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DEVELOPMENT OF MICROBICIDE EQUIPMENT AND RESEARCH IN PATHOGEN INACTIVATION BY COLD ARGON PLASMA
Aleksandr Petrovich SEMENOV1, Bair Batoevich BALDANOV1, Cyrempil Valerevich RANZHUROV1, Erdem Olegovich NIKOLAEV1, Sayana Vladimirovna GOMBOEVA2
1 Institute of Physical Material Science of SB RAS 670047, Ulan-Ude, Sakhyanova's str., 6
2 East Siberia State University of Technology and Management 670013, Ulan-Ude, Klyuchevskaya str., 40B, bldg. 1
The high bactericidal effect of cold argon plasma generated by low-current spark discharge plasma jets at atmospheric pressure has been revealed. It has been found that the direct contact of low-current spark plasma jets helps perform effective inactivation of bacteria.
Keywords: argon plasma, microorganisms, low-current spark, plasma jets, plasma inactivation, atmospheric pressure glow discharge.
Currently, an increasing focus rests on the researches in the gas-discharge process properties that determine their sterilizing and disinfecting abilities for protection of industrial materials, equipment and electronics from biodegradation and microbiologi-cally induced corrosion [5, 12]. Plasma treatment of living tissues has the desired therapeutic sterilization, wound healing, hemostasis, and skin disease curing effects [4, 6]. This area has become of particular importance in recent years due to the increasing demand for new low-temperature efficient, reliable and high-performance sterilization and decontamination technologies.
A special place among plasma methods is occupied by the study of discharges generating the low-temperature (cold) non-equilibrium plasma at atmospheric pressure [1, 7-11, 13]. As the sources of low-temperature non-equilibrium atmospheric pressure plasma various types of gas discharges are viewed, among which the atmospheric pressure creeping, corona, barrier and pulsed discharges should be noted. Despite the broad range of works [9, 10], dedicated to the study of different discharge characteristics, and proven high efficiency of discharges in
biomedical laboratory applications, the atmospheric pressure cold plasma antiseptic treatment has not been practiced widely. This is due, firstly, to the fact that the cold plasma sources currently represent technically sophisticated equipment with low economic efficiency. Secondly, for the treatment of bioobjects, living tissues of animals and humans atmospheric pressure discharges are used at high voltage of 10-40 kV, which requires a high security level. Therefore, the choice of discharge parameters under which a safe and non-destructive effect is observed, is one of the main physical problems of plasma medicine.
The aim of this paper is the study of bactericidal properties of low-temperature non-equilibrium argon plasma generated by high-voltage low-current discharges.
EXPERIMENTAL PROCEDURE
To generate low-temperature (cold) argon plasma two types of non-equilibrium plasma generators have been developed on the basis of low-current high-voltage discharges.
Semyonov A.P. - doctor of technical sciences, professor, director, e-mail: semenov@ipms.bscnet.ru
Baldanov B.B. - candidate of technical sciences, senior researcher of laboratory for plasma power processes and
technologies, mail: baibat@mail.ru
Ranzhurov Ts. V. - junior researcher of laboratory for plasma power processes and technologies Nikolaev E.O. - postgraduate student
Gomboeva S.V. - candidate of biological sciences, docent of the chair for biotechnology
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Fig. 1. Schematic representation of electrode structure. A: 1 - cathode; 3 - ballast resistances; 3 - anode; 4 - power supply; B: 1 - point cathode; 2 - cylindrical anode; 3 - ballast resistance; 4 - power supply
An approach is based on the use of atmospheric pressure glow discharge. A discharge is generated in a special electrode construction (Fig. 1A) with a multipoint divided cathode and flat metal anode [2]. The stability of discharge in the transition of negative corona to discharge gap spark breakdown is achieved via low gas flow through the discharge gap.
The other approach (Fig. 1B) is based on the use of atmospheric pressure low-current spark discharge plasma jets formed in an argon flux [11]. Cathode 1 (point with a radius of curvature r = 30 ^m) is placed in a cylindrical nonconducting housing R = = 2 cm. Anode 2 is a metal cylinder 1.5 cm long and 2 cm in diameter. To stabilize the discharge the point was loaded with adjustable ballast resistance 3 Rb > 1MQ. The housing has through holes for argon flux supply arranged so that cold argon plasma formed by a system of low-current spark plasma jets is spread via the argon flux in a direction away from the interelectrode spacing, like a flame. Argon consumption G = 5 x 10-5 kg/s was measured using a flowmeter PM - A - 0,16 GUZ.
The study involved the Escherichia coli M17 strains in the natural association and vegetative form. The microbial treatment efficiency in an atmospheric pressure glow gas-discharge chamber was evaluated using metal wafers (test strips). The decontamination completeness was determined by placing the test strips in an indicator medium.
To evaluate the sensitivity of microorganisms to cold argon plasma generated by low-current spark discharge plasma jets a method was used based on measuring the diameter of the microbial environment affected areas. Therefore, a medium was inoculated with test microorganisms. 100 (j,l of working suspension were added into a Petri dish with nutrient medium Agar GRM or RPA, and carefully rubbed with a spreader. The plates with medium inoculated with microorganisms were placed in a discharge chamber under plasma jets. Plasma-treated media were incubated in an incubator within 24
hours at 37 °C, and then the diameters of formed affected areas were measured.
RESULTS AND DISCUSSION
The bactericidal efficacy of diffuse argon plasma formed in the repetitively pulsed negative corona and atmospheric pressure glow discharge mode (Fig. 1A) was tested in the bacteria of natural association of microorganisms. Test strips with microorganisms were placed on a flat anode, the plasma treatment time of wafers ranged from 2 to 5 min.
The study of bacterial survival in the repetitively-pulsed negative corona plasma showed that after treating the wafers for 2 and 5 min (discharge current I = 250 ^A) bacteria perish totally (tubes 1, 2 Fig. 2). In the atmospheric pressure glow discharge mode (discharge current I = 700 ^A, treatment time t = 2 and 5 min), where the discharge is homogeneous self-sustained discharges covering the
Fig. 2. Results of bactericidal effect of cold argon plasma. N - untreated wafer; K - control; 1, 2 - pulsed corona treatment, t = 5 and 2 min, respectively; 3, 4 - atmospheric pressure glow discharge treatment, t = 5 and 2 min, respectively
A
Ü
p
Fig. 3. Cold argon plasma generator based on low-current spark plasma jets: A - microorganism inactivation in a Petri dish, B - side view of discharge
Fig. 5. Cold argon plasma influence on the vegetative form of the Escherichia coli strain. Exposure time = 30 s. Test medium on the left
entire interelectrode spacing, bacteria perish totally as well (tubes 3, 4 Fig. 2). Microbial growth after cold argon plasma treatment is absent for the seven days of test strips cultivation in the liquid medium.
The bactericidal properties of low-current spark discharge plasma jets were studied for their effects on the vegetative form of the Escherichia coli
strains. Fig. 3 shows a picture of low-current spark discharge cold argon plasma jet generator. Low-current spark discharge plasma jetting time ranged from 5 to 60 s. Distance h from the plasma source to the surface of microorganism growth ranged from 0.5 to 3 cm.
The current-voltage characteristic of discharge is dropping, and the nature of current flow in the plasma channel is an established mode of periodical current pulses (Fig. 4). In the formation of discharge current pulse two specific areas can be marked out: the initial narrow peak with amplitude Im ~ 280 ^A (area 1) and the second longer area (T ~ 70 ^s) that essentially determines the period T of discharge current pulse. The low-current spark plasma jetting influence on microorganisms is registered as round transparent areas, which are the microorganism growth inactivation zones (Fig. 5).
The bactericidal properties of low-temperature non-equilibrium argon plasma generated by plasma jets have been studied for the influence on the Escherichia coli strain vegetative forms compared to antibiotics of various inhibitory effect.
It has been shown that the effect of different antibiotics on the Escherichia coli bacteria for 18 h results in different affected areas depending on the toxicity of antibiotic. In the case of plasma inactivation, a 30-second treatment causes complete destruction of bacteria on an area (S = 2 cm2) substantially equal to that referring to the most toxic antibiotics.
The data obtained show high sensitivity of microorganisms to cold argon plasma treatment (Fig. 6) [11]. Minimal plasma inactivation time of the E. coli cells is 5 s at 0.5 cm from the side section of electrode structure. At a distance increased to 3 cm the number of survived microorganism macrocolonies is considerably greater. Increasing the plasma treatment time up to 40 s at a distance of 3 cm causes a considerable reduction (by 74 %) of survived microorganisms (Fig. 7, 2). The determination of argon plasma inactivation ability carried out by counting the colonies shows that after a minute of treatment only a few grown-up microorganism macrocolonies survive.
It should be noted that the inactivation area is not restricted by the anode diameter, within which low-current spark plasma jet are formed. As it can be seen in Fig. 8, increasing the time of microbial environment treatment with low-current spark plasma jets allows efficient inactivation of a much larger area. At that, the diameter of inactivation area in direct contact of plasma jets with the surface of mi-crobial environment increases more than in remote exposure. The results obtained correspond with the results shown in [3]. So the diameter of inactivation area increases with increasing time of treatment of
Fig. 4. Low-voltage spark discharge current pulses Interelectrode spacing d = 1,25 cm, amperage I = 500 pA, R6 = 21 UQ; [I] = 100 pA/div, [t] = = 0.2 ms/div
Fig. 6. Bacterial growth inactivation areas h = 0.5 cm
Treatment time t = 4 min, distance
Pseudomonas aeruginosa bacteria on solid agar with atmospheric pressure cold plasma jets (99.5 % helium and 0.5 % oxygen).
CONCLUSION
The high bactericidal efficiency of low-current high-voltage discharge cold argon plasma is displayed. The study of bacterial survival in the repetitively-pulsed negative corona plasma and atmospheric pressure glow discharge showed that after treating the wafers for 2 min in each mode the microorganisms are totally inactivated. It has been found, that increasing the time of microbial environment treatment with low-current spark plasma jets allows efficient inactivation of a much larger area than the anode electrode cross section.
This work was supported by the Russian Foundation for Basic Research (project no. 15-44-04209 r_sibir_a).
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LJ
Fig. 7. E. coli growth inactivation areas in relation to h: a - h = 0.5 cm, t = 30 s; b - h = 3 cm, t = 40 s
S, cm2
20-
15-
10-
0 20 40 60 t,s
Fig. 8. The relationship of inactivation area and exposure time, h = 0.5 cm
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РАЗРАБОТКА БАКТЕРИЦИДНОГО ОБОРУДОВАНИЯ И ИССЛЕДОВАНИЕ ПРОЦЕССОВ ОБЕЗЗАРАЖИВАНИЯ ПАТОГЕННЫХ МИКРООРГАНИЗМОВ ХОЛОДНОЙ АРГОНОВОЙ ПЛАЗМОЙ
Александр Петрович СЕМЕНОВ1, Баир Батоевич БАЛДАНОВ1, Цыремпил Валерьевич РАНЖУРОВ1, Эрдэм Олегович НИКОЛАЕВ1, Саяна Владимировна ГОМБОЕВА2
1 Институт физического материаловедения СО РАН 670047, г. Улан-Удэ, ул. Сахъяновой, д. 6
2 Восточно-Сибирский государственный университет технологий и управления 670013, г.Улан-Удэ, ул. Ключевская, д. 40В, стр. 1
Показана высокая эффективность бактерицидного действия холодной аргоновой плазмы генерируемой плазменными струями слаботочного искрового разряда при атмосферном давлении. Установлено, что прямой контакт плазменных струй слаботочной искры позволяет проводить эффективную инактивацию бактерий микроорганизмов.
Ключевые слова: аргоновая плазма, микроорганизмы, слаботочная искра, плазменные струи, плазменная инактивация, тлеющий разряд атмосферного давления.
Семенов А.П. - д.т.н., профессор, директор, e-mail: semenov@ipms.bscnet.ru
Балданов Б.Б. - к.т.н., с.н.с. лаборатории плазменно-энергетических процессов и технологий,
e-mail: baibat@mail.ru
Ранжуров Ц.В. - м.н.с. лаборатории плазменно-энергетических процессов и технологий Николаев Э.О. - аспирант
Гомбоева С.В. - к.б.н., доцент, кафедра биотехнологии
