Biofilm Removal Using Carbon Dioxide Aerosols without Nitrogen Purge

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 chl....

Protocol

1. Preparation of Surface for Biofilm Formation

1. Cut 1-mm-thick 304 stainless steel plates into chips (10 × 10 mm²) with a mechanical cutter.
2. Perform ultrasonic cleaning of the chips in acetone, methanol, and deionized (DI) water sequentially for 10 min each. Use a solvent-resistant container, made of substances such as glass, to remove organic contamination.
3. Rinse the chips with flowing DI water for 3-5 sec.
4. Dry the chips using N₂ gas flow for 3-5 sec.

2. Preparation of Bacterial Culture

1. Take a P. putida KT2440 (kindly provided by Prof. Sung Kuk....

Results

CO₂ aerosols were used to remove the P. putida biofilms from SUS304 surfaces (Figure 1). Most of the surfaces were covered with the biofilm after 24 hr of growth. Most of the biofilm was removed using the CO₂ aerosols (Figure 2). As expected, Figure 3 shows an increase in biofilm removal efficiency as the CO₂ aerosol treatment time increased. For the treatment time of 88 sec, the removal efficiency was determined to be as high as.......

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.......

Materials

List of materials used in this article

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