去除生物膜使用二氧化碳气溶胶没有氮气吹扫

摘要

Biofilms on surfaces can be effectively and rapidly removed by using a periodic jet of carbon dioxide aerosols without a nitrogen purge.

摘要

Biofilms can cause serious concerns in many applications. Not only can they cause economic losses, but they can also present a public health hazard. Therefore, it is highly desirable to remove biofilms from surfaces. Many studies on CO₂ aerosol cleaning have employed nitrogen purges to increase biofilm removal efficiency by reducing the moisture condensation generated during the cleaning. However, in this study, periodic jets of CO₂ aerosols without nitrogen purges were used to remove Pseudomonas putida biofilms from polished stainless steel surfaces. CO₂ aerosols are mixtures of solid and gaseous CO₂ and are generated when high-pressure CO₂ gas is adiabatically expanded through a nozzle. These high-speed aerosols were applied to a biofilm that had been grown for 24 hr. The removal efficiency ranged from 90.36% to 98.29% and was evaluated by measuring the fluorescence intensity of the biofilm as the treatment time was varied from 16 sec to 88 sec. We also performed experiments to compare the removal efficiencies with and without nitrogen purges; the measured biofilm removal efficiencies were not significantly different from each other (t-test, p > 0.55). Therefore, this technique can be used to clean various bio-contaminated surfaces within one minute.

引言

Biofilms are complex bacterial community structures in which bacterial cells are embedded within self-produced matrices of extracellular polymeric substances, held together, and protected from the external environment. Biofilms can present a public health risk and cause economic losses because they can form on various surfaces, including medical implant materials and devices, food processing equipment, and heat exchangers. In fact, biofilms have been found to be associated with 65% of all bacterial infectious diseases in humans, according to the Centers for Disease Control and Prevention¹.

Autoclaves and disinfectants such as chlorine have generally been used for the inactivation of biofilms. However, the use of an autoclave is limited for surfaces that can neither withstand high temperature steam nor be placed into the autoclave. Disinfectants are not suitable for surfaces sensitive to chemical treatment or prone to collecting toxic oxidation products². In addition, the biofilm should not only be inactivated, but also removed in order to prevent the attachment of new cells onto the surface, thereby forming a new biofilm³. However, it is difficult to remove biofilms using methods based on viscous fluid shear because the flow velocity near the surface is almost zero and the shear force usually cannot overcome the adhesive forces of micron- and submicron-sized substances. Moreover, the biofilm matrix is known to act as a physical and chemical barrier¹.

Many physical and mechanical techniques have been developed to remove biofilms from surfaces, including ultrasonic vibration¹, electric currents⁴, laser irradiation⁵, and high-pressure water sprays⁶. Each technique has its own pros and cons. Ultrasonic vibration and electric current can be used to control biofilm formation; however, they require a particular configuration and conductive surfaces, respectively, requiring additional shear stress^{1, 4}. Laser irradiation can be applied to a limited area and to hard surfaces; however, some live and dead cells remain on the surface⁵. High-pressure water sprays effectively remove biofilms; however, their high momentum can cause damage to soft substrates⁶.

Biofilm removal using CO₂ aerosols has been previously proposed. It has shown promising results, with high removal efficiencies within a short time^7-11. CO₂ aerosols are generated by adiabatic expansion of a high-pressure CO₂ gas through a nozzle, and they are applied to the surfaces contaminated with a biofilm. This cleaning technique utilizes the momentum transfer of solid CO₂ particles and the solvent action of the melted CO₂ liquids, followed by the aerodynamic shear force of the CO₂ gas¹². Compared with high-pressure N₂ gas jets, CO₂ aerosol jets at the same stagnation pressure are much more effective in removing E. coli biofilms⁷. Moreover, although the momentum of the solid CO₂ that is delivered to the bacteria is considerably high, the momentum of the total aerosol jet applied to the solid surface is substantially lower than that of water jets. Therefore, damage-free cleaning is possible using this CO₂ aerosol technique.

In this protocol, periodic jets of CO₂ aerosols without nitrogen purges were used to remove Pseudomonas putida biofilms from a polished stainless steel surface. In fact, nitrogen purges have been used in many CO₂ aerosol studies to increase the removal efficiency by reducing the moisture condensation generated during the cleaning, even though heating with a hot plate or infrared lamps and employing dry boxes have also been adopted¹². The surface biofilm formation and the optimized cleaning procedures are described below. The removal efficiency was evaluated by measuring the fluorescence intensity of the biofilm on the surface.

研究方案

1.表面的制备生物膜形成

1. 切断用机械切刀1毫米厚的304不锈钢板成芯片（10×10毫米^2）。
2. 每10分钟进行的丙酮，水依次甲醇和去离子（DI）芯片的超声波清洗。使用的耐溶剂容器，制成的物质如玻璃，以除去有机污染物。
3. 有3-5秒的流动DI水冲洗芯片。
4. 使用3-5秒_的 N ₂气流干燥该芯片。

2.细菌培养的制备

1. 以一个P.恶臭假单胞菌 KT2440储存在-80℃深冷冻股票（由宋谷李教授，UNIST，韩国友情提供）。
2. 解冻在室温下1分钟后，与冷冻储液的顶层变为融雪。浸入一个循环到原液的熔化层。
3. 使用此循环连胜细菌到卢里亚含有1.5％琼脂-Bertani（LB）板上。
4. 过夜孵育所述板在30℃下生长的细菌菌落。
5. 使用一个新的循环板挑取单个菌落。
6. 接种10毫升LB肉汤中在与含有单菌落环路50ml锥形管中。
7. 孵育在振荡培养箱的肉汤在30℃下16小时和160的转速。

3.表面生物膜的形成

1. 拿起所制备的各芯片用镊子和在70％的乙醇蘸5次，每次1-2秒，以消毒每个芯片的表面上。确保芯片保持与浸渍期间镊子。
2. 浸在蒸压DI水和肉汤LB依次每一个芯片，5次，每次1-2秒，以去除残留的乙醇。
3. 放置在6孔培养板，这些芯片具有2芯片和每孔5ml的LB肉汤。
4. 稀释细菌培养，直到在LB肉汤达到的浓度8×10 ⁸个细胞/ ml（在600nm波长的光密度:〜0.8）。
5. 用50μl稀释的细菌培养物接种每个孔中。
6. 孵育在30℃下将板不摇动用于形成生物膜的24小时。

4. _二氧化碳汽清洗

1. 浸生物膜形成芯片在10mM乙酸铵缓冲液（挥发性的）5次，以去除松散附着和浮游细菌。
2. 在生物安全柜，那里的空气流动婉转干这些芯片。
3. 立即干燥后，放置在装载位置，这是由沿射流轴线的CO ₂的喷嘴20mm的芯片。倾斜射流轴线以40°角。
4. 设置CO ₂和N ₂气体的停滞压力分别5.3兆帕和0.7兆帕，利用气体压力调节器。
5. 应用该气溶胶喷射到芯片的中心部分。白色的气溶胶包括固体_二氧化碳应是可见的。转"的"为_二氧化碳 5秒电磁阀，然后将其"关闭"3秒（周期:8秒）定期使用手动控制开关。如果氮气吹扫需要使用，打开该电磁阀的N ₂的连续供给。
6. 用CO处理芯片₂气溶胶16，40，和88秒的使用和不使用氮气清洗。芯片保留不经处理作为阴性对照。

5.分析去除效率

1. 制备1μM的绿色荧光核酸染料（激发/发射波长:500分之480纳米）在DI水中进行染色上的控制和气溶胶处理的芯片的细菌细胞。
2. 放置芯片在染色溶液。
3. 孵育在培养箱芯片没有光在37℃下30分钟。
4. 孵育后，轻轻地用流动的去离子水去除过量的荧光染料冲洗芯片。
5. 擦干芯片无线第N ₂气的流动。
6. 取荧光用落射荧光显微镜，40X物镜，和CCD照相机对每个芯片的视图5随机视野（321×240微米^2）的显微图像。
7. 获得如ImageJ的每个图像使用图像处理软件的荧光强度。在ImageJ的，使用"扣除背景"功能，在"进程"菜单，在"分析"菜单中的"设置测量"窗口中选择"集成密度"。执行"测量"中的"分析"菜单获得的荧光强度。
8. 根据下列公式计算生物膜去除效率:100％×（的控制芯片我 - 我处理芯片）/（Ⅰ的控制芯片），其中I是计算的荧光强度。
9. 获得平均去除率和标准偏差。在使用至少4芯片每种情况。

结果

_二氧化碳气溶胶被用来去除P.恶臭从SUS304表面（ 图1）生物膜。大部分表面被后生长24小时覆盖有生物膜。大多数生物膜的是使用_二氧化碳气溶胶（ 图2）除去。正如预期的那样， 图3示出生物膜去除效率为_二氧化碳气溶胶处理时间增加的增加。为88秒的处理时间，除去效率被确定为高达97.74％和有和没有氮气吹扫，分别98.29％。?...

讨论

Previously, we conducted optimization studies on CO₂ gas pressure, jet angle, and distance to the solid surface in CO₂ aerosol cleaning⁷. Unlike our previous studies, in the present study, a nitrogen purge was not included in the aerosol (Figure 1). Moreover, 304 stainless steel was used in this protocol, since it is one of the most common stainless steels and is widely used in the food industry. The polished surface is beneficial for fluorescence analysis because of a un...

材料

Name	Company	Catalog Number	Comments
304 stainless steel	Steelni (South Korea)		Polished and diced ones
Ultrasonic cleaner	Branson (USA)	5510E-DTH
Luria-Bertani (LB)	Becton, Dickinson and Company (USA)	244620	500 g
Agar	Becton, Dickinson and Company (USA)	214010	500 g
6-well culture plate	SPL Life Sciences (South Korea)	32006
Ammonium acetate buffer	Sigma-Aldrich (USA)	667404	10 mM
Dual gas unit	Applied Surface Technologies (USA)	K6-10DG	One nozzle for CO2 gas & 8 nozzles for N2 gas
SYTO9	Thermo Fissher Scientific (USA)	Invitrogen	Excitaion: 480 nm Emission: 500 nm
Epifluorescence microscope	Nikon (Japan)	Eclipse 80i
40X objective lens	Nikon (Japan)	Plan Fluor	NA: 0.75
CCD camera	Photometrics (USA)	Cool SNAP HQ2	Monochrome