Summary

Удаление биопленки с использованием диоксида углерода Аэрозоли без продувки азотом

Published: November 06, 2016
doi:

Summary

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

Abstract

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.

Introduction

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 stress1, 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 time7-11. 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.

Protocol

1. Подготовка поверхности для биопленки Вырезать 1 мм толщиной 304 пластины из нержавеющей стали в щепу (10 × 10 мм 2) с механическим резаком. Выполнение ультразвуковой очистки щепы в ацетоне, метаноле, и деионизированную (ДИ) воды последовательно в течение 10 минут каждый. Ис?…

Representative Results

CO 2 аэрозолей были использованы для удаления P. putida биопленки из SUS304 поверхностей (рисунок 1). Большая часть поверхности были покрыты биопленки после 24 часов роста. Большая часть биопленки была удалена с помощью СО 2 аэрозолей (рисунок 2). Как и следовало ?…

Discussion

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…

Disclosures

The authors have nothing to disclose.

Acknowledgements

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT, and Future Planning (# 2015R1A2A2A01006446).

Materials

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
40× objective lens Nikon
(Japan)
Plan Fluor NA: 0.75
CCD camera  Photometrics
(USA)
Cool SNAP HQ2 Monochrome

References

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Cite This Article
Hong, S., Jang, J. Biofilm Removal Using Carbon Dioxide Aerosols without Nitrogen Purge. J. Vis. Exp. (117), e54827, doi:10.3791/54827 (2016).

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