WO2023140747A1 - Method of degrading antibiotics from aqueous solutions by using cold atmospheric pressure plasma generated in a flowing plasma brush and a plasma brush intended for this method - Google Patents

Abstract

The subject of the invention is a method of degrading antibiotics in aqueous solutions by using cold atmospheric pressure plasma - pm-rf-APGD or DBD, characterized in that by the following steps: a) the analyzed aqueous antibiotic solution or a mixture of antibiotics selected from the group of fluoroquinolones and/or tetracyclines and/or trimethoprims and/or chloramphenicols and/or β-lactams, are introduced into the flow plasma brush by a connection (8) attached to the chamber (7), b) the discharge gas is introduced through a wire (1) equipped with a gas valve (2), c) the discharge gas is further transported in a pipe (1) separated into at least 3 parts in the root (3), d) then a current is applied to the metal tubes (5) with the use of a current generator, leading as a result to generation of cold atmospheric plasma (6) in the form of 4 or 5 cones of pm-rf-APGD cold plasma or DBD, respectively. e) after treatment of the aqueous solution of antibiotics or the mixture of antibiotics with the pm-rf-APGD- or DBD-type cold plasma (6), respectively, the solution is collected in a chamber (7), delivered outside through a stub pipe (10) and subjected to further analysis. The subject of the invention is also the flow plasma brush characterized in that it contains a root (3) into whose upper part a gas valve (2) and a discharge gas supply conduit (1), divided in the middle part of the root (3) into at least 3 parts, are connected, the lower part of the root (3) is equipped with metal tubes (5) from which, as a result of applying a current from a current generator, cold atmospheric pressure plasma (6) is generated, the generated plasma (6) is in the form of 4 or 5 pm-rf-APGD- or DBD-type cones, metal pipes (5) are attached to the root (3) with rubber hoses (4) and link the chamber (7) with a connection (8) and a stub pipe (10), where the metal pipes (5) are braided with copper wires (9).

Description

Method of degrading antibiotics from aqueous solutions by using cold atmospheric pressure plasma generated in a flowing plasma brush and a plasma brush intended for this method

The subject of the invention is a method of degrading antibiotics from aqueous solutions by using cold atmospheric pressure plasma, i.e. a radio-frequency pulse- modulated atmospheric pressure glow discharge generated in contact with a liquid (pm-rf- APGD) or dielectric barrier discharge (DBD), in flow plasma brushes, i.e. in the pm-rf- APGD plasma brush or DBD type plasma brush, intended for this method.

The invention also covers a plasma brush for decomposing antibiotics from aqueous solutions.

Magureanu, M., Piroi, D., Mandache, N. B., David, V., Medvedovici, A., Bradu, C., & Parvulescu, V. I., in the publication entitled: "Degradation of antibiotics in water by non-thermal plasma treatment ’’(Water Research, 2011, 45, 3407-3416) describe decomposition of three P-lactam antibiotics, i.e. amoxcillin, oxacillin, and ampicillin, from aqueous solutions by using a DBD pulse discharge generated in a coaxial configuration in a circulating plasma reactor. Oxygen was used as the discharge gas, which was introduced to the plasma reactor at a rate of 600 mL min ¹. A two hundred milliliters of an aqueous solution of the analyzed antibiotic (amoxcillin, oxacillin or ampicillin at concentration of 100 mg L ¹) were placed in the plasma reactor, hence it is known that each reaction-discharge system utilized by the researchers worked in a stationary mode. Antibiotic solutions have been prepared in tap water to simulate the natural aquatic environment. With the use of liquid chromatography with tandem mass spectrometry (LC-MS/MS), as well as total organic carbon (TOC) and chemical oxygen demand (COD) analyses, the scientists determined the effectiveness of the DBD-type pulsed plasma method developed by them for degradation of the above -listed P-lactam antibiotics. As reported by the authors, degradation of amoxicillin from a 100 mg L ¹ concentrated solution was achieved after 10 minutes of plasma treatment. In the case of ampicillin and oxacillin solutions, the treatment time of the analyzed aqueous solutions of these antibiotics was longer, i.e. it amounted to 20 and 30 minutes, respectively, and led not to degradation, but to decomposition of the analyzed drugs. As noted by the researchers, despite the fact that the tested antibiotics belong to the same group, i.e. P- lactams, the achieved degradation degrees were different, which correlates with diverse treatment times with DBD-type pulsed discharge generated in a stationary system. In all three cases, the degradation mechanism of these organic compounds was similar, as evidenced by the total organic carbon content (TOC) and chemical oxygen demand (COD). This proves that the applied cold atmospheric pressure plasma cannot be regarded as a universal tool for degradation of antibiotics. Moreover, the transfer of the proposed technology to the industrial sector seems doubtful, taking into account the small volumes of the treated aqueous solutions of antibiotics, i.e. 200 mL of each. It is also worth emphasizing that the researchers analyzed only the impact of cold atmospheric pressure plasma treatment on degradation of antibiotics belonging to one chemical group. In order to determine whether the proposed system can be used to degrade other antibiotics present in wastewaters of pharmaceutical or medical origin, it is worth including in the research other antibiotics belonging to different groups basing on the chemical structure and/or their mixtures so as to imitate the composition of the actual municipal or postindustrial effluents. Unfortunately, the authors did not study this issue. Furthermore, they did not investigate the antibacterial properties of the analyzed aqueous antibiotics solutions both before and after the cold atmospheric plasma treatment, so the effectiveness of the described degradation method in terms of reducing/increasing multidrug resistance among microorganisms occurring in the natural environment is unknown.

Work by Sokolov, A., Krakstrom, M., Eklund, P., Kronberg, L., & Louhi- Kultanen, M. entitled: "Abatement of amoxicillin and doxycycline in binary and ternary aqueous solutions by gas-phase pulsed corona discharge oxidation " (Chemical Engineering Journal, 2018, 334, 673-681), describes the use of pulsed corona discharge (PCD) as a source of cold atmospheric pressure plasma, to reduce the contents of antibiotics, i.e. amoxicillin and doxycycline, in the analyzed aqueous solutions containing 50 mg L ¹ of antibiotics. PCD-type cold plasma was generated at the atmospheric pressure, while the current-voltage characteristics point to the energy of a single pulse 0.12 J at 22 kV and 180 A at the amplitude peak with a duration of 100 ns. This corresponds to an average energy density of 16.8 J m³. For generation of cold atmospheric plasma, a circulating plasma reactor was used, i.e. a given volume of the drug aqueous solution circulated in the system at a specified flow rate, in this case 4.5 L min ¹. The other parameters regulated by the researchers were both pH, i.e. neutral and alkaline, as well as the pulse repetition frequency, i.e. 50 pps, 200 pps, and 500 pps. The presented experiment was carried out on two-component aqueous antibiotic solutions, i.e. water-antibiotic systems, and on three -component aqueous antibiotic solutions, containing two different antibiotics beside water (water-antibiotic l-antibiotic2). The researchers rightly carried out analyzes on both single antibiotic solutions and ternary mixtures, which more closely reflect the composition of medical or pharmaceutical wastewaters, thus responding to the need of a real market. Then, based on the results of liquid chromatography coupled with mass spectrometry (LC-MS), it was found that all the degradation products of antibiotics could be easily oxidized by the proposed PCD plasma reactor. The frequency of 50 pps and pH 12 were considered as the optimal parameters for conducting treatment with a PCD-type cold atmospheric pressure plasma. Though, too high pH of the cold atmospheric pressure plasma treated-solutions may pose problems during implementation of this plasma system to the industry. Subsequent neutralization of such a high pH may prove to be too expensive precluding widespread use of the plasma system developed by Sokolov and others. Besides, in these studies as in the work of Magureanu et al. (2011), no attempt was made to assess whether the PCD- type atmospheric cold plasma treatment of aqueous solutions of antibiotics, i.e. amoxicillin and doxycycline, led to the loss of their biological activity. Therefore, the question whether it is possible to apply this cold atmospheric PCD-type plasma for deactivation of wastewaters that shall subsequently be released to the natural environment without any safety risks associated with spread of multi-drug resistance among microorganisms pathogenic for humans and animals, has not been addressed.

On the other hand, publication by Zhang, T., Zhou, R., Wang, P., Mai-Prochnow, A., McConchie, R., Li, W., Zhou, R., Thompson, EW, Ostrikov, K., & Cullen, PJ, entitled: "Degradation of cefixime antibiotic in water by atmospheric plasma bubbles: Performance, degradation pathways and toxicity evaluation" (Chemical Engineering Journal, 2021, 421, 127730), presents the possibility of using cold atmospheric pressure plasma generated inside vesicles with enlarged gas -liquid interfaces for degradation of antibiotic from aqueous solutions being in this case cefixime. Researchers used DBD as a source of cold atmospheric pressure plasma, generated between dielectric tubes in an atmosphere of air, oxygen, argon, or nitrogen. The number of bubbles produced using the plasma bubble reactor augmented with the increase in the number of microholes that regulated the interactions between cold atmospheric pressure plasma and the liquid. The authors observed that the strong electric field generated around the microholes contributes to a high electron ionization coefficient, as well as to increasing the electron energy in bubbles, igniting and sustaining the discharge, and thus elevating the efficiency of reactive oxygen and nitrogen species (RONS) production in these vesicles. The degradation of the studied antibiotic, i.e., cefixime, from the aqueous solution was largely dependent on the number of microholes, the discharge gas flow rate in the plasma system, the power of the discharge, the plasma exposure time as well as the concentration of the antibiotic in the solution. The scientists achieved a 94.8% degree of degradation of cefixime in a 10-micro-hole reactor, with an energy efficiency of 1.5 g kWh ¹, after treating this aqueous antibiotic solution for 30 minutes with DBD-type cold atmospheric pressure plasma. The highest degradation rate i.e. 99.8% and 94.8%, respectively, of the analyzed antibiotic was achieved by using DBD generated under the atmosphere of oxygen and air. Implementation of air as the discharge gas reduces the total cost of generating cold atmospheric pressure plasma and thus allows the proposed system to be applied in the industry. Unfortunately, the relatively long exposure time of such small volumes of antibiotic solutions to the plasma may turn out to cause too many difficulties in order to apply the presented method for degrading antibiotics at an industrial scale. Taking into account the stationary nature of that plasma reactor allowing for simultaneous exposure of only 50 mL of the antibiotic solution, in spite of the proven effectiveness, there are numerous scale -related limitations prior to implementation of this method into industry. Referring to the assessment of biological activity, Zhang et al. investigated the antibacterial and cytotoxic properties of the cold atmospheric pressure plasma-treated antibiotic solution. As a result of the conducted research, it was found that antibacterial properties of the cefixime solution were reduced as proven on Escherichia coli species when 1 pg and 3 pg of cefixime had been exposed for just one minute to the plasma source. The analysis of the cytotoxic properties of the plasma treated antibiotic solutions was carried out by scientists on 3 human breast cell lines, both cancer cell lines, i.e. MDA-MB-231 (human metastatic breast cancer line) and MCF-7 (human non-metastatic human breast cancer line) and non-tumor lines, i.e. MCF-10A (mammary epithelial line). Aqueous solution of cefixime at a dose of 1 pg showed cytotoxicity against the MDA- MB-231 and MCF-7 cell lines, with cell viability after incubation reaching approximately 74% and 82%, respectively. This may indicate that the antibiotic degradation products show stronger cytotoxic properties than the untreated antibiotic solutions. Thus, the above-mentioned research results disqualify the proposed method of antibiotic degradation from aqueous solutions from further considerations.

Bearing in mind the literature reports cited above related to the possibility of using cold atmospheric pressure plasma technology to degrade antibiotics from aqueous solutions, to the best of our knowledge, most of these scientific works concern the use of the developed methodologies to degrade only one or several single antibiotics from aqueous solutions. Scientific papers on the cold atmospheric pressure plasma treatment of antibiotic mixtures from aqueous solutions or sewage sludge have not been published yet. Furthermore, there are notable limitations related to the character of the current plasma systems, including their stationary modes, which significantly impede the possibility of their implementation into the wastewater treatment-associated industry. In addition, only few researchers attempted to estimate the antibacterial properties of the plasma-treated antibiotic solutions, thus the problem of widespread multi-drug resistance among pathogenic microorganisms towards humans and animals remains unresolved.

Referring to the patent literature, in the invention entitled: "Plasma treatment device with brush head" (W02020083992A1, 30.04.2020), the authors presented a plasma device dedicated to the treatment of surfaces. In the described plasma generation system, putatively a DBD barrier discharge initiated inside the device was utilized as the plasma source. In spite of the fact that the structure of the device at first glance seems to be similar to that of our invention, they are significantly different in terms of the construction details. Importantly, the applied atmosphere for plasma generation has not been defined, i.e. the discharge gases utilized for plasma ignition were not revealed. In addition, the device was designed by the authors in such a way to produce as many plasma cones as possible. In our solution, the number of plasma cones equals from four to five, in order to simultaneously expose the largest possible surface area of single antibiotic solutions or their mixtures to the plasma source. It is also worth mentioning that in the case of the invention of our design, it works in the form of flow reaction-discharge system, which quantifies its further applications.

Considering the invention entitled: "Plasma brush apparatus and method" (US2007116891A1, 24.05.2007), it is dedicated to, inter alia, plasma cutting, plasma cleaning, plasma deposition, and plasma sterilization. The device is not intended for use in environmental engineering, e.g. for waste water treatment. The construction scheme of the device, as well as its operating principle, differs significantly from the invention developed by our research group.

Taking into consideration both the literature and patent-related reports cited above, the methods presented in this patent application intended for decomposition of antibiotics with the use of unique flow plasma brushes relying either on pm-rf-APGD- or DBD-type discharges as cold atmospheric pressure plasma sources are herein proposed for the first time in the world. No other research group has developed or used flow- through plasma brushes to degrade antibiotics, or their mixtures, from aqueous solutions.

The essence of the invention is a method of decomposing antibiotics from aqueous solutions by using cold atmospheric pressure plasma - pm-rf-APGD or DBD, characterized in that by the following steps: a) the analyzed aqueous antibiotic solution or a mixture of antibiotics selected from the group of fluoroquinolones and/or tetracyclines and/or trimethoprims and/or chloramphenicols and/or P-lactams are introduced into the flow plasma brush by a connection attached to the chamber, b) the discharge gas is introduced through a wire equipped with a gas valve, c) the discharge gas is further transported in a pipe separated into at least 3 parts in the root, d) then a current is applied to the metal tubes with the use of a current generator, leading as a result to generation of cold atmospheric pressure plasma in the form of 4 or 5 cones of pm-rf-APGD or DBD, respectively. e) after treatment of the aqueous solution of antibiotics or the mixture of antibiotics with the pm-rf-APGD- or DBD-type cold atmospheric plasma, respectively, the solution is collected in a chamber, delivered outside through a stub pipe and subjected to further analysis.

Preferably, pm-rf-APGD-type cold atmospheric pressure plasma is generated at the atmospheric pressure under a helium atmosphere.

Preferably, a DBD-type cold atmospheric pressure plasma is generated at the atmospheric pressure under a helium-nitrogen atmosphere.

Preferably, the process is carried out in a chamber at a 2-20 mm distance, most preferably at a 10 mm distance, from the surface of the treated aqueous single antibiotic solution or their mixture.

Preferably, the process is carried out with the use of a pm-rf-APGD-type glow discharge generated by applying a current to metal tubes, with a current with a frequency of modulation of 500-2300 Hz and a duty cycle in the range of 10-90%.

Most preferably, a current with a frequency of modulation of 1700 Hz and a duty cycle of 30% is used.

Preferably, the process is carried out by using a DBD, generated by applying a current to metal tubes a current with a frequency of modulation of 1500-2300 Hz and duty cycle in the range of 10-90%. Most preferably, a current with a frequency of modulation of 1300 Hz and a duty cycle of 51.8% is utilized.

Preferably, the flow rate of the introduced aqueous antibiotic solution or the antibiotics mixture ranges from 0.5 to 20 mL min ¹.

Most preferably, the aqueous antibiotic solution or the antibiotics mixture is introduced at a flow rate of 3.0 mL min ¹ into the pm-rf-APGD-type plasma brush.

Most preferably, the aqueous antibiotic solution or the mixture of antibiotics is introduced at a flow rate of 1.0 mL min ¹ into a DBD-type plasma brush.

Preferably, the helium discharge gas is introduced at a flow rate of 1-10 L min ¹, for a pm-rf-APGD-type brush.

Most preferably, the helium discharge gas is introduced at a flow rate of 5.0 L min ¹, for a pm-rf-APGD-type brush.

Preferably, the helium discharge gas is introduced at a flow rate of 1-10 L min ¹ and nitrogen at a flow rate of 0.1-1 L min ¹, for a DBD-type brush.

Most preferably, the helium discharge gas is introduced at a flow rate of 7.0 L min ¹ and a nitrogen gas at a flow rate of 0.1 L min ¹ for a DBD-type brush.

Preferably, cold atmospheric pressure plasma in a DBD-type plasma brush is generated under the helium-nitrogen gases atmosphere in a ratio of 98.6% : 1.4%.

Preferably, the concentration of the antibiotic in an aqueous solution or the mixture of antibiotics ranges from 1 to 100 mg L '.

Preferably, a root made of epoxy resin is used.

Preferably, the metal tubes are made of brass.

Preferably, the chamber made of quartz is utilized.

The subject of the invention is also the flow plasma brush, characterized by a root into whose upper part a gas valve and a discharge gas supply conduit, divided in the middle part of the root into at least 3 parts, are connected, the lower part of the root is equipped with metal tubes from which, as a result of applying a current from a current generator, cold atmospheric pressure plasma is operated, the generated plasma is in the form of 4 or 5 pm-rf-APGD- or DBD-type cones, metal pipes are attached to the root with rubber hoses and link the chamber with a connection and a stub pipe, where the metal pipes are braided with copper wires, and the connection is braided with a wire.

Preferably, the root is made of epoxy resin.

Preferably the metal tubes are made of brass. Preferably, the chamber is made of quartz.

The advantage of the plasma flow brushes and the method, according to the invention, is the possibility of continuous degradation of antibiotics from aqueous solutions, which enables the implementation of this technology, for example, for treatment of wastewaters from the pharmaceutical or medical industry. Moreover, the possibility of generating more than one cold atmospheric pressure plasma cone significantly increases the reduction percentages of the analyzed pharmaceuticals.

The method according to the invention allows for degradation of antibiotics from single-component solutions or their mixtures with the use of pm-rf-APGD- or DBD-type cold atmospheric pressure plasma, leading to the reduction in the antibacterial properties of the plasma-treated solutions towards microorganisms from the genera Staphylococcus, Salmonella, Escherichia, Bacillus, Enterobacter, and Serratia.

The subject of the invention is presented in the form of five examples showing decomposition of antibiotics from aqueous solutions or their mixtures by using the pm-rf- APGD- or DBD-type plasma brushes, operating continuously under a helium (or heliumnitrogen) atmosphere at an atmospheric pressure. The subject of the invention is also disclosed in Table 1, which presents the conditions for treatment of a tetracycline antibiotic (doxycycline) with a pm-rf-APGD-type plasma brush and the results of the total organic carbon (TOC) and total nitrogen (TN) analyses. In addition, the subject of the invention is depicted in the Figure, in which:

- Fig. 1 presents a scheme of a pm-rf-APGD- or DBD-type plasma brush used for degradation of antibiotics, or their mixtures, from aqueous solutions.

- Fig. 2 presents a chart showing reduction in the antibacterial activity of ofloxacin towards Salmonella typhimurium ATCC 13311, Enterobacter cloacae ATCC 13047, Staphyloccocus aureus 25904, Staphyloccocus haemolyticus ATCC 29970, Escherichia coli ATCC 25922, and Bacillus subtilis 168, resulting from the treatment of the ofloxacin-containing solution with a pm-rf-APGD-type plasma brush. The reduction in the antibacterial properties of the pm-rf-APGD-treated antibiotic solution in comparison to the non-plasma-treated control solution was determined basing on the measured growth inhibition zones of the studied pathogens in a standard disc-diffusion assay.

- Fig. 3 presents a chart showing reduction in the antibacterial activity of ciprofloxacin towards S. typhimurium ATCC 13311, E. cloacae ATCC 13047, S. aureus 25904, S. haemolyticus ATCC 29970, E. coli ATCC 25922 and B. subtilis 168, resulting from the treatment of the ciprofloxacin-containing solution with a pm-rf-APGD-type plasma brush. The reduction in the antibacterial properties of the pm-rf-APGD-treated antibiotic solution in comparison to the non-plasma-treated control solution was determined basing on the measured growth inhibition zones of the studied pathogens in a standard disc-diffusion assay.

- Fig. 4 presents a chart showing reduction in the antibacterial activity of a mixture of ciprofloxacin, ofloxacin, enrofloxacin and doxycycline towards .S'. typhimurium ATCC 13311, E. cloacae ATCC 13047, S. aureus 25904, S. haemolyticus ATCC 29970, E. coll ATCC 25922 and B. subtilis 168, resulting from the treatment of the ciprofloxacin, ofloxacin, enrofloxacin, trimethoprim and doxycycline-containing solution with a pm-rf-APGD-type plasma brush. The reduction in the antibacterial properties of the pm-rf-APGD-treated antibiotics mixture in comparison to the non- plasma-treated control solution was determined basing on the measured growth inhibition zones of the studied pathogens in a standard disc-diffusion assay.

- Fig. 5 presents a chart showing reduction in the antibacterial activity of a mixture of chloramphenicol, doxycycline, ampicillin and ofloxacin towards E. cloacae ATCC 13047, E. coll ATCC 25922, Serratia marcescens ATCC 14756 and B. subtilis 168, resulting from the treatment of the chloramphenicol, doxycycline, ampicillin and ofloxacin -containing solution with a DBD-type plasma brush. The reduction in the antibacterial properties of the DBD-treated antibiotics mixture in comparison to the non- plasma-treated control solution was determined basing on the measured growth inhibition zones of the studied pathogens in a standard disc-diffusion assay.

Example 1. Method for degradation of a tetracycline antibiotic, i.e. doxycycline, with a use of a pm-rf-APGD-type plasma brush.

To degrade an antibiotic from the tetracycline group, i.e. doxycycline (in a concentration from 10 to 100 mg L '), with the use of a pm-rf-APGD-type cold atmospheric pressure plasma and, at the same time, carry out a multivariate optimization of the degradation process for this drug (according to Table 1), a doxycycline is introduced to a pm-rf-APGD-type plasma brush at a flow rate ranging from 3.0 to 10 mL min ¹ by a four-channel peristaltic pump (Masterflex L/S, Cole -Palmer, USA), using a silicone tube of 13 mm inner diameter. The above-mentioned silicone tube is connected to a port 8 supplying an antibiotic solution and to a quartz chamber 7. The receipt of the pm-rf-APGD-type cold plasma-treated antibiotic solution is led through a stub pipe 10, which collects the doxycycline aqueous solution. The main part of the pm-rf-APGD-type brush consists of a root 3 made of epoxy resin of 34.60 mm in diameter and provided with a 19.20 mm connection that is located in its upper part: the gas valve 2 and the 1 conduit supplying the discharge gas, i.e. helium. The helium is introduced into the pm-rf- APGD-type plasma brush at a flow rate of 5 L min ¹. There are four brass tubes 5 of 4.0 mm in diameter and 40.51 mm length each, which are fastened to the root 3. As a result of provision of a current, with a frequency of modulation of 1700 Hz and a duty cycle of 30-50%, from the current generator (Dora Electronic Equipment, Poland), 4 cones of pm- rf-APGD cold atmospheric pressure plasma 6 are generated. The above-mentioned brass tubes 5 are additionally attached to the root 3 with rubber hose lines 4. Additional elements of the system, without which initiation of pm-rf-APGD-type cold atmospheric pressure plasma would not be possible, are copper wires 9 entwining the brass tubes 5 and a connection 8 for supplying the doxycycline solution. After treatment of the doxycycline solution 4 with cold atmospheric pressure plasma cones 6, the solution of this antibiotic is collected in a quartz chamber 7, received through a port 10 and subjected to further analyze to determine the effectiveness of the cold atmospheric pressure plasma action for decomposition of this antibiotic.

To determine the decomposition efficiency of doxycycline caused by pm-rf- APGD-type cold atmospheric pressure plasma treatment according to the Box-Behnken design factor plan (Table 1), the Total Organic Carbon (TOC) and Total Nitrogen Contents (TN) analyses are to be performed. Table 1. The conditions applied for the treatment of doxycycline aqueous solutions with pm-rf-APGD-type cold atmospheric pressure plasma, ignited in the pm-rf-APGD-type plasma brush, according to the Box-Behnken design together with the determined TOC and TN concentrations (solutions treated and non-treated by plasma).

TOC: Total Organic Carbon in the analyzed sample [mg L'¹]

TN: Total Nitrogen Contents in the analyzed sample [mg L'¹]

The conducted research revealed that the pm-rf-APGD-type cold atmospheric pressure plasma treatment of doxycycline solutions results in changing of the values of TOC and TN (Table 1), which may indicate decomposition of this antibiotic. In most cases, we observe a decrease in the concentration of TOC in the pm-rf-APGD-type cold atmospheric pressure plasma-treated doxycycline solutions in comparison to the non- plasma-treated doxycycline solutions, which confirms degradation of this antibiotic. Referring to the concentration of TN, in all cases we noted an increase in this value when plasma-treated doxycycline solutions were contrasted with the non-treated ones, which may point to the formation of nitric acid and nitrous acid, i.e. HNO3 or/and HNO2 due to the impact of the pm-rf-APGD-type cold atmospheric pressure plasma application. Example 2. Method of degradation of a fluoroquinolone antibiotic, i.e. ofloxacin, at the concentration of 37 mg L¹ from an aqueous solution by application of a pm-rf-APGD- type plasma brush.

To degrade an antibiotic from a fluoroquinolone group, i.e. ofloxacin, with the use of a pm-rf-APGD-type cold atmospheric pressure plasma, this drug is introduced to a pm-rf-APGD-type plasma brush at a flow rate 3.0 mL min ¹ by a four-channel peristaltic pump (Masterflex L/S, Cole -Palmer, USA), using a silicone tube of 13 mm inner diameter. The above-mentioned silicone tube is connected to a port 8 supplying an antibiotic solution and to a quartz chamber 7. The receipt of the pm-rf-APGD-type cold plasma-treated antibiotic solution is led through a stub pipe 10, which collects the ofloxacin aqueous solution. The main part of the pm-rf-APGD-type brush consists of a root 3 made of epoxy resin of 34.60 mm in diameter and provided with a 19.20 mm connection that is located in its upper part: the gas valve 2 and the 1 conduit supplying the discharge gas, i.e. helium. The helium is introduced into the pm-rf-APGD-type plasma brush at a flow rate of 5 L min ¹. There are four brass tubes 5 of 4.0 mm in diameter and 40.51 mm length each, which are fastened to the root 3. As a result of provision of a current, with a frequency of modulation of 1700 Hz and duty cycle of 30-50%, from the current generator (Dora Electronic Equipment, Poland), 4 cones of pm-rf-APGD cold atmospheric pressure plasma 6 are generated. The above-mentioned brass tubes 5 are additionally attached to the root 3 with rubber hose lines 4. Additional elements of the system, without which initiation of pm-rf-APGD-type cold atmospheric pressure plasma would not be possible, are copper wires 9 entwining the brass tubes 5 and a connection 8 for supplying the ofloxacin solution. After treatment of the ofloxacin solution 4 with cold atmospheric pressure plasma cones 6, the solution of this antibiotic is collected in a quartz chamber 7, received through a port 10 and subjected to further analyzes to determine the decomposition rate and antibacterial properties of this cold atmospheric pressure plasma- treated antibiotic.

In order to determine the degree of ofloxacin degradation post treatment with cold atmospheric pressure plasma generated in a pm-rf-APGD plasma brush, high performance liquid chromatography (HPLC-DAD) is utilized.

Degradation efficiency of a fluoroquinolone antibiotic, i.e. ofloxacin, at a concentration of 37 mg L ¹ from an aqueous solution with the use of a pm-rf-APGD-type plasma brush: By application of the HPLC-DAD technique (Nexera XR, Shimadzu, Japan), it was confirmed that the use of a pm-rf-APGD-type plasma brush leads to 93.5% degradation of ofloxacin.

Aiming to determine the impact of a treatment with cold atmospheric pressure plasma generated in a pm-rf-APGD-type plasma brush on the antibacterial properties of an aqueous solution of ofloxacin, a standard disk diffusion assay is performed. The antibacterial properties of an antibiotic solution are determined towards human bacterial pathogens, i.e. S. typhimurium on the example of ATCC 13311 strain, E. cloacae on the example of ATCC 13047 strain, S. aureus on the example of ATCC 25904 strain, S. haemolyticus on the example of ATCC 29970, E. coll ATCC 25922, and B. subtilis on the example of strain 168. The investigated bacterial strains are kept in 40% (v/v) sterile glycerol solution at -80 °C. In order to obtain a single bacterial colony, bacterial biomass collected from a stock kept at -80 °C is streaked on Muller-Hinton Agar. The inoculated plate is incubated at 37 °C for 24 h. Then, by using a sterile loop, a single bacterial colony is picked to inoculate 5 ml of Muller-Hinton Broth liquid medium, which is incubated with shaking (140 rpm) for 16-20 h at 37 °C. The resultant overnight culture of the tested strain is centrifugated for 10 min at a rotation speed of 6500 rpm. The obtained bacterial pellet present at the bottom of the test tube is rinsed twice with sterile distilled water and centrifuged at 6500 rpm for 10 min, prior to resuspending bacterial cells in 200 mL of sterile distilled water. The optical density of the bacterial suspension is adjusted to 0.5 in McFarland (McF) scale (approx. 1.5 x 10⁸ cells per ml) with the use of a densitometer. The bacterial suspensions of S. typhimurium ATCC 13311, E. cloacae ATCC 13047 strain, S. aureus 25904, S. haemolyticus ATCC 29970, E. coli ATCC 25922, and B. subtilis 168 of the optical density of 0.5 McF prepared in the abovedescribed way are utilized to perform disc diffusion assays in order to determine antibacterial properties of 35 mg L ¹ ofloxacin solution treated with cold atmospheric plasma that was generated in a pm-rf-APGD-type plasma brush. A sterile swab is introduced into the previously prepared bacterial suspension. The excess of bacterial suspension is removed by pressing the swab towards the inside wall of the glass tube containing bacterial suspension and, subsequently, the collected on a swab bacterial suspension is spread three times over the whole surface of the Muller-Hinton Agar. Sterile paper discs of 5 mm in diameter, in a number of 2-4 discs per plate, are placed on the surface of the plate inoculated with bacteria, while a minimum distance of 2 cm between the discs is kept. On each plate there is one disc on which 10 pL of ofloxacin aqueous solution, treated by cold atmospheric pressure plasma that was generated in a pm-rf-APGD-type plasma brush, was poured. Also on each plate there is one disc that plays the role of a control sample and contains 10 pL of ofloxacin aqueous solution provided at the same concentration, which was not treated by cold atmospheric pressure plasma obtained in a pm-rf-APGD-type plasma brush. Afterwards, the so-prepared Muller-Hinton Agar plates are incubated at 4 °C for 1 h to allow for diffusion of the active ingredient into the medium. Post 1 hour, the plates are placed at 37 °C for 24 h. The presented experiment is carried out in three biological replications, each of them involving two technical repeats. The efficiency of ofloxacin deactivation is exhibited by a reduction in the diameter of the growth inhibition zones of the tested microorganisms, in this case the strains S. typhimurium ATCC 13311, E. cloacae ATCC 13047 strain, S. aureus 25904, S. haemolyticus ATCC 29970, E. coll ATCC 25922, and B. subtilis 168. The degradation efficiency of ofloxacin by using cold atmospheric pressure plasma generated in a pm-rf-APGD-type plasma brush in comparison to the control sample is shown in Fig. 2:

Decrease in the antibacterial properties of an aqueous ofloxacin solution at the concentration of 37 mg L ¹, which was treated with cold atmospheric pressure plasma, generated in a plasma brush of the pm-rf-APGD type, in comparison to the control sample.

After conducting the described experiments, it was found that the antibacterial properties of the aqueous solutions of ofloxacin treated by cold atmospheric pressure plasma dropped in relation to the analyzed microorganisms, i.e. S. typhimurium ATCC 13311 - a reduction of 51.5% was noted, E. cloacae ATCC 13047 - a reduction of 76.4% was observed, S. aureus 25904 - a reduction of 100% was acquired, S. haemolyticus ATCC 29970 - a reduction of 100% was obtained, E. coli ATCC 25922 - a reduction of 46.5% was revealed and B. subtilis 168 - a reduction of 80.3% was observed. Example 3. Method of degradation of a fluoroquinolone antibiotic, i.e. ciprofloxacin, at the concentration of 28 mg L¹ from an aqueous solution by application of a pm-rf- APGD-type plasma brush.

To degrade an antibiotic from a fluoroquinolone group, i.e. ciprofloxacin, with the use of a pm-rf-APGD-type cold atmospheric pressure plasma, this drug is introduced to a pm-rf-APGD-type plasma brush at a flow rate 3.0 mL min ¹ by a four-channel peristaltic pump (Masterflex L/S, Cole -Palmer, USA), using a silicone tube of 13 mm inner diameter. The above-mentioned silicone tube is connected to a port 8 supplying an antibiotic solution and to a quartz chamber 7. The receipt of the pm-rf-APGD-type cold plasma-treated antibiotic solution is led through a stub pipe 10. The main part of the pm- rf-APGD-type brush consists of a root 3 made of epoxy resin of 34.60 mm in diameter and provided with a 19.20 mm connection that is located in its upper part: the gas valve 2 and the 1 conduit supplying the discharge gas, i.e. helium. The helium is introduced into the pm-rf-APGD-type plasma brush at a flow rate of 5 L min ¹. There are four brass tubes 5 of 4.0 mm in diameter and 40.51 mm length each, which are fastened to the root 3. As a result of provision of a current, with a frequency of modulation of 1700 Hz and a duty cycle of 30-50%, from the current generator (Dora Electronic Equipment, Poland), 4 cones of pm-rf-APGD cold atmospheric pressure plasma 6 are generated. The above- mentioned brass tubes 5 are additionally attached to the root 3 with rubber hose lines 4. Additional elements of the system, without which initiation of pm-rf-APGD-type cold atmospheric pressure plasma would not be possible, are copper wires 9 entwining the brass tubes 5 and a connection 8 for supplying the ciprofloxacin solution. After treatment of the ciprofloxacin solution 4 with cold atmospheric pressure plasma cones 6, the solution of this antibiotic is collected in a quartz chamber 7, received through a port 10 and subjected to further analyzes to determine the decomposition rate and antibacterial properties of this cold atmospheric pressure plasma-treated antibiotic. In order to determine the degree of ciprofloxacin degradation post treatment with cold atmospheric pressure plasma generated in a pm-rf-APGD plasma brush, high performance liquid chromatography (HPLC-DAD) is utilized.

Degradation efficiency of a fluoroquinolone antibiotic, i.e. ciprofloxacin, at a concentration of 28 mg L ¹ from an aqueous solution with the use of a pm-rf-APGD-type plasma brush: By application of the HPLC-DAD technique (Nexera XR, Shimadzu, Japan), it was confirmed that the use of a pm-rf-APGD-type plasma brush leads to 95.9% degradation of ciprofloxacin.

Aiming to determine the impact of a treatment with cold atmospheric pressure plasma generated in a pm-rf-APGD-type plasma brush on the antibacterial properties of an aqueous solution of ciprofloxacin, a standard disk diffusion assay is performed. The antibacterial properties of an antibiotic solution are determined towards human bacterial pathogens, i.e. S. typhimurium on the example of ATCC 13311 strain, E. cloacae on the example of ATCC 13047 strain, S. aureus on the example of ATCC 25904 strain, S. haemolyticus on the example of ATCC 29970, E. coll ATCC 25922, and B. subtilis on the example of strain 168. The investigated bacterial strains are kept in 40% (v/v) sterile glycerol solution at -80 °C. In order to obtain a single bacterial colony, bacterial biomass collected from a stock kept at -80 °C is streaked on Muller-Hinton Agar. The inoculated plate is incubated at 37 °C for 24 h. Then, by using a sterile loop, a single bacterial colony is picked to inoculate 5 ml of Muller-Hinton Broth liquid medium, which is incubated with shaking (140 rpm) for 16-20 h at 37 °C. The resultant overnight culture of the tested strain is centrifugated for 10 min at a rotation speed of 6500 rpm. The obtained bacterial pellet present at the bottom of the test tube is rinsed twice with sterile distilled water and centrifuged at 6500 rpm for 10 min, prior to resuspending bacterial cells in 200 mL of sterile distilled water. The optical density of the bacterial suspension is adjusted to 0.5 in McFarland (McF) scale (approx. 1.5 x 10⁸ cells per ml) with the use of a densitometer. The bacterial suspensions of S. typhimurium ATCC 13311, E. cloacae ATCC 13047 strain, S. aureus 25904, S. haemolyticus ATCC 29970, E. coli ATCC 25922, and B. subtilis 168 of the optical density of 0.5 McF prepared in the abovedescribed way are utilized to perform disc diffusion assays in order to determine antibacterial properties of 28 mg L ¹ ciprofloxacin solution treated with cold atmospheric plasma that was generated in a pm-rf-APGD-type plasma brush. A sterile swab is introduced into the previously prepared bacterial suspension. The excess of bacterial suspension is removed by pressing the swab towards the inside wall of the glass tube containing bacterial suspension and, subsequently, the collected on a swab bacterial suspension is spread three times over the whole surface of the Muller-Hinton Agar. Sterile paper discs of 5 mm in diameter, in a number of 2-4 discs per plate, are placed on the surface of the plate inoculated with bacteria, while a minimum distance of 2 cm between the discs is kept. On each plate there is one disc on which 10 pl of ciprofloxacin aqueous solution, treated by cold atmospheric pressure plasma that was generated in a pm-rf-APGD-type plasma brush, was poured. Also on each plate there is one disc that plays the role of a control sample and contains 10 pL of ciprofloxacin aqueous solution provided at the same concentration, which was not treated by cold atmospheric pressure plasma obtained in a pm-rf-APGD-type plasma brush. Afterwards, the so-prepared Muller-Hinton Agar plates are incubated at 4 °C for 1 h to allow for diffusion of the active ingredient into the medium. Post 1 hour, the plates are placed at 37 °C for 24 h. The presented experiment is carried out in three biological replications, each of them involving two technical repeats. The efficiency of ciprofloxacin deactivation is exhibited by a reduction in the diameter of the growth inhibition zones of the tested microorganisms, in this case the strains S. typhimurium ATCC 13311, E. cloacae ATCC 13047 strain, S. aureus 25904, S. haemolyticus ATCC 29970, E. coll ATCC 25922, and B. subtilis 168. The degradation efficiency of ciprofloxacin by using cold atmospheric pressure plasma generated in a pm-rf-APGD-type plasma brush in comparison to the control sample is shown in Fig. 3:

Decrease in the antibacterial properties of an aqueous ciprofloxacin solution at the concentration of 28 mg L ¹, which was treated with cold atmospheric pressure plasma, generated in a plasma brush of the pm-rf-APGD type, in comparison to the control sample.

After conducting the described experiments, it was found that the antibacterial properties of the aqueous solutions of ciprofloxacin treated by cold atmospheric pressure plasma dropped in relation to the analyzed microorganisms, i.e. S. typhimurium ATCC 13311 - a reduction of 36.3% was noted, E. cloacae ATCC 13047 - a reduction of 39.4% was observed, S. aureus 25904 - a reduction of 100% was acquired, S. haemolyticus ATCC 29970 - a reduction of 100% was obtained, E. coli ATCC 25922 - a reduction of 38.2% was revealed, and B. subtilis 168 - a reduction of 62.8% was observed.

Example 4. Method of degradation of a mixture of antibiotics, including ciprofloxacin, ofloxacin, enrofloxacin, doxycycline and trimethoprim, from an aqueous solution by application of a pm-rf-APGD-type plasma brush.

To degrade antibiotics from a fluoroquinolone group, i.e. ciprofloxacin, ofloxacin, enrofloxacin, from a tetracycline group, i.e. doxycycline, and trimethoprim (at a concentration of: 7.5 mg L ¹- ciprofloxacin, 10.7 mg L 'ofloxacin, 10.5 mg L ¹ enrofloxacin, 7.4 mg L¹- doxycycline, and 5.3 mg L ¹ - trimethoprim), this mixture is introduced to a pm-rf-APGD-type plasma brush at a flow rate 3.0 mL min ¹ by a four- channel peristaltic pump (Masterflex L/S, Cole-Palmer, USA), using a silicone tube of 13 mm inner diameter. The above-mentioned silicone tube is connected to a port 8 supplying a mixture of antibiotics and to a quartz chamber 7, which collects the cold atmospheric pressure plasma-treated aqueous solution of ciprofloxacin, ofloxacin, enrofloxacin, doxycycline and trimethoprim. The receipt of the pm-rf-APGD-type cold plasma-treated antibiotics-containing solution is led through a stub pipe 10. The main part of the pm-rf- APGD-type brush consists of a root 3 made of epoxy resin of 34.60 mm in diameter and provided with a 19.20 mm connection that is located in its upper part: the gas valve 2 and the 1 conduit supplying the discharge gas, i.e. helium. The helium is introduced into the pm-rf-APGD-type plasma brush at a flow rate of 5 L min ¹. There are four brass tubes 5 of 4.0 mm in diameter and 40.51 mm length each, which are fastened to the root 3. As a result of provision of a current, with a frequency of modulation of 1700 Hz and a duty cycle of 30-50%, from the current generator (Dora Electronic Equipment, Poland), 4 cones of pm-rf-APGD cold atmospheric pressure plasma 6 are generated. The above- mentioned brass tubes 5 are additionally attached to the root 3 with rubber hose lines 4. Additional elements of the system, without which initiation of pm-rf-APGD-type cold atmospheric pressure plasma would not be possible, are copper wires 9 entwining the brass tubes 5 and a connection 8 for supplying the antibiotics-containing solution. After treatment of the antibiotics mixture 4 with cold atmospheric pressure plasma cones 6, the antibiotics-containing solution is collected in a quartz chamber 7, received through a port 10 and subjected to further analyze to determine the decomposition rate and antibacterial properties of these cold atmospheric pressure plasma-treated drugs. In order to determine the degree of degradation of each antibiotic in the mixture post treatment with cold atmospheric pressure plasma generated in a pm-rf-APGD plasma brush, high performance liquid chromatography (HPLC-DAD) is utilized. Degradation efficiency of fluoroquinolone antibiotics, i.e. ciprofloxacin, ofloxacin, enrofloxacin, tetracycline antibiotic, i.e. doxycycline, and trimethoprim with the use of a pm-rf-APGD-type plasma brush:

By application of the HPLC-DAD technique (Nexera XR, Shimadzu, Japan), it was confirmed that the use of a pm-rf-APGD-type plasma brush leads to degradation of ciprofloxacin, ofloxacin, enrofloxacin, doxycycline, trimethoprim from an antibiotics mixture in 95.9%, 95.5%, 97.0%, 100%, and 91.4%, respectively.

Aiming to determine the impact of a treatment with cold atmospheric pressure plasma generated in a pm-rf-APGD-type plasma brush on the antibacterial properties of an antibiotics mixture, containing ciprofloxacin, ofloxacin, enrofloxacin, doxycycline and trimethoprim in concentrations as listed above, a standard disk diffusion assay is performed. The antibacterial properties of an antibiotics mixture are determined towards human bacterial pathogens, i.e. S. typhimurium on the example of ATCC 13311 strain, E. cloacae on the example of ATCC 13047 strain, S. aureus on the example of ATCC 25904 strain, S. haemolyticus on the example of ATCC 29970, E. coli ATCC 25922 and B. subtilis on the example of strain 168. The investigated bacterial strains are kept in 40% (v/v) sterile glycerol solution at -80 °C. In order to obtain a single bacterial colony, bacterial biomass collected from a stock kept at -80 °C is streaked on Muller-Hinton Agar. The inoculated plate is incubated at 37 °C for 24 h. Then, by using a sterile loop, a single bacterial colony is picked to inoculate 5 ml of Muller-Hinton Broth liquid medium, which is incubated with shaking (140 rpm) for 16-20 h at 37 °C. The resultant overnight culture of the tested strain is centrifugated for 10 min at a rotation speed of 6500 rpm. The obtained bacterial pellet present at the bottom of the test tube is rinsed twice with sterile distilled water and centrifuged at 6500 rpm for 10 min, prior to resuspending bacterial cells in 200 mL of sterile distilled water. The optical density of the bacterial suspension is adjusted to 0.5 in McFarland (McF) scale (approx. 1.5 x 10⁸ cells per ml) with the use of a densitometer. The bacterial suspensions of S. typhimurium ATCC 13311, E. cloacae ATCC 13047 strain, S. aureus 25904, S. haemolyticus ATCC 29970, E. coli ATCC 25922, and B. subtilis 168 of the optical density of 0.5 McF prepared in the above -described way are utilized to perform disc diffusion assays in order to determine antibacterial properties of an antibiotics mixture, containing ciprofloxacin, ofloxacin, enrofloxacin, doxycycline and trimethoprim each at a concentration of 10 mg L ¹ in an aqueous solution, treated with cold atmospheric plasma that was generated in a pm-rf- APGD-type plasma brush. A sterile swab is introduced into the previously prepared bacterial suspension. The excess of bacterial suspension is removed by pressing the swab towards the inside wall of the glass tube containing bacterial suspension and, subsequently, the collected on a swab bacterial suspension is spread three times over the whole surface of the Muller-Hinton Agar. Sterile paper discs of 5 mm in diameter, 4 discs per plate, are placed on the surface of the plate inoculated with bacteria, while a minimum distance of 2 cm between the discs is kept. On each plate there is one disc on which 10 pL of an aqueous antibiotics mixture, including ciprofloxacin, ofloxacin, enrofloxacin, doxycycline and trimethoprim, treated by cold atmospheric pressure plasma that was generated in a pm-rf-APGD-type plasma brush, was poured. Also on each plate there is one disc that plays the role of a control sample and contains 10 pL of an aqueous antibiotics mixture, including ciprofloxacin, ofloxacin, enrofloxacin, doxycycline and trimethoprim, which was not treated by cold atmospheric pressure plasma obtained in a pm-rf-APGD-type plasma brush. Afterwards, the so-prepared Muller-Hinton Agar plates are incubated at 4 °C for 1 h to allow for diffusion of the active ingredients into the medium. Post 1 hour, the plates are placed at 37 °C for 24h. The presented experiment is carried out in three biological replications, each of them involving two technical repeats. The efficiency of antibiotics deactivation from a mixture is exhibited by a reduction in the diameter of the growth inhibition zones of the tested microorganisms, in this case the strains S. typhimurium ATCC 13311, E. cloacae ATCC 13047 strain, S. aureus 25904, S. haemolyticus ATCC 29970, E. coll ATCC 25922, and B. subtilis 168. The degradation efficiency of antibiotics from a mixture by using cold atmospheric pressure plasma generated in a pm-rf-APGD-type plasma brush in comparison to the control sample is shown in Fig. 4:

Decrease in the antibacterial properties of an antibiotics mixture, involving ciprofloxacin, ofloxacin, enrofloxacin, doxycycline and trimethoprim, which was treated with cold atmospheric pressure plasma, generated in a plasma brush of the pm-rf-APGD type, in comparison to the control sample.

After conducting the described experiments, it was found that the antibacterial properties of the mixture of antibiotics, containing ciprofloxacin, ofloxacin, enrofloxacin, doxycycline and trimethoprim, treated by cold atmospheric pressure plasma dropped in relation to the analyzed microorganisms, i.e. S. typhimurium ATCC 13311 - a reduction of 41.1% was noted, E. cloacae ATCC 13047 - a reduction of 54.9.4% was observed, S. aureus 25904 - a reduction of 71.7% was acquired, S. haemolyticus ATCC 29970 - a reduction of 64.4% was obtained, E. coli ATCC 25922 - a reduction of 38.7% was revealed and B. subtilis 168 - a reduction of 56.9% was observed.

Example 5. Method of degradation of antibiotics from an aqueous solution, containing ofloxacin, doxycycline, chloramphenicol and ampicillin at a concentration of 10 mg L ¹ by application of a DBD-type plasma brush.

To degrade antibiotics from an aqueous solution containing ofloxacin, doxycycline, chloramphenicol and ampicillin each at the concentration of 10 mg L ', this mixture is introduced to a DBD-type plasma brush at a flow rate 1.0 mL min ¹ by a four- channel peristaltic pump (Masterflex L/S, Cole-Palmer, USA), using a silicone tube of 13 mm inner diameter. The above-mentioned silicone tube is connected to a port 8 supplying a mixture of antibiotics and to a quartz chamber 7, which collects the cold atmospheric pressure of DBD type plasma-treated antibiotics mixture containing ofloxacin, doxycycline, chloramphenicol and ampicillin. The main part of the DBD-type brush consists of a root 3 made of epoxy resin of 34.60 mm in diameter and provided with a 19.20 mm connection that is located in its upper part: the gas valve 2 and the 1 conduit supplying the discharge gas, mixture of helium and nitrogen in 98.6% : 1.4% ratio. The helium is introduced into the DBD-type plasma brush at a flow rate of 7 L min ¹, while nitrogen at a flow rate of 0.1 L min ¹. There are four brass tubes 5 of 4.0 mm in diameter and 40.51 mm length each, which are fastened to the root 3. As a result of provision of a current, with a frequency of modulation of 1300 Hz and a duty cycle of 51.8%, from the current generator (Dora Electronic Equipment, Poland), 5 cones of DBD-type cold atmospheric pressure plasma 6 are generated. The above-mentioned brass tubes 5 are additionally attached to the root 3 with rubber hose lines 4. Additional elements of the system, without which initiation of DBD-type cold atmospheric pressure plasma would not be possible, are copper wires 9 entwining the brass tubes 5 and a connection 8 for supplying the antibiotics-containing solution. After treatment of the antibiotics mixture with five cold atmospheric pressure plasma cones of DBD-type 6, the solution is collected in a quartz chamber 7, received through a port 10 and subjected to further analyzes to determine the decomposition rate and antibacterial properties of these cold atmospheric pressure plasma-treated drugs. In order to determine the degree of degradation of each antibiotic in the mixture post treatment with cold atmospheric pressure plasma generated in a DBD-type plasma brush, analyses based on high performance liquid chromatography (HPLC-DAD) or liquid chromatography with mass detector (LC-MS) are performed.

Degradation efficiency of antibiotics from a mixture containing ofloxacin, doxycycline, chloramphenicol and ampicillin, with the use of a DBD-type plasma brush:

By application of the HPLC-DAD technique (Nexera XR, Shimadzu, Japan), it was confirmed that the use of a DBD-type plasma brush leads to degradation of antibiotics, i.e. ofloxacin, doxycycline and chloramphenicol, from a 3-component mixture in 62.4%, 65.7%, and 49.8%, respectively.

By application of the LC-MS (Agilent Technologies, USA) technique, it was confirmed that the use of a DBD-type plasma brush leads to degradation of ampicillin in 62.6% from an antibiotics mixture. In the case of ampicillin, it was necessary to apply the analytical technique LC-MS instead of HPLC-DAD, as ampicillin yields no absorbance in the UV/Vis range.

Aiming to determine the impact of a treatment with cold atmospheric pressure plasma generated in a flow-through DBD-type plasma brush on the antibacterial properties of an antibiotics mixture, containing ofloxacin, doxycycline, chloramphenicol and ampicillin in concentrations as listed above, a standard disk diffusion assay is performed. The antibacterial properties of an antibiotics mixture are determined towards human bacterial pathogens, i.e. E. cloacae on the example of ATCC 13047 strain, S. marcescens on the example of ATCC 14756 strain, E. coli ATCC 25922, and B. subtilis on the example of strain 168. The investigated bacterial strains are kept in 40% (v/v) sterile glycerol solution at -80 °C. In order to obtain a single bacterial colony, bacterial biomass collected from a stock kept at -80 °C is streaked in a reductive manner on Muller-Hinton Agar. The inoculated plate is incubated at 37 °C for 24 h. Then, by using a sterile loop, a single bacterial colony is picked to inoculate 5 mL of Muller-Hinton Broth liquid medium, which is incubated with shaking (140 rpm) for 16-20 h at 37 °C. The resultant overnight culture of the tested strain is centrifugated for 10 min at a rotation speed of 6500 rpm. The obtained bacterial pellet present at the bottom of the test tube is rinsed twice with sterile distilled water and centrifuged at 6500 rpm for 10 min, prior to resuspending bacterial cells in 200 mL of sterile distilled water. The optical density of the bacterial suspension is adjusted to 0.5 in McFarland (McF) scale (approx. 1.5 x 10⁸ cells per ml) with the use of a densitometer. The bacterial suspensions of E. cloacae ATCC 13047, S. marcescens ATCC 14756, E. coli ATCC 25922 and B. subtilis 168 of the optical density of 0.5 McF prepared in the above -described way are utilized to perform disc diffusion assays in order to determine antibacterial properties of an antibiotics mixture, containing ofloxacin, doxycycline, chloramphenicol and ampicillin each at a concentration of 10 mg L ¹ in an aqueous solution, treated with cold atmospheric plasma that was generated in a DBD-type plasma brush. A sterile swab is introduced into the previously prepared bacterial suspension. The excess of bacterial suspension is removed by pressing the swab towards the inside wall of the glass tube containing bacterial suspension and, subsequently, the collected on a swab bacterial suspension is spread three times over the whole surface of the Muller-Hinton Agar. Sterile paper discs of 5 mm in diameter, 4 discs per plate, are placed on the surface of the plate inoculated with bacteria, while a minimum distance of 2 cm between the discs is kept. On each plate there is one disc on which 10 pL of an aqueous antibiotics mixture, including ofloxacin, doxycycline, chloramphenicol and ampicillin, treated by cold atmospheric pressure plasma that was generated in a DBD-type plasma brush, was poured. Also on each plate there is one disc that plays the role of a control sample and contains 10 pL of an aqueous antibiotics mixture, including ofloxacin, doxycycline, chloramphenicol and ampicillin, which were not treated by cold atmospheric pressure plasma obtained in a DBD-type plasma brush. Afterwards, the so-prepared Muller-Hinton Agar plates are incubated at 4 °C for 1 h to allow for diffusion of the active ingredients into the medium. Post 1 hour, the plates are placed at 37 °C for 24 h. The presented experiment is carried out in three biological replications, each of them involving two technical repeats. The efficiency of antibiotics deactivation from a mixture is exhibited by a reduction in the diameter of the growth inhibition zones of the tested microorganisms, in this case the strains E. cloacae ATCC 13047, S. marcescens ATCC 14756, E. coll ATCC 25922 and B. subtilis 168. The degradation efficiency of antibiotics from a mixture by using cold atmospheric pressure plasma generated in a DBD-type plasma brush in comparison to the control sample is shown in Fig. 5:

Decrease in the antibacterial properties of an antibiotics mixture, involving ofloxacin, doxycycline, chloramphenicol and ampicillin each at an initial concentration of 10 mg L¹, which was treated with cold atmospheric pressure plasma, generated in a plasma brush of the DBD-type, in comparison to the control sample.

After conducting the described experiments, it was found that the antibacterial properties of the mixture of antibiotics, containing ofloxacin, doxycycline, chloramphenicol and ampicillin, treated by cold atmospheric pressure plasma dropped in relation to the analyzed microorganisms, i.e. E. cloacae ATCC 13047 strain - a reduction of 77.8% was noted, S. marcescens ATCC 14756 - a reduction at the level of 100% was observed, E. coll ATCC 25922 - a reduction of 38.2% was acquired and B. subtilis 168 - a reduction of 39.6% was revealed.

Claims

Patent claims

1. The method of decomposing antibiotics from aqueous solutions by using cold atmospheric pressure plasma - pm-rf-APGD or DBD, characterized in that by the following steps: a) the analyzed aqueous antibiotic solution or a mixture of antibiotics selected from the group of fluoroquinolones and/or tetracyclines and/or trimethoprims and/or chloramphenicols and/or P-lactams, are introduced into the flow plasma brush by a connection (8) attached to the chamber (7), b) the discharge gas is introduced through a wire (1) equipped with a gas valve (2), c) the discharge gas is further transported in a pipe (1) separated into at least 3 parts in the root (3), d) then a current is applied to the metal tubes (5) with the use of a current generator, leading as a result to generation of cold atmospheric plasma (6) in the form of 4 or 5 cones of pm-rf-APGD cold plasma or DBD, respectively. e) after treatment of the aqueous solution of antibiotics or the mixture of antibiotics with the pm-rf-APGD- or DBD-type cold plasma (6), respectively, the solution is collected in a chamber (7), delivered outside through a stub pipe (10) and subjected to further analysis.

2. The method according to Claim 1, characterized in that the cold atmospheric pressure plasma (6) of pm-rf-APGD type is generated at the atmospheric pressure under a helium atmosphere.

3. The method according to Claim 1, characterized in that the cold atmospheric pressure plasma (6) of DBD type is generated at the atmospheric pressure under a helium-nitrogen atmosphere.

4. The method according to Claim 1, characterized in that the process is carried out in a chamber (7) at a 2-20 mm distance from the surface of the treated aqueous single antibiotic solution or their mixture.

5. The method according to Claim 4, characterized in that the process is carried out at a 10 mm distance from the surface of the treated aqueous single antibiotic solution or their mixture.

6. The method according to Claim 1, characterized in that the process is carried out with the use of a pm-rf-APGD-type glow discharge generated by applying a current to metal tubes (5) with a current with a frequency of modulation in the range 500-2300 Hz and duty cycle in the range of 10-90%. The method according to Claim 6, characterized in that a current with a frequency of modulation of 1700 Hz and a duty cycle of 30% is used. The method according to Claim 1, characterized in that the process is carried out by using a DBD-type glow discharge, generated by applying a current to metal tubes (5) with a current with a frequency of modulation of 1500-2300 Hz and duty cycle in the range of 10-90%. The method according to Claim 8, characterized in that the current with a frequency of modulation of 1300 Hz and a duty cycle of 51.8% is utilized. The method according to Claim 1, characterized in that the flow rate of the introduced aqueous antibiotic solution or the antibiotics mixture ranges from 0.5 to 20 mL min ¹. The method according to Claim 10, characterized in that the aqueous antibiotic solution or the antibiotics mixture is introduced at a flow rate of 3.0 mL min ¹ into the pm-rf-APGD-type plasma brush The method according to Claim 10, characterized in that the aqueous antibiotic solution or the mixture of antibiotics is introduced at a flow rate of 1.0 mL min ¹ into a DBD-type plasma brush. The method according to Claim 1, characterized in that the helium discharge gas is introduced at a flow rate of 1-10 L min ¹ for a pm-rf-APGD-type brush. The method according to Claim 13, characterized in that the helium discharge gas is introduced at a flow rate of 5.0 L min ¹, preferably. The method according to Claim 1, characterized in that the helium discharge gas is introduced at a flow rate of 1-10 L min ¹ and nitrogen at a flow rate of 0.1- 1 L min ¹ for a DBD-type brush. The method according to Claim 15, characterized in that the helium discharge gas is introduced preferably at a flow rate of 7.0 L min ¹ and a nitrogen gas at a flow rate from 0.1 L min ¹. The method according to Claim 1, characterized in that the concentration of the antibiotic in an aqueous solution or the mixture of antibiotics ranges from 1 to 100 mg L '. The method according to Claim 1, characterized in that a root (3) made of epoxy resin is used. The method according to Claim 1, characterized in that the metal tubes (5) are made of brass. The method according to Claim 1, characterized in that the chamber (7) made of quartz is utilized. The flow plasma brush, characterized in that it contains a root (3) into whose upper part a gas valve (2) and a discharge gas supply conduit (1), divided in the middle part of the root (3) into at least 3 parts, are connected, the lower part of the root (3) is equipped with metal tubes (5) from which, as a result of applying a current from a current generator, cold atmospheric pressure plasma (6) is generated, the generated plasma (6) is in the form of 4 or 5 pm-rf-APGD- or DBD-type cones, metal pipes (5) are attached to the root (3) with rubber hoses (4) and link the chamber (7) with a connection (8) and a stub pipe (10), where the metal pipes (5) are braided with copper wires (9). The flow plasma brush according to Claim 21, characterized in that the root (3) is made of epoxy resin. The flow plasma brush according to Claim 21, characterized in that the metal tubes (5) are made of brass. The flow plasma brush according to Claim 21, characterized in that the chamber (7) is made of quartz.