plasma Discharges

The spread of multidrug-resistant bacteria poses a significant threat to human health. Plasma activated liquids (PAL) could be a promising alternative for microbial decontamination, where different PAL can possess diverse antimicrobial efficacies and cytotoxic profiles, depending on the range and concentration of their reactive chemical species. In this research, the biological activity of plasma activated water (PAW) on different biological targets including both microbiological and mammalian cells was investigated in vitro. The aim was to further an understanding of the specific role of distinct plasma reactive species, which is required to tailor plasma activated liquids for use in applications where high antimicrobial activity is required without adversely affecting the biology of eukaryotic cells. PAW was generated by glow and spark discharges, which provide selective generation of hydrogen peroxide, nitrite and nitrate in the liquid. The PAW made by either spark or glow discharges showed similar antimicrobial efficacy and stability of activity, despite the very different reactive oxygen species (ROS) and reactive nitrogen species profiles (RNS). However, different trends were observed for cytotoxic activities and effects on enzyme function, which were translated through the selective chemical species generation. These findings indicate very distinct mechanisms of action which may be exploited when tailoring plasma activated liquids to various applications. A remarkable stability to heat and pressure was noted for PAW generated with this set up, which broadens the application potential. These features also suggest that post plasma modifications and post generation stability can be harnessed as a further means of modulating the chemistry, activity and mode of delivery of plasma functionalised liquids. Overall, these results further understanding on how PAL generation may be tuned to provide candidate disinfectant agents for biomedical application or for bio-decontamination in diverse areas.

In vitro antimicrobial effect and mechanism of action of plasma-activated liquid on planktonic Neisseria gonorrhoeae

ABSTRACT Neisseria gonorrhoeae (Ng) is highly resistant to treatment, and there is an urgent need for new treatments to alleviate gonococcal resistance caused by antibiotic monotherapy. The antimicrobial effect and mechanism of plasma-activated liquid (PAL) on Ng were evaluated in this study. Upon PAL treatment, extensively analyses on cell culturability, metabolic capacity, intracellular reactive oxygen species (ROS),membrane integrity and nucleic acids for Ng were carried out and significant antimicrobial effects observed.PAL exerted antibacterial effect on Ng and induced bacterial death (6.71-log) following immersion for 30 min and treatment for 120 s. However, bacterial viability test revealed that after immersion in the same PAL, 10.17% of bacteria retained their metabolic capacity. This indicates that bacteria enter a physiological viable but non-culturable state to protect themselves from environmental stress. Confocal fluorescence microscopy and transmission electron microscopy demonstrated that PAL exerts bactericidal effect on Ng and disrupts its morphological structure. PAL may upregulate inflammatory factors and genes to modulate the resistance of Ng and affect the immune status of the host during infection.

Temperature Stability and Effectiveness of Plasma-Activated Liquids over an 18 Months Period

Non-buffered plasma-activated liquids such as water and saline have shown bactericidal effects. In the present study, we investigated the anti-bacterial efficacy and stability of plasma-activated water (PAW) and plasma-activated saline (PAS), generated using a high voltage dielectric barrier discharge system. This study compares the potential of non-buffered plasma-activated liquids (PAL) for the inactivation of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) after storage of the solutions at five different temperatures for a storage time up to 18 months after their generation. The temperatures used were room temperature, 4 °C, −16 °C, −80 °C, −150 °C. Both PAW and PAS achieved 6 log reduction for both bacteria on the first day of their generation after 60 min contact time and they retained these effects after 18 months when stored at the lowest temperatures. Chemical analysis of PAL showed that plasma caused a drop in pH, generation of reactive oxygen species and nitrates, whereas no nitrites are detected in the system used. The concentrations of chemical species were affected by the storage at different temperatures and a thermocouple probe was used to investigate the freezing behaviour of the PAL.

Potential Agricultural and Biomedical Applications of Cold Atmospheric Plasma-Activated Liquids With Self-Organized Patterns Formed at the Interface

Cold atmospheric plasma (CAP) has become a topical research area due to its diverse applications in agriculture, medicine, environment, materials, energy, nanotechnology, and other fields. Plasmas in contact with liquids form marked sensitivity patterns at the interface depending on controlling parameters, including gas species, driving current, gas flow rate, gap length, and electrolyte conductivity. This review overviews basic aspects of plasmas inducing self-organization including computational and experimental studies and potential applications of such plasmas-treated liquids in agriculture and medicine. Representative experimental evidence of self-organized pattern (SOP) in diverse types of plasma discharges is reviewed. Generation and transport of reactive species in SOP plasma and SOP plasma interacting with liquids are introduced and discussed from their potential applications in agriculture and medicine.

Cancer treatment with gas plasma and with gas plasma-activated liquid: positives, potentials and problems of clinical translation.

Gas plasmas, created in atmospheric pressure conditions, both thermal (hot) and non-thermal (cold) are emerging as useful tools in medicine. During surgery, hot gas plasmas are useful to reduce thermal damage and seal blood vessels. Gas plasma pens use cold gas plasma to produce reactive chemical species with selective action against cancers, which can be readily exposed in surgery or treated from outside of the body. Solutions activated by cold gas plasma have potential as a novel treatment modality for treatment of less readily accessible tumours, or those with high metastatic potential. This review summarises the preclinical and clinical trial evidence currently available, as well as the challenges for translation of direct gas plasma and gas plasma-activated solution treatment into regular practice.

Oxidative stress responses of pathogen bacteria in poultry to plasma-activated lactic acid solutions

Investigation of the plasma-activated liquid has seen some success, yet no studies have systematically explored the potential damage from oxidative stress against the bacteria. In this study, the oxidative stress response of Salmonella Enteritidis suffered from plasma-activated lactic acid (PALA) solutions was assessed. PALA solution was made by treating 300-mL of lactic acid (LA, 0.05–0.20%) solution with a plasma jet for 40–100 s. The results revealed that a 6.86 log-reduction of S. Enteritidis was detected when 0.20% LA solution was treated with plasma for 80 s. The reactive oxygen species (ROS) in PALA solutions enhanced the intracellular ROS level that cannot be rescued by major anti-oxidative enzymes, such as superoxide dismutase (SOD) and catalase (CAT). Gel electrophoresis and liquid chromatography-tandem mass spectrometry (LC-MS/MS) indicated the generated ROS resulted in the increase of carbonyls content and the fragmentation of outer membrane protein A (OmpA) of S. Enteritidis. Further, such property makes PALA a superior sanitizer for poultry meat compared to widely used sodium hypochlorite (NaClO) and lactic acid (LA). This study not only provides a theoretical basis for developing novel sanitizer but also enables PALA to find a way for practical application.

Anti-Cancer Potential of Two Plasma-Activated Liquids: Implication of Long-Lived Reactive Oxygen and Nitrogen Species.

Cold atmospheric plasma-exposed culture medium may efficiently kill cancer cells in vitro. Due to the complexity of the medium obtained after plasma exposure, less complex physiological liquids, such as saline solutions and saline buffers, are gathering momentum. Among the plethora of reactive oxygen and nitrogen species (RONS) that are produced in these plasma-activated liquids, hydrogen peroxide, nitrite and nitrate appear to be mainly responsible for cytotoxic and genotoxic effects. Here, we evaluated the anti-cancer potential of plasma-activated phosphate-buffered saline (P-A PBS) and sodium chloride 0.9% (P-A NaCl), using a three-dimensional tumor model. Two epithelial cancer cell lines were used to evaluate cellular effects of either P-A PBS or P-A NaCl. Human colorectal cancer cells HCT 116 and human ovarian carcinoma, SKOV-3 were used to investigate the manner by which different cell types respond to different plasma-activated liquids treatments. Our investigations indicate that P-A PBS is more efficient than P-A NaCl mainly because RONS are produced in larger quantities. Indeed, we show that the cytotoxicity of these liquids directly correlates with the concentration of hydrogen peroxide and nitrite. Moreover, P-A PBS induced a faster-occurring and more pronounced cell death, which arose within deeper layers of the 3D multicellular spheroid models.

Plasma-activated Ringer's Lactate Solution Displays a Selective Cytotoxic Effect on Ovarian Cancer Cells.

Epithelial Ovarian Cancer (EOC) is one of the leading causes of cancer-related deaths among women and is characterized by the diffusion of nodules or plaques from the ovary to the peritoneal surfaces. Conventional therapeutic options cannot eradicate the disease and show low efficacy against resistant tumor subclones. The treatment of liquids via cold atmospheric pressure plasma enables the production of plasma-activated liquids (PALs) containing reactive oxygen and nitrogen species (RONS) with selective anticancer activity. Thus, the delivery of RONS to cancer tissues by intraperitoneal washing with PALs might be an innovative strategy for the treatment of EOC. In this work, plasma-activated Ringer’s Lactate solution (PA-RL) was produced by exposing a liquid substrate to a multiwire plasma source. Subsequently, PA-RL dilutions are used for the treatment of EOC, non-cancer and fibroblast cell lines, revealing a selectivity of PA-RL, which induces a significantly higher cytotoxic effect in EOC with respect to non-cancer cells.

Plasma activated liquids: New decontamination solutions

In the field of new decontaminants, there is an increasing consensus that improved disinfection of environmental surfaces is needed in patient care facilities as an important component in the overall strategy for prevention of HAI. The interaction of cold atmospheric plasma, i.e. an ionized gas, with liquids results in complex physical and chemical processes, which offer a source of short-lived and long-lived reactive chemical species that are critical for microbial inactivation. These solutions may fulfil the urgent need for new decontaminant solutions for special purposes such as disinfection of surfaces, or use as an antiseptic for body surfaces. In this study, we explored the bactericidal effects of plasma activated liquids (PALs) on E.coli and S. aureus strains and investigated factors which influence PALs stability over time. Liquids of interest were non-complex solutions such as water and saline. An atmospheric cold plasma system using air was employed for the generation of PALs. The solutions were compared with respect to their content of long-lived reactive chemical species and bactericidal effects. Our results documented that PALs may carry different concentrations of chemical species and maintain diverse antimicrobial properties. The bactericidal activity of these solutions demonstrated high thermal stability and could be preserved over a 6 month period through specific sub-ambient storage conditions. Investigation of the inactivation processes in relation to the PALs’ chemical composition will enhance our knowledge on how prokaryotic and eukaryotic cells respond to these, and demonstrate how PALs could be a promising treatment method for future applications, with chemical and antimicrobial stability.

Ability of plasma-activated acetated Ringer's solution to induce A549 cell injury is enhanced by a pre-treatment with histone deacetylase inhibitors.

Non-thermal plasma (NTP) is applicable to living cells and has emerged as a novel technology for cancer therapy. NTP affect cells not only by direct irradiation, but also by an indirect treatment with previously prepared plasma-activated liquid. Histone deacetylase (HDAC) inhibitors have the potential to enhance susceptibility to anticancer drugs and radiation because these reagents decondense the compact chromatin structure by neutralizing the positive charge of the histone tail. The aim of the present study was to demonstrate the advantage of the combined application of plasma-activated acetated Ringer’s solution (PAA) and HDAC inhibitors on A549 cancer cells. PAA maintained its ability for at least 1 week stored at any temperature tested. Cell death was enhanced more by combined regimens of PAA and HDAC inhibitors, such as trichostatin A (TSA) and valproic acid (VPA), than by a single PAA treatment and was accompanied by ROS production, DNA breaks, and mitochondria dysfunction through a caspase-independent pathway. These phenomena induced the depletion of ATP and elevations in intracellular calcium concentrations. The sensitivities of HaCaT cells as normal cells to PAA were less than that of A549 cells. These results suggest that HDAC inhibitors synergistically induce the sensitivity of cancer cells to PAA.

Inactivation of Single Strains of Listeria monocytogenes and Salmonella Typhimurium Planktonic Cells Biofilms With Plasma Activated Liquids.

Recent research has proven the ability of cold atmospheric plasma (CAP) for assuring food safety. A more flexible and transportable alternative is the use of plasma activated liquids (PAL), which are also known to have antimicrobial properties. However, within the context of food safety, little is known on its potential regarding decontamination. This research therefore focusses on identifying the impact of (i) the microbial species and its cell type (planktonic cells or biofilms), (ii) the CAP settings (i.e., gas composition and generation time) and (iii) PAL related factors (treatment time and PAL age) on the technologies efficacy. Cell densities were monitored using the plate counting technique for which the results were analyzed by means of predictive inactivation models. Moreover, the pH and the concentrations of long-lived species (i.e., hydrogen peroxide, nitrite, and nitrate) were measured to characterize the PAL solutions. The results indicated that although the type of pathogen impacted the efficacy of the treatment, mainly the cell mode had an important effect. The presence of oxygen in the operating gas ensured the generation of PAL solutions with a higher antimicrobial activity. Moreover, to ensure a good microbial inactivation, PAL generation times needed to be sufficiently long. Both CAP related factors resulted in a higher amount of long-lived species, enhancing the inactivation. For 30 min. PAL generation using O2, this resulted in log reductions up to 3.9 for biofilms or 5.8 for planktonic cells. However, loss of the PAL activity for stored solutions, together with the frequent appearance of a tailing phase in the inactivation kinetics, hinted at the importance of the short-lived species generated. Different factors, related to (i) the pathogen and its cell mode, (ii) the CAP settings and (iii) PAL related factors, proved to impact the antimicrobial efficacy of the solutions and should be considered with respect to future applications of the PAL technology.