低温等离子体和脉冲电场灭菌技术
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摘要
等离子体和脉冲电场是新兴环境友好的低温灭菌技术。在两个电极之间施加高压脉冲电时,产生较为均匀的脉冲电场但并不发生放电或击穿,是为脉冲电场处理。两个高压电极之间由于局部场强过大而发生放电或击穿并伴随活性粒子、光辐射、脉冲电场、冲击波等复合物理化学效应,则为等离子体处理。本文介绍了三种不同的等离子体/脉冲电场工艺在净化水中微生物的应用,以开发成套的低温净化装备。
沿面脉冲等离子体系统采用自制脉冲电源和2.5L不锈钢反应器。处理1.6L去离子水时,脉冲放电从悬于水面上方的高压针尖沿水面击穿到接地的筒壁。典型的脉冲电压、脉冲电流、单脉冲能量和脉冲频率分别为18kV、2.5kA、10J和2pps。等离子体的灭菌效率与注入水中的能量密度呈正相关。当能量密度小于2J/mL时,等离子体可将水中的微生物降低4-6个对数,包括大肠杆菌、枯草芽孢杆菌、细菌孢子和白色假丝酵母。细胞的初始密度和水的电导率是影响等离子体灭菌效率的重要因素。水中的初始细胞密度越低,所有细胞越容易被杀光。初始细胞密度低于7×104cfu/mL时,所有细菌在60个脉冲处理之后被全部杀光,能耗约为0.1kWh/m3.细胞密度高于106cfu/mL之后,由于死细胞对活细胞的保护作用,300个脉冲处理之后还有少量残余,在活菌中加入高压蒸汽灭活的死菌可证实这一过程。水的电导率提高后,脉冲电压电流波形发生改变,脉冲等离子体的紫外发射光谱变弱,在水相形成的活性粒子如过氧化氢、臭氧和硝酸也相应减少。当电导率高于2mS/cm之后,等离子体的灭菌效率显著下降。结合紫外发射光谱,以及水下石英管中的灭菌效果,可知NOβ和NOγ自由基产生的紫外辐射是等离子体灭菌的重要原因。水中含与DNA有相似紫外吸收光谱的尿苷时,等离子体的灭菌过程受到抑制。细胞在等离子体处理过程中保持形态完整,但是通过观察含胞内GFP的大肠杆菌的荧光变化可知,细胞内部的蛋白质遭到了快速的破坏。
沿面脉冲等离子体灭菌系统实现了500L/h的连续水处理,但是,连续处理的灭菌效率远低于静态处理。减小水的流量或者提高脉冲频率可以增大注入水中的能量密度,从而提高等离子体的灭菌效率。等离子体处理自来水的灭菌效率高于去离子水,这与自来水中的微量有机物有关。当大肠杆菌初始细胞密度在103-106cfu/mL,能量密度小于2J/mL时,单次通过反应器细胞密度下降1.2个对数。
与传统的脉冲电场灭菌工艺相比,我们在极低的脉冲场强下实现了淡水的低温灭菌。采用双极性高频脉冲电源,电压为2-6kV,脉宽为25μs,频率为100-3000Hz,1.2L轴筒式不锈钢反应器内获得的场强为0.6-1.7kV/cm。水的电导率对脉冲电压电流波形和灭菌效率有很大影响,电导率为25μS/cm时获得脉冲方波和最佳灭菌效果。场强为1.7kV/cm时,120000个脉冲处理后大肠杆菌密度下降2-4个对数,消耗的能量密度为70J/mL,水温小于25℃。
利用双极性高频脉冲电源产生的氩气等离子体射流可高效杀灭琼脂平板表面的大肠杆菌,形成直径数厘米的杀菌斑。当脉冲电压为2-4kV,脉冲频率为1.3kHz时,氩气等离子体射流的功率约为1.5W。用来处理酶标板小孔内的250μL菌液时,在0.5-2.5min内可将大肠杆菌细胞密度下降6个对数。等离子体射流的灭菌效率随脉冲电压、脉冲频率和等离子体能量密度的增大而提高,随处理间距和液体厚度的减小而提高。虽然水中的细菌可被高效的杀光,但消耗的等离子体能量密度在400-2000J/mL之间。可见低温等离子体射流只适合于表面处理,而不适于大规模水处理。
As emerging disinfection techniques, Pulsed Electric Field (PEF) and Non-Thermal Plasma (NTP) are environmentally friendly and of low temperature. PEF is realized by applying a high voltage pulse over two electrodes inside water to generate electric field without electrical breakdown. NTP is generated by high voltage discharges inside water or above water surface. During the NTP process, physical and chemical effects such as reactive species, ultraviolet radiation, strong electromagnetic fields, shock wave, and etc. are produced. In this dissertation, we studied three NTP/PEF processes for water disinfection. The aim of this work is to develop non-thermal decontamination devices.
A homemade pulsed plasma system includes a pulsed power source and a2.5L stainless steel reactor filled with1.6L deionized water. Air plasma is generated from the tip of high voltage electrodes to the grounded reactor wall via the water surface. The typical vales of peak voltage, peak current, energy per pulse, and repetition rate are18kV,2.5kA,10J, and2pulses per second (pps), respectively. There is a positive correlation between injected energy density and plasma disinfection efficiency. When energy density is2J/mL, the4-6order reduction can be obtained for microorganism such as Escherichia coli, Bacillus subtilis, Bacillus subtilis spores and Candida albicans. Initial cell density and water conductivity are other important factors. Cells are easily sterilized with a lower initial density. For instance, when the cell density is below7×104cfu/mL, all of them can be disinfected after60pulses with an energy consumption of about0.1kWh/m3. However, for the initial density higher than106cfu/mL, there is a so called "shield effect" of the dead cells to the live, which can lead the high-density autoclaved cells to inhibit the plasma treatment. Water conductivity can significantly affects the characteristics of pulsed discharges. When the conductivity is higher, the intensity of light emission decreases and corresponding aqueous products such as hydrogen peroxide, ozone and nitric acid becomes less. According to UV emission spectrum and disinfection of cell suspension inside quartz tubes, it is deduced that germicidal UVC of NOβ and NOγ radicals plays an important role in plasma disinfection. UV absorbents such as uridine can inhibit the process. Inactivated cell morphology almost remains the same shape, their intracellular protein like green fluorescent protein (GFP), however, is destructed rapidly according to fluorescence observation.
The homemade pulsed plasma system can be operated continuously with a water flow rate of up to500L/h. It is found that the disinfection efficiency is much lower than that for the stationary processing. The efficiency can be improved by increasing the pulse repetition rate or decreasing the water flow rate, since both of them can increase the injected energy density. Compared to the deionized water, it gives a less efficiency to the tap water due to its organic impurities. The order reduction of E. coli by single pass the reactor is1-2, with an energy density of less than2J/mL and cell density of103-106cfu/mL.
A low intensity PEF device is developed for freshwater disinfection. A repetitive bipolar pulsed power source is applied. The values of peak voltage, pulse width and pulse repetition rate are2-6kV,25μs and200-6000pps, respectively. The PEF intensity is0.6-1.7kV/cm in a1.2L rod-cylinder reactor. It is observed that water conductivity has a significant effect on disinfection efficiency. When conductivity is25μS/cm, it shows best efficiency with a square waveform. After120000pulses, the order reduction of E. coli is2-4with an energy density of70J/mL. Water temperature is below25℃during the PEF processing.
Argon (Ar) plasma jet is also realized by the bipolar pulsed power source. It can rapidly inactivate E. coli bacteria on the agar plate, with bactericidal spot of few centimeters. When the peak voltage is2-4kV and repetition rate is2000-6000pps, the power of generated Ar plasma jet is1-5W. For250μL of aqueous E. coli in microtiter plates, up to6orders reduction is obtained after0.5-2.5minute treatment. The efficiency of plasma jet disinfection is enhanced by raising the pulsed voltage or frequency as well as inducing the treatment distance and liquid depth. Although the E. coli cells in water are rapidly inactivated by plasma jet, the required energy density is as high as400-2000J/mL. Therefore the presented plasma jet is suitable for surface decontamination, but not for water treatment.
沿面脉冲等离子体系统采用自制脉冲电源和2.5L不锈钢反应器。处理1.6L去离子水时,脉冲放电从悬于水面上方的高压针尖沿水面击穿到接地的筒壁。典型的脉冲电压、脉冲电流、单脉冲能量和脉冲频率分别为18kV、2.5kA、10J和2pps。等离子体的灭菌效率与注入水中的能量密度呈正相关。当能量密度小于2J/mL时,等离子体可将水中的微生物降低4-6个对数,包括大肠杆菌、枯草芽孢杆菌、细菌孢子和白色假丝酵母。细胞的初始密度和水的电导率是影响等离子体灭菌效率的重要因素。水中的初始细胞密度越低,所有细胞越容易被杀光。初始细胞密度低于7×104cfu/mL时,所有细菌在60个脉冲处理之后被全部杀光,能耗约为0.1kWh/m3.细胞密度高于106cfu/mL之后,由于死细胞对活细胞的保护作用,300个脉冲处理之后还有少量残余,在活菌中加入高压蒸汽灭活的死菌可证实这一过程。水的电导率提高后,脉冲电压电流波形发生改变,脉冲等离子体的紫外发射光谱变弱,在水相形成的活性粒子如过氧化氢、臭氧和硝酸也相应减少。当电导率高于2mS/cm之后,等离子体的灭菌效率显著下降。结合紫外发射光谱,以及水下石英管中的灭菌效果,可知NOβ和NOγ自由基产生的紫外辐射是等离子体灭菌的重要原因。水中含与DNA有相似紫外吸收光谱的尿苷时,等离子体的灭菌过程受到抑制。细胞在等离子体处理过程中保持形态完整,但是通过观察含胞内GFP的大肠杆菌的荧光变化可知,细胞内部的蛋白质遭到了快速的破坏。
沿面脉冲等离子体灭菌系统实现了500L/h的连续水处理,但是,连续处理的灭菌效率远低于静态处理。减小水的流量或者提高脉冲频率可以增大注入水中的能量密度,从而提高等离子体的灭菌效率。等离子体处理自来水的灭菌效率高于去离子水,这与自来水中的微量有机物有关。当大肠杆菌初始细胞密度在103-106cfu/mL,能量密度小于2J/mL时,单次通过反应器细胞密度下降1.2个对数。
与传统的脉冲电场灭菌工艺相比,我们在极低的脉冲场强下实现了淡水的低温灭菌。采用双极性高频脉冲电源,电压为2-6kV,脉宽为25μs,频率为100-3000Hz,1.2L轴筒式不锈钢反应器内获得的场强为0.6-1.7kV/cm。水的电导率对脉冲电压电流波形和灭菌效率有很大影响,电导率为25μS/cm时获得脉冲方波和最佳灭菌效果。场强为1.7kV/cm时,120000个脉冲处理后大肠杆菌密度下降2-4个对数,消耗的能量密度为70J/mL,水温小于25℃。
利用双极性高频脉冲电源产生的氩气等离子体射流可高效杀灭琼脂平板表面的大肠杆菌,形成直径数厘米的杀菌斑。当脉冲电压为2-4kV,脉冲频率为1.3kHz时,氩气等离子体射流的功率约为1.5W。用来处理酶标板小孔内的250μL菌液时,在0.5-2.5min内可将大肠杆菌细胞密度下降6个对数。等离子体射流的灭菌效率随脉冲电压、脉冲频率和等离子体能量密度的增大而提高,随处理间距和液体厚度的减小而提高。虽然水中的细菌可被高效的杀光,但消耗的等离子体能量密度在400-2000J/mL之间。可见低温等离子体射流只适合于表面处理,而不适于大规模水处理。
As emerging disinfection techniques, Pulsed Electric Field (PEF) and Non-Thermal Plasma (NTP) are environmentally friendly and of low temperature. PEF is realized by applying a high voltage pulse over two electrodes inside water to generate electric field without electrical breakdown. NTP is generated by high voltage discharges inside water or above water surface. During the NTP process, physical and chemical effects such as reactive species, ultraviolet radiation, strong electromagnetic fields, shock wave, and etc. are produced. In this dissertation, we studied three NTP/PEF processes for water disinfection. The aim of this work is to develop non-thermal decontamination devices.
A homemade pulsed plasma system includes a pulsed power source and a2.5L stainless steel reactor filled with1.6L deionized water. Air plasma is generated from the tip of high voltage electrodes to the grounded reactor wall via the water surface. The typical vales of peak voltage, peak current, energy per pulse, and repetition rate are18kV,2.5kA,10J, and2pulses per second (pps), respectively. There is a positive correlation between injected energy density and plasma disinfection efficiency. When energy density is2J/mL, the4-6order reduction can be obtained for microorganism such as Escherichia coli, Bacillus subtilis, Bacillus subtilis spores and Candida albicans. Initial cell density and water conductivity are other important factors. Cells are easily sterilized with a lower initial density. For instance, when the cell density is below7×104cfu/mL, all of them can be disinfected after60pulses with an energy consumption of about0.1kWh/m3. However, for the initial density higher than106cfu/mL, there is a so called "shield effect" of the dead cells to the live, which can lead the high-density autoclaved cells to inhibit the plasma treatment. Water conductivity can significantly affects the characteristics of pulsed discharges. When the conductivity is higher, the intensity of light emission decreases and corresponding aqueous products such as hydrogen peroxide, ozone and nitric acid becomes less. According to UV emission spectrum and disinfection of cell suspension inside quartz tubes, it is deduced that germicidal UVC of NOβ and NOγ radicals plays an important role in plasma disinfection. UV absorbents such as uridine can inhibit the process. Inactivated cell morphology almost remains the same shape, their intracellular protein like green fluorescent protein (GFP), however, is destructed rapidly according to fluorescence observation.
The homemade pulsed plasma system can be operated continuously with a water flow rate of up to500L/h. It is found that the disinfection efficiency is much lower than that for the stationary processing. The efficiency can be improved by increasing the pulse repetition rate or decreasing the water flow rate, since both of them can increase the injected energy density. Compared to the deionized water, it gives a less efficiency to the tap water due to its organic impurities. The order reduction of E. coli by single pass the reactor is1-2, with an energy density of less than2J/mL and cell density of103-106cfu/mL.
A low intensity PEF device is developed for freshwater disinfection. A repetitive bipolar pulsed power source is applied. The values of peak voltage, pulse width and pulse repetition rate are2-6kV,25μs and200-6000pps, respectively. The PEF intensity is0.6-1.7kV/cm in a1.2L rod-cylinder reactor. It is observed that water conductivity has a significant effect on disinfection efficiency. When conductivity is25μS/cm, it shows best efficiency with a square waveform. After120000pulses, the order reduction of E. coli is2-4with an energy density of70J/mL. Water temperature is below25℃during the PEF processing.
Argon (Ar) plasma jet is also realized by the bipolar pulsed power source. It can rapidly inactivate E. coli bacteria on the agar plate, with bactericidal spot of few centimeters. When the peak voltage is2-4kV and repetition rate is2000-6000pps, the power of generated Ar plasma jet is1-5W. For250μL of aqueous E. coli in microtiter plates, up to6orders reduction is obtained after0.5-2.5minute treatment. The efficiency of plasma jet disinfection is enhanced by raising the pulsed voltage or frequency as well as inducing the treatment distance and liquid depth. Although the E. coli cells in water are rapidly inactivated by plasma jet, the required energy density is as high as400-2000J/mL. Therefore the presented plasma jet is suitable for surface decontamination, but not for water treatment.
引文
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